Embed Size (px)
Citation preview
1
CHAPTER ONE
1.1 Introduction
In many parts of the world, water supplies for domestic consumption, agriculture and industrial
uses are no longer able to keep up with demand. Water reuse is becoming more commonly
considered as a viable option for addressing these needs. Indirect reuse of wastewater have been
reported in many places, however direct potable reuse may present a number of treatment
challenges; in terms of needing to achieve high levels of pathogen reduction and elimination of
micro-pollutants, as well as public acceptance issues, thus water quantity and quality issues are
both of concern (Michael and David, 2011).
Wastewater may contain millions of bacteria per milliliter including coliforms, Streptococci,
Staphylococci, anaerobic spore forming bacilli, Salmonella, Proteus, and many other types of
organisms. Wastewater is also a potential source of many human pathogenic forms including
bacteria, viruses, and protozoa (Younis et al., 2003).
Despite large advances in water and wastewater treatment, waterborne diseases still poses a
major worldwide threat to public health (UN-HABITAT, 2010). The presence of microbial
pathogens in polluted, untreated and treated water presents a considerable health risk to both
humans and animals. Furthermore, some studies have shown that the conventional wastewater
treatment do not guarantee their complete elimination (Howard et al., 2004; Espigares et al.,
2006). This results in increased incidences of waterborne diseases with far reaching socio-
economic and public health implications.
Selection of wastewater treatment process depends on the wastewater composition, such as
biological oxygen demand, pH, suspended solids, presence of toxic compounds and nutrients
(Negm et al., 1995). However, there is an increasing concern over a range of microbial
pathogens which may escape conventional wastewater treatment and pass into receiving water
(Michael and David, 2011).
Salmonella species have been frequently isolated from wastewater and are known to cause
severe disease symptoms that range from self-limiting diarrhea to bacteremia. Salmonella species
are the etiological agents of a wide range of diseases such as salmonellosis and typhoid fever and
2
are among the leading causes of gastroenteritis worldwide. There are 16 million annual cases of
typhoid fever, 1.3 billion cases of gastroenteritis and 3 million deaths worldwide due to
Salmonella (CDC, 2005; Bhunia, 2008). More than 2,540 Salmonella serotypes have been
identified, based on somatic (O), flagellar (H) and capsular (Vi) antigenic profile (Popoff et al.,
2004).
Traditionally, detection and enumeration of pathogenic bacteria including Salmonella have been
largely based on the use of selective culture and standard biochemical methods. This approach
requires confirmatory test of all typical and atypical colonies on selective plates and it becomes
very cumbersome to identify these suspected Salmonella isolates (Rakesh et al., 2009). Although
these methods are simple and inexpensive but such methods suffer from a number of drawbacks.
Despite the fact that most bacterial pathogens can be easily cultured, there are a number of
problems associated with attempts to detect and quantify them in wastewater samples. Problems
associated with the direct culture of bacterial species include difficulties in identification of
bacterial pathogens, the time and expense involved in identifying and typing of bacterial isolates
and the effect of selective media and/or selective isolation methods. Another dilemma frequently
encountered is that viable bacterial strains in the environment can enter a dormancy state where
they are non-culturable (viable-but-non-culturable state) which can cause an underestimation of
pathogen numbers or a failure to isolate a pathogen from wastewater (Alexandrino et al., 2004).
Consequently, rapid, accurate and culture-independent alternatives are being investigated to
enable monitoring of pathogens in water and wastewater. Different array of tests have been
developed in the form of miniaturized biochemical kits, immunoassays and DNA-based tests for
rapid screening of large number of wastewater samples (Straub and Chandler, 2003).
In epidemiological studies, biotyping, serotyping, phage typing and antimicrobial typing
methods have been frequently used for characterization of Salmonella serotypes from different
environments. Microbial typing methods have been used for a wide range of microorganisms,
but none of these typing methods offers an ideal approach for subtyping microbial species
(Rakesh et al., 2009).
Different molecular typing methods based on variations in genetic makeup have been now used
in complement with traditional typing methods for fingerprinting of Salmonella serotypes.
3
Nucleic acid, protein and lipoppolysaccharides are the only macromolecules that carry
information in their sequences and compositions to allow the study of microbial diversity
(Rakesh et al., 2009). Hence, the combination of different typing methods may be the best
approach to characterize Salmonella isolates.
1.1.1 Statement of Problem
Wastewater generally contains significantly high concentration of pathogens. Salmonella are
enteric pathogens and have also been associated with most waterborne diseases. The presence
and survival of Salmonella in wastewater despite treatment poses a serious public health concern
to wildlife and human health.
In view of the global prevalence of salmonellosis, there is an urgent need to employ techniques
that not only identify and characterize Salmonella species but also facilitate epidemiological
studies to trace the source of infection, thereby facilitating the formulation of effective control
strategies.
1.1.2 Aim of the study
The aim of this research is to investigate the occurrence and identity of Salmonella serovars
present in the University of Nigeria, Nsukka wastewater treatment plant, and to evaluate the
sensitivity and precision of different microbial (conventional and molecular) typing methods to
detect and characterize Salmonella species in wastewater.
1.1.3 Research Objectives
i. Isolation and Characterization of Salmonella serovars from the wastewater treatment
plant, UNN.
ii. Serotyping of Salmonella serovars found.
iii. Isolation of specific Salmonella phages from wastewater.
iv. RAPD based fingerprinting of Salmonella serovars present.
v. Identification of Salmonella serovars with invA gene.
4
1.2 Literature Review
1.3 Wastewater and Wastewater Treatment
Water is crucial for all aspects of life, the defining feature of our planet. It plays a vital role in the
sustenance of all life and it is a source of economic and political power (Narasimhan, 2008).
Fresh, accessible water is a scarce and unevenly distributed resource, not matching patterns of
human development. At the beginning of the 21st century, the world faces a water quality crisis,
caused by continuous population growth, industrialization, food production practices, increased
living standards and poor water use strategies (UNDESA 2009).
Ninety seven and a half per cent of all water is found in the oceans; only one per cent of the
remaining freshwater is accessible for extraction and use. Worldwide, nearly 900 million people
still do not have access to safe water (UNDESA 2009), and some 2.6 billion, almost half the
population of the developing world do not have access to adequate sanitation (WHO/UNICEF,
2010). Furthermore, over 80 per cent of people with unimproved drinking water and 70 per cent
of people without improved sanitation live in rural areas (DFID, 2008).
Estimates of the global burden of water-associated human diseases provide a simple index hiding
a complex reality. At least 1.8 million children under five years die every year due to water
related disease, or one every 20 seconds (WHO, 2008). WHO estimates that worldwide some 2.2
million people die each year from diarrhoeal disease, 3.7 per cent of all deaths and at any one
time, over half of the world’s hospitals beds are filled with people suffering from water related
diseases (UNDP 2006).
It is difficult to tease out which fraction of the disease burden can be attributed to the poor
management of wastewater. The burden of disease is about more than just mortality; it also takes
into account the proportion of healthy life years lost.
The role of wastewater in human ill-health can pass through one of two transmission pathways:
the faecal-oral pathway (i.e. disease-causing microbes originating from faecal contamination that
find their way into the body when water is ingested); or the ecosystem, where wastewater
collects, providing an ecological niche for the propagation of certain human diseases vectors
(DFID, 2008).
5
1.3.1 Wastewater
Wastewater can mean different things to different people with a large number of definitions in
use. However, wastewater can be defined as a combination of one or more of: domestic effluent
consisting of blackwater (excreta, urine and faecal sludge) and greywater (kitchen and bathing
wastewater); water from commercial establishments and institutions, including hospitals;
industrial effluent, stormwater and other urban run-off; agricultural, horticultural and aquaculture
effluent, either dissolved or as suspended matter (Raschid-Sally and Jayakody, 2008).
Wastewater, also called sewage is mostly water by mass (99.9%). The contaminants in
wastewater include suspended solids, biodegradable dissolved organic compounds, inorganic
solids, nutrients, metals and pathogenic microorganisms. The suspended solids in wastewater are
primarily organic particles composed of body wastes (i.e. faeces), food waste, and toilet paper,
while inorganic solids in wastewater include surface sediments and soil, as well as salts and
metals (Michael and David, 2011).
As water travels through the hydrological system from the mountain summit to the sea, the
activities of human society capture, divert and extract, treat and reuse water to sustain
communities and economies throughout the watershed (agricultural, industrial and municipal).
These activities do not return the water they extract in the same condition. A staggering 80–90
per cent of all wastewater generated in developing countries is discharged directly into surface
water bodies (UN Water, 2008).
Wastewater can be contaminated with a myriad of different components; pathogens, organic
compounds, synthetic chemicals, nutrients, organic matter and heavy metals. They are either in
solution or as particulate matter and are carried along in the water from different sources and
affect water quality. These components can have (bio-) cumulative, persistent and synergistic
characteristics affecting ecosystem health and function, food production, human health and
wellbeing, and undermining human security (Appelgren, 2004; Pimentel and Pimentel, 2008).
Unmanaged wastewater can be a source of pollution, a hazard for the health of human
populations and the environment alike. The Millennium Ecosystem Assessment (2005) reported
that 60 per cent of global ecosystem services are being degraded or used unsustainably, and
highlighted the inextricable links between ecosystem integrity, human health and wellbeing.
6
1.3.1.1 Sources and variability in wastewater flow
Wastewater originates from domestic, commercial, and industrial sources. In many networks, the
domestic component is the largest. The defining variable is domestic water consumption, which
is linked to human behavior and habits. Domestic water use is linked to a number of variables
including; climate, demography, development type, and socio-economic factors (Viessman and
Hammer, 1998).
The toilet is the appliance which contributes the most to household water demand, accounting for
almost a third of total domestic water use, followed by showers/baths, sinks and laundry
machines; even though toilets do not have as large a volume compared to other appliances, they
have a higher frequency of usage. Per capita water consumption may well decrease in future
given the increased emphasis on water conservation and efficiency (Tebbutt, 1998; Michael and
David, 2011).
Commercial water use includes the water used in the shops, offices and light industrial units, as
well as restaurants, laundries, public houses, and hotels. Water demand is mainly generated from
drinking, washing and sanitary facilities. Toilets/urinal usage is an even higher component of the
total water consumption than in households, accounting for nearly half of the total water demand
in commercial buildings (Michael and David, 2011).
Industrial water use can be a very important contributor of wastewater flow depending on the
region and the nature of the industry. Industrial wastewater effluent may originate from
processing (e.g. manufacturing, waste and by-product removal, transportation), cooling,
cleaning, as well as sanitary uses. The rate of discharge varies significantly from industry to
industry and is generally expressed in terms of water volume used per mass of product (e.g.
papermaking is 50-150 m3/tonne, dairy products are 3-35 m
3/tonne) . The timing of generation of
industrial effluents can be highly variable depending on operational start-ups and shutdowns,
batch discharges, and working hours (Michael and David, 2011).
Water may also ingress into a foul sewer as infiltration, which is when extraneous groundwater
or water from other nearby pipes enters the sewer through defective drains and sewers (e.g.
cracked/leaking sewer pipes), pipe joints and couplings, or manholes. In addition, water may
ingress into a foul sewer by what is called inflow, which is when stormwater enters the foul
7
sewer through either accidental or illegal misconnections, yard gullies, roof downpipes, or
through manholes covers (Tebbutt, 1998; Viessman and Hammer, 1998).
1.3.2 Wastewater treatment
The aim of wastewater treatment is to enable wastewater to be disposed safely, without
constituting a danger to public health and without polluting watercourses or causing other
nuisance. Increasingly, another important aim of wastewater treatment is to recover energy,
nutrients, water, and other valuable resources from wastewater (Michael and David, 2011).
The choice of unit processes to include in the treatment train takes into account a number of
criteria including; energy requirements, effectiveness in removing a particular target
contaminant, sludge generation and disposal requirements, complexity, reliability/robustness,
flexibility/adaptability, operation and maintenance cost, personnel requirements, construction
costs and total costs (Metcalf and Eddy, 2002; Hammer and Hammer, 2011).
1.3.2.1 Unit processes in wastewater treatment
Unit processes are individual treatment options for treating wastewater using either physical
forces (e.g. gravity settling), biological reactions (e.g. aerobic, anaerobic degradation), or
chemical reactions (e.g. precipitation) (Hammer and Hammer, 2011).
A treatment train consists of a combination of unit processes designed to reduce wastewater
contaminants to acceptable levels. Many different configurations and combinations of unit
processes are possible to make up a treatment train, but a number of standard approaches have
evolved (Metcalf and Eddy, 2002; Hammer and Hammer, 2011).
1.3.2.2 Preliminary treatment
The aim of this treatment process is to remove large and/or heavy debris which would otherwise
interfere with subsequent unit processes or damage pumps and other mechanical equipment in
the treatment works. Typically, preliminary treatment includes screening and grit removal steps
(Butler and Davis, 2011).
Screening
Screening is the first step of treatment in the wastewater treatment works. The objective of
screens is to remove large floating debris, such as rags (~60%), paper (~25%), and plastics
8
(~5%). The materials that are removed from the water by the screens are referred to as
screenings. Screenings have a bulk density of approximately 600-1000 kg/m3, moisture content
of 75-90%, and volatile content of 80-90%. Common types of screens are bar screens, drum
screens, cutting screens, and band screens (Qasim, 1998).
Grit removal
The second step of preliminary treatment immediately downstream of screening is normally grit
removal. Grit includes heavy inorganic particles such as sand, gravel, and other heavy particulate
matter (e.g. corn kernels, bone fragments). For design purposes, grit is normally considered as
fine sand with a diameter of 0.2 mm, specific gravity of 2.65 mm, and a settling velocity of 20
mm/s. As with screenings, there are no established standard test procedures to determine grit
characteristics, however the bulk density is approximately 800 kg/m3, the moisture content
ranges between 10-85%, and the volatile content is typically 10-30%. Grit has the physical
characteristics of saturated sand (i.e. heavy, moderately cohesive, and should be low in organic
content (Qasim, 1998; Butler and Davis, 2011).
Grit is removed by settling in grit channels. The two main types of grit channels are constant
velocity grit channels and aerated grit channels. The quantity of grit removed varies widely,
depending on the type of sewer network (combined versus separate), local geology (i.e. dictating
the type of sand/gravel that may occur as grit in the wastewater) and other factors (Qasim, 1998).
1.3.2.3 Sedimentation
Wastewater contains impurities which in flowing water will remain in suspension but in
quiescent water will settle under the influence of gravity. The sedimentation process, also called
‘settling’ or ‘clarification’, exploits this phenomenon and is used for the separation of solids
from water and the concentration of separated solids. Sedimentation is used in both the primary
and secondary treatment stages of wastewater treatment. There are four classes of particle
settling; discrete settling, flocculent settling, hindered settling, and compression settling (Metcalf
and Eddy, 2002).
9
1.3.2.4 Biological treatment
The aim of biological treatment is to transfer dissolved organic contaminants (e.g. BOD) from a
soluble form into suspended matter in the form of cell biomass, which can then be subsequently
removed by particle-separation processes.
The most effective biological processes for removing dissolved organics in this way are aerobic
processes, since they are fastest and their products are relatively inoffensive (H2O, CO2).
Typically oxygen must be added to the wastewater to support the aerobic process, either through
bubbling air into the water or through mixing (Tebbutt, 1998).
Conceptually, the aerobic process can be simplified as:
Organic Matter + Bacteria + O2 New Cells (Biomass) + CO2, H2O, NH3
Biological treatment processes can be classified as either ‘suspended growth’ processes, wherein
the bacterial cells are suspended in the water column in a tank, or ‘attached growth’ (also called
fixed film) processes, wherein the cells are attached onto a surface as a biofilm and the water is
passed over the surface. Suspended growth biological treatment is commonly referred to as
‘activated sludge’, whereas attached growth process can take various forms, including trickling
filters and rotating biological contactors (Michael and David, 2011).
1.3.2.5 Disinfection
Wastewater treatment often includes a disinfection step at the end of the process (after secondary
clarification). Disinfection may be included to protect particularly sensitive receiving water. The
most commonly used disinfectants in wastewater treatment are chlorination and ultraviolet (UV)
disinfection (Tebbutt, 1998).
Chlorination involves the addition of either chlorine gas or sodium hypochlorite solution into the
wastewater in sufficient quantities to overcome the chlorine demand of the wastewater, and then
allowing the residual chlorine (normally a few milligrams per litre) to remain in contact with the
water for a set period of time (normally on the order of several minutes) in a baffled chlorine
contact tank. The residual chlorine is then normally eliminated by a de-chlorination step prior to
discharge of the effluent, to prevent chlorine release into the aquatic environment. A drawback of
chlorination is the formation of a number of toxic chemical by-products due to the reaction of
chlorine with organic compounds in the wastewater (Tebbutt, 1998).
10
UV disinfection is a popular disinfection alternative in wastewater treatment since it does not
require a contact tank, and does not require a step to neutralize the active disinfecting agent (as in
de-chlorination). Furthermore, it produces few by-products compared to chlorination (Keller et
al., 2003).
UV disinfection reactors are typically open channel, with the water spending only seconds in
contact with the UV light. The UV light is generated by mercury arc-discharge lamps which are
contained in quartz sleeves and oriented either perpendicular or parallel to the flow. UV reactors
for wastewater treatment are typically designed to deliver a UV fluence of between 20-100
mJ/cm2, which provides high levels of inactivation of indicator organisms (e.g. total and faecal
coliform bacteria) as well as a range of waterborne pathogens (Michael and David 2011).
Suspended solids in the effluent can limit the performance of both chemical and UV disinfection
processes, by shielding target pathogens from the disinfecting agent. This explains why the
disinfection process is normally applied at the end of the wastewater treatment process, when
suspended solids concentrations are lowest (Keller et al., 2003).
1.3.2.6 Sludge treatment
Sewage sludge is collected from primary and secondary treatment and must be treated. In its
untreated state it is malodorous and contains pathogens, toxic elements and compounds. Sludge
treatment is aimed at reducing the volume and bulk of the sludge, reducing pathogens in the
sludge, minimizing the cost of disposal and transport of the sludge, reducing odor and vector
attraction to the otherwise putrescible solids, satisfy environmental requirements and public
concerns and to generate energy (Metcalf and Eddy, 2002).
Primary sludge contains inorganic solids as well as coarse organic solids. It is more granular and
concentrated than secondary sludge. Primary sludge is typically 2-6% dry solids by mass
(Qasim, 1998).
Secondary sludge is mainly composed of biological solids. Its composition is more variable than
primary sludge; depending on process variables (e.g. attached growth processes produce more
particulate sludge, whereas activated sludge produces light, flocculent sludge). Secondary sludge
is typically 0.5-2% dry solids for activated sludge processes, or 4-7% for humus from attached
growth processes (Qasim, 1998).
11
Sludge treatment involves a number of steps and process options. The selection and extent
depends on factors such as the site-specific composition of the sludge, the available budget for
sludge treatment, and the intended fate of the final treated sludge. Generally these can be
considered in the following common steps: thickening, pre-treatment, digestion (also called
stabilization), conditioning, de-watering, thermal reduction and end-use or disposal (Metcalf and
Eddy, 2002). Other processes for treating sewage sludge which may be used in combination with
or in place of anaerobic digestion include; incineration, composting, thermal drying, pyrolysis,
gasification, alkali (lime) treatment, aerobic digestion, pasteurization, thermal hydrolysis and
novel processes (e.g. gamma irradiation) (Michael and David, 2011).
12
1.4 Genus Salmonella
1.4.1 Background-historical
The study of Salmonella began with Eberth’s first recognition of the organism in 1880, and the
subsequent isolation of the bacillus responsible for human typhoid fever by Gaffky (Le Minor,
1991). Further investigations by European workers characterized the bacillus and developed a
serodiagnostic test for the detection of this human disease agent (Tindall et al., 2005). Thereafter,
D.E. Salmon isolated the bacterium then thought to be the etiological agent of hog cholera, but
later disproved. The genus was named Salmonella by Lignieres in 1900 in honour of D.E.
Salmon. Further investigations led to the isolation of other Salmonella species (Su and Chiu,
2006).
It soon became a common practice to name each new isolate based on the disease it caused or by
the species of animal from which it was isolated. In the early 20th century, great advances
occurred in the serological detection of somatic and flagella antigens within Salmonella group.
An antigenic scheme for the classification of Salmonella was first proposed by White and
subsequently expanded by Kauffmann into Kauffmann-White scheme, which currently includes
more than 2540 serovars (Popoff and Le Minor, 2005).
1.4.2 Taxonomy and nomenclature
Salmonella nomenclature is very complex and scientists used different systems to refer to and
communicate about this genus. Unfortunately, current usage often combines several
nomenclature systems that divide the genus into species, subspecies, subgenera, groups,
subgroups, and serotypes (serovars), and all these usages caused lots of confusion among
researchers (Rakesh et al., 2009).
Salmonella nomenclature has progressed through a succession of taxonomical and serological
characteristics and on the principles of numerical taxonomy and DNA homology (Tindall et al.,
2005). The nomenclature for the genus Salmonella has evolved from the initial one serotype-one
species concept proposed by Kauffmann in 1966 on the basis of somatic (O), flagellar (H) and
capsular (Vi) antigens. In the early development of the taxonomic scheme, biochemical reactions
were used to separate Salmonella into subgroups and the Kauffmann-White scheme was the first
attempt to systematically classify Salmonella using scientific parameters. Thus, the effort
13
culminated into the development of five biochemically defined subgenera (I-V) where individual
serovars were designated status of a species. Subsequently, three species nomenclature system
was proposed using 16 discriminating tests to identify Salmonella typhi, Salmonella
choleraesuis, and Salmonella enteritidis and later this scheme recognized members of Arizona
group as a distinct genus (Ewing, 1972; Su and Chiu, 2006).
The scientific development in Salmonella taxonomy occurred in 1973 when Crosa et al. (1973)
demonstrated using DNA-DNA hybridization that all serotypes and sub-genera I, II, and IV of
Salmonella and all serotypes of Arizona were related at the species level. Thus, they belonged to
a single species, and the exception later described was called Salmonella bongori, previously
known as subspecies-V. However, based on the multilocus enzyme electrophoretic pattern,
Salmonella enterica subsp. bongori was designated into a new species called Salmonella bongori
(Reeves et al., 1989). Thereafter, Salmonella choleraesuis was designated as species name.
Salmonella choleraesuis, causative agent of swine salmonellosis, appeared on the “Approved
List of Bacterial Names” as the type species of Salmonella; it therefore had priority as the
species name. The name “Choleraesuis”, however, refers to both a species and a serotype, which
caused more confusion for bacteriologist. In addition, the serovar Choleraesuis is not
representative of the majority of serotypes because it is biochemically distinct, being arabinose
and trehalose negative. Other taxonomic proposals have been made based on the clinical role of a
strain and biochemical characteristics that divided the serovars into subgenera (Brenner et al.,
2000; Ezaki et al., 2000).
The antigenic formulae of Salmonella serovars are defined and maintained by the World Health
Organization (WHO) Collaborating Centre for Reference and Research on Salmonella at the
Pasteur Institute, Paris (Popoff et al., 2004). Presently, Salmonella genus consists of two species:
Salmonella enterica and Salmonella bongori. Salmonella enterica is further divided into six
subspecies; S. enterica subsp. enterica (I), S. enterica subsp. salamae (II), S. enterica subsp.
arizonae (Illa), S. enterica subsp. diarizonae (lllb), S. enterica subsp. houtenae (IV), and S.
enterica subsp. indica (VI) (Popoff and Le Minor, 2005).
14
According to the recommendation of Popoff and Le Minor (1997), laboratories have to report the
names of Salmonella serovars under the different subspecies of enterica. The names of the
serovars are no longer italicized and first letter of the serovar should be written in a capital letter.
1.5 Characteristics of Salmonella
1.5.1 Morphology and isolation
Salmonella are Gram negative, facultative anaerobic, rod shaped bacteria belonging to family
Enterobacteriaceae. Members of this genus are motile by peritrichous flagella, except Salmonella
enterica serovar Pullorum and Salmonella enterica serovar Gallinarum. Salmonella are 2-3 x
0.4-0.6 µm in size and they are chemoorganotrophs, with ability to metabolize nutrients by both
respiratory and fermentative pathways (D’Aoust et al., 2001; Popoff and Le Minor, 2005).
Hydrogen sulphide (H2S) is produced by most Salmonella but a few serovars like Salmonella
paratyphi A and Salmonella choleraesuis do not produce H2S. Most Salmonellae are aerogenic;
however, Salmonella typhi does not produce gas. Members of the genus have a % G+C content
of 50-53. They are urease and Voges-Proskauer negative and citrate utilizing (Montville and
Matthews, 2008).
Most Salmonella do not ferment lactose and this property has been the basis for the development
of numerous selective and differential media for culture and presumptive identification of
Salmonella spp; they include xylose lysine decarboxycholate agar, Salmonella-Shigella agar,
brilliant green agar, Hektoen enteric agar, MacConkey’s agar, lysine iron agar and triple sugar
iron agar. Isolation of Salmonella from food and environmental samples with culture method
utilizes the multiple steps of pre-enrichment and enrichment on the selective and differential
media in order to increase the sensitivity of the detection assay (Andrews and Hammack, 2001;
Anderson and Ziprin, 2001).
Pre-enrichment is a process in which the sample is first cultured in a non-selective growth
medium such as buffered peptone water or lactose broth with the intent of allowing the growth of
any viable bacteria, and the recovery of injured cells. Subsequently, pre-enriched samples are
cultured on enrichment media to restrict the growth of undesirable bacteria. Enrichment media
commonly used include tetrathionate broth, selenite cystine broth and Rappaport Vassiliadis
broth.
15
Following the enrichment period, the enriched cultures are spread onto selective and differential
agar plate and then typical colonies for Salmonella has to be identified. Final confirmation of
typical colonies is determined by series of biochemical and serological tests (Rakesh et al.,
2009).
A few Salmonella serovars do not exhibit the typical biochemical characteristics of the genus and
these strains pose problem diagnostically because they may not easily be recovered on the
commonly used differential media. About 1% of the Salmonella serovars submitted to Centre for
Disease Control (CDC) ferment lactose; hydrogen sulphide production too was quite variable
(Ziprin, 1994). Salmonella chrome agar medium has been described very promising for detection
of both lactose positive and lactose negative Salmonella isolates from food samples (Dick et al.,
2005).
1.5.2 Physiology and biochemical characteristics
The biochemical properties of Salmonella spp show that almost all Salmonella serovars do not
produce indole, hydrolyze urea, nor deaminate phenylalanine or tryptophan. Most of the serovars
readily reduce nitrate to nitrite and most ferment a variety of carbohydrates with the production
of acid, and have been reported to be negative for Voges-Proskauer (VP) reaction. Other
prominent characteristics of this genus include the ability of most serovars to produce hydrogen
sulfide (H2S) and decarboxylate lysine, arginine and ornithine with a few exceptions (Popoff and
Le Minor, 2005).
Most Salmonella serovars utilize citrate with a few exceptions such as Salmonella Typhi,
Salmonella paratyphi A and a few Salmonella choleraesuis serovars. Dulcitol is generally
utilized by all serovars except Salmonella enterica subsp. arizonae (Illa) and Salmonella enterica
subsp. diarizonae (lllb) (Popoff and Le Minor, 2005). Lactose may not be utilized by most
Salmonella serovars, however, it has been reported that less than 1% of all Salmonella spp
ferment lactose (Ewing, 1986). Furthermore, Salmonella isolation from different sources with
routine selective and differential media utilizes non-lactose fermentation as a key biochemical
property and commonly used differential plating media for isolation of Salmonella contains
lactose.
16
Salmonella serovars are considered resilient microorganisms that readily adapt to extreme
environmental conditions. Optimum temperature for growth is in the range of 35 – 37ºC but
some can grow at temperatures as high as 54ºC and as low as 2ºC (Gray and Fedorka-Cray,
2002). Salmonella grow in a pH range of 4 - 9 with the optimum being 6.5 – 7.5. They require
high water activity (aw) for growth (> 0.94) but can survive at aw of < 0.2 such as in dried foods.
Inhibition of growth occurs at temperatures < 7ºC, pH < 3.8 or aw < 0.94 (Hanes, 2003; D’Aoust
and Maurer, 2007).
The outer membrane (OM) of Salmonella, as with almost all Gram-negative bacteria, is
composed of outer membrane proteins (OMPs) and lipopolysaccharides (LPS). LPS plays an
essential role in maintaining the cell’s structural integrity and protection from chemicals. In the
host organisms, they act as endotoxins and as a pyrogen displaying a strong immune response.
Structurally, they are composed of three distinct components: lipid A, core oligosaccharide and
O-polysaccharide (Bell and Kyriakides, 2002; Raetz and Whitfield, 2002). Table 1.1 below
shows the morphological, growth and biochemical characteristics of Salmonella species.
17
Table 1.1: Comparison of characteristics of Salmonella species.
Characteristics Salmonella enterica subsp. Salmonella
bongori enterica salamae arizonae diarizonae houtenae Indica
Classification
(roman
numeral)
Usual
Habitat
I
Warm
blooded
animals
II
Warm
blooded
animals
IIIa
Cold
blooded
animals
and
environm
ent
IIIb
Cold
blooded
animals
and
environme
nt
IV
Cold
blooded
animals
and
environme
nt
VI
Cold
blooded
animals
and
environme
nt
V
(formerly)
Cold
blooded
animals and
environment
Morphological
characteristics
Gram stain - - - - - - -
Motility + (except
pullorum
and
gallinaru
m)
+ + + + + +
Shape Rod Rod Rod Rod Rod Rod Rod
Size (width, µm) 0.7-1.5 0.7-1.5 0.7-1.5 0.7-1.5 0.7-1.5 0.7-1.5 0.7-1.5
Colony
morphologies
Bismuth sulphite
Agar
Black colonies surrounded by a brown to black zone that casts a metallic sheen
Eosin-methylene
blue agar
Translucent amber to colourless colonies
Hektoen enteric
agar
Blue to blue-green colonies mostly with black centers (H2S producers)
Salmonella-
Shigella agar
Colourless colonies on a pink background
Growth characteristics
Optimum
temperature (oC)
35-37 35-37 35-37 35-37 35-37 35-37 35-37
Optimum pH 6.5-7.5 6.5-7.5 6.5-7.5 6.5-7.5 6.5-7.5 6.5-7.5 6.5-7.5
Biochemical
characteristics
α-
glutamyltransfera
se
D + - + + + +
β-
Gluocufronidase
D d - + - D -
Dulcitol + + - - - D +
Galacturonate - + - + + + +
Gelatinase - + + + + + -
Glucose + + + + + + +
Hydrogen sulfide + + + + + + +
Indole test - - - - - - -
Lactose - - - + - + d
Lysine
decarboxylase
+ + + + + + +
18
L(+)-tartrate + - - - - - -
Malonate - + + + - - -
Methyl red test + + + + + + +
Murate + + + - - + +
Ortho-
nitrophenyl-β-D-
Galactopyranosid
e test
- - + + - D +
Phage 01
susceptible
+ + - + - + d
Potassium
cyanide broth
- - - - - - -
Salicine - - - + - + d
Sorbitol + + + + + - +
Urease - - - - - - -
Voges-Proskauer
test
- - - - - - -
Note: +, more than 90% positive reactions, -, less than 10% positive reactions, d, different
reactions given by different serovars (source: Pui et al., 2011).
19
1.6 Rapid detection method for Salmonella
1.6.1 Polymerase chain reaction (PCR)
Nucleic acid (DNA or RNA) based methods have become very popular for rapid detection of
pathogens. The first in vitro amplification of mammalian genes using the Klenow fragment of
Escherichia coli DNA polymerase was carried out by Kary Mullis (Mullis and Faloona, 1987).
This assay is now popularly known as polymerase chain reaction (PCR).
The polymerase chain reaction is a method which produces multiple copies of a target DNA. The
PCR method uses a thermostable polymerase enzyme (Taq polymerase) to create multiple copies
of target DNA. Detection of target DNA is achieved through the use of short sections of
synthetic, single stranded DNA known as oligonucleotide primers. These primers can be
designed to be specific for an individual organism, or for a group of organisms (Simon, 1999).
PCR also works by using a cycling of different temperatures. It also requires the target template
DNA, primers, dNTPs and Taq polymerase (Tenover et al., 1997). This large number of a target
DNA segment can then be detected using standard detection methods such as agarose gel
electrophoresis or membrane hybridization. The ability of PCR to produce extremely large
numbers of copies of a specific nucleic acid segment provides the requirements for the rapid,
very sensitive and specific detection of desired microorganisms in a water sample.
PCR holds great potential for the direct detection of microbial pathogens and detection of
virulence genes in water and wastewater (Malorny et al., 2003a; del Cerro et al., 2002). Specific
nucleic acid primers already exist for most of the major waterborne pathogens and have been
proven to be specific for these organisms. It is both highly specific and sensitive and is capable
of detecting very small numbers of microorganisms in a sample. In addition, multiple primers
can be used to detect different pathogens in one multiplex reaction (Ziemer and Steadham, 2003;
Moganedi et al., 2007; El-Lathy et al., 2009).
PCR based methods have been found to be very sensitive for detection of Salmonella spp in
environmental water samples and other sources (Way et al., l993; Pathmanathan et al., 2003;
Aurelie et al., 2005). PCR based detection assays for rapid and specific detection of Salmonella
in wastewater were compared with conventional method and reported; PCR method was
comparable to the culture method (Fricker and Fricker, 1995; Simon, 1999).
20
PCR does not require the culturing of microorganisms and therefore can improve detection
efficiency, time and labor. It negates the requirement for indicator organisms as pathogenic
microorganisms can be directly detected from a wastewater sample. PCR also has the advantage
of being able to be used to determine the viability of a microorganism and thus, is not restricted
by dormancy status or the ability to culture the microorganism (Simon, 1999).
There are, however, a number of outstanding problems associated with PCR which can seriously
affect its performance. The polymerase enzyme which is central to the PCR method is sensitive
to a number of environmental contaminants which can be commonly found in water and
wastewater. Some of these environmental contaminants, such as humic compounds, high
divalent cation concentrations and salt can also reduce the extraction efficiency of nucleic acids
from water samples (Wilson, 1997). Also, the great sensitivity of PCR also makes it susceptible
to false positive results due to contamination from extraneous naked nucleic acid, either from the
water sample or from the laboratory (Tsai et al., 1993).
Another major problem associated with the direct detection of microbial pathogens in water and
wastewater, which can have an effect on the efficiency of PCR, is the recovery of microbial
pathogens from water samples. While, small numbers of target nucleic acids can be detected in a
sample using PCR, the detection limit can be affected by the extraction or concentration
efficiencies for different pathogenic microorganisms from water or wastewater samples (Straub
et al., 1995b).
The use of PCR as a routine surveillance tool in the water industry still remains a potential for
the future. At present, the costs and expertise required to use these techniques remain prohibitive
for most laboratories. Rapid advances, however, have recently been made in a number of these
problem areas, promising the potential for viable solutions in the near future.
1.7 Salmonella typing methods
1.7.1 Biotyping
Salmonella strains in a particular serovar may be differentiated into biotypes by their utilization
pattern of selected substrates such as carbohydrates and amino acids. In many serovars there are
few biochemical tests in which significant numbers of strains behave differently and so the
number of identifiable biotypes within a serovar can be obtained. The organisms expressing
21
different phenotypes of a given serotype are considered a different biotype, and these differences
can be associated with differences in virulence properties (Anderson and Ziprin, 2001).
Duguid et al. (1975) developed a scheme for biotyping to study the epidemiology of infections
with Salmonella typhimurium. This scheme was based on the use of 15 biochemical characters.
Thirty-two potential primary biotypes were defined by the combinations of positive and negative
reactions shown in 5 tests (d-xylose, m-inositol, l-rhamnose, d- tartrate and m-tartrate) most
discriminating in Salmonella typhimurium. These primary biotypes were designated by numbers
(1-32) and the full biotypes were developed by an additional 10 secondary tests and finally a
total of 24 primary and 184 full biotypes have been identified.
Recently, de la Torre (2005) used the biochemical kinetic data to determine strain relatedness
among Salmonella enterica subsp. enterica isolates. Different biochemical tests results were
used in the determination of strain relatedness among different serovars of Salmonella enterica
subsp. enterica (59 Salmonella typhimurium strains, 25 Salmonella typhimurium monophasic
variant strains, 25 Salmonella anatum strains, 12 Salmonella tilburg strains, 7 Salmonella
virchow strains, 6 Salmonella choleraesuis strains, and l Salmonella enterica (4,5,l2::)
(Hoszowski and Wasyl, 2001).
1.7.2 Serotyping
The basis of Salmonella serotyping depends upon the complete determination of the different
antigens; somatic (O), flagellar (H) and capsular (Vi) antigens.
The aim of the serological testing procedure is to determine the complete antigenic formula of
the individual Salmonella isolate. Commercially available polyvalent somatic antisera kits
consist of mixtures of antibodies specific for major antibodies (Herikstad et al., 2002).
Antigen-antibody complexes are formed (agglutination) when a bacterial culture is mixed with a
specific antiserum directed against bacterial surface components. The complexes are usually
visible to the naked eye which allows for easy determination of O and H antigens by slide
agglutination. Some cultures are monophasic and may be directly H-typed, whereas the second
phase in a diphasic culture is determined after phase inversion. After full serotyping of the
Salmonella culture the name of the serotype can be determined by using the Kauffmann-White
Scheme. The serological typing of Salmonella has led to identification of large number of
22
Salmonella serovars. Currently, Kauffmann-White scheme recognizes 2610 Salmonella serovars,
the majority (2587) belongs to S. enterica, while the remaining (23 serovars) are assigned to S.
bongori (Guibourdenche et al., 2010).
1.7.2.1 Somatic (O) antigens
These are heat stable antigens which are composed of phospholipid-polysaccharide complexes.
Analysis of O antigens revealed polysaccharide (60%), lipid (20 to 30%), and hesomione (3.5-
4.5%). The nature of terminal groups and the order in which they occur in the repeating units of
the polysaccharide chain provide the specificity to the numerous kinds of O antigens (Hu and
Kpoecko, 2003).
Somatic antigens are resistant to alcohol and dilute acid. Different variants (smooth, rough) are
prevalent in Salmonella spp and these variations affect the serological typing of Salmonella. In
addition, smooth (S) to rough (R) variations occur in Salmonella (Yousef and Carlstrom, 2003).
The heat stable O antigen consist of lipopolysaccharide-protein chain exposed on the cell surface
and are classified as major and minor antigens. The major category consists of antigens such as
somatic factors O:4 and O:3, which are specific determinants of serogroups like B and E
respectively. In contrast, minor somatic antigenic components, such as O:l2 are
nondiscriminatory, as evidenced by their presence in different serogroups (D’Aoust et al., 2001).
These are heterogeneous structures and the antigenic specificity is determined by the
composition and the lineage of the O group sugars and sometimes mutation affect the sugars
leading to new O antigen (Grimont and Weill, 2007).
1.7.2.2 Flagellar (H) antigens
H-antigens are heat labile proteins associated with the peritrichous flagella and can be expressed
in one of two phases. These are heat labile antigens that are present in the flagella of Salmonella
are proteineous in nature and are called flagellin (Yousef and Carlstrom, 2003). The flagellin is a
keratinomyosin epiderm-fibrinogen group protein of 40 kDa in molecular weight. The amino
acid content and the order in which these acids present in the flagellins determine the specificity
of the different H antigens. The flagellar agglutination occurs very rapidly and the aggregates
formed are loosely knit and floccular forms (Raetz and Whitfield, 2002).
23
The phase 1 H-antigens are specific and associated with the immunological identity of the
particular serovars, whereas phase 2 antigens are non-specific antigens containing different
antigenic subunit proteins which can be shared by many serovars. These homologous surface
antigens are chromosomally encoded by the H1 (phase 1) and H2 (phase 2) of the vh2 locus. By
convention each serotype has been denoted by an antigenic formula with the major O antigen,
followed by phase l H-antigen(s), and then phase 2 H-antigen(s). The phase l H-antigens are
designated by lowercase letters and then phase 2 H-antigens by Arabic numerals or some
instances by components of e or z series (Brenner et al., 2000; Grimont and Weill, 2007).
1.7.2.3 Capsular (Vi) antigens
The capsular antigens are present in Salmonella Typhi, Salmonella dublin and Salmonella
paratyphi A. The Vi antigen could be purified by chemical method. The thermal solubilization of
capsular antigen (Vi) antigen is necessary for the immunological detection of serotypes
containing capsular antigens (Fluit, 2005).
More than 99% of Salmonella strains causing human infections belong to Salmonella enterica
subspecies enterica. Although not common, cross-reactivity between O antigens of Salmonella
and other genera of Enterobacteriaceae do occur. Therefore, further classification of serotypes is
based on the antigenicity of the flagellar H antigens which are highly specific for Salmonella
(Scherer and Miller, 2001). Officially recognized by the World Health Organization (WHO), the
Kauffmann-White diagnostic scheme involves the primarily subdivision of Salmonella into
serogroups and further delineated into serotypes based on the O, H and Vi antigenic formula
(Popoff and LeMinor, 2005).
1.7.3 Phage typing
Bacteriophages are the most abundant entities on earth and have contributed a lot to the field of
molecular biology and biotechnology. Many mysteries of molecular biology were solved using
bacteriophages. Bacteriophages are getting enormous amount of attention due to their potential
to be used as antibacterials, phage display systems, and vehicles for vaccines delivery. They have
also been used for diagnostic purposes (phage typing) as well (Clark and March, 2006).
These bacterial viruses have genetic material in the form of either DNA or RNA, encapsulated
by a protein coat (Clark and March, 2006). The capsid is attached to a tail which has fibers, used
24
for attachments to receptors on bacterial cell surface. Most of the phages have polyhedral capsid
except filamentous phages (Ackerman, 1998).
Phages infect bacteria and can propagate in two possible ways; lytic life cycle and lysogenic life
cycle. When phages multiply vegetatively they kill their hosts and the life cycle is referred to as
lytic life cycle. On the other hand, some phages known as temperate phages can grow
vegetatively and can integrate their genome into host chromosome replicating with the host for
many generations (Inal, 2003). If induction to some harsh conditions like ultraviolet (UV)
radiations occurs then the prophage will escape via lysis of bacteria (Inal, 2003).
The specificity of phages for bacterial cells enables them to be used for the typing of bacterial
strains and the detection of pathogenic bacteria. Phage typing is also known as the use of
sensitivity patterns to specific phages for precisely identifying the microbial strains. The
sensitivity of the detection would be increased if the phages bound to bacteria are detected by
specific antibodies. For the detection of unknown bacterial strain its lawn is provided with
different phages, and if the plaque (clear zones) appears then it means that the phage has grown
and lysed the bacterial cell, making it easy to identify the specific bacterial strain (Clark and
March, 2006).
There are certain other methods which can be employed to detect pathogenic bacteria such as the
use of phages that can deliver reporter genes (e.g. lux) specifically (Kodikara et al., 1991) or
using green fluorescent protein (Funatsu et al., 2002) that would express after infection of
bacteria. Similarly, phages having a fluorescent dye covalently attached to their coats can be
used to detect specific adsorption (Hennes et al., 1995; Goodridge et al., 1999). The detection of
some of the released components such as adenylate kinase (Corbitt et al., 2000) after the specific
lysis of bacteria and the use of antibodies and peptides that are displayed by phages which bind
to toxins and bacterial pathogens specifically can also be used, (Petrenko and Vodyanoy, 2003).
Dual phage technology is another application of phages in detection of bacteria, in which phages
are used to detect the binding of antibodies to specific antigens (Sulakvelidze and Kutter, 2005).
Phage amplification assay can also be used to detect pathogenic bacteria. The technique has most
extensively been used for the detection of Mycobacterium tuberculosis, E.coli, Pseudomonas,
Salmonella, Listeria, and Campylobacter species (Barry et al., 1996).
25
The applications of phages range from the diagnosis of the disease, through phage typing, and its
prevention (phage vaccine), to the treatment (phage therapy).There is the hope that phages could
be useful to humans in many ways.
1.7.4 Molecular typing of Salmonella
Conventional culture methods have been popularly employed in identifying and isolating
microorganisms present in wastewater. Unfortunately, as a result of inconsistently expressed
phenotypic traits, these classical typing approaches are often unable to discriminate between
related outbreak strains.
The ability to characterize and determine the genetic relatedness among bacterial isolates
involved in a waterborne outbreak is a prerequisite for epidemiological investigations. Detailed
strain identification is essential for the successful epidemiological investigation of Salmonella
outbreaks. Investigations have relied traditionally on serological and antibiogram techniques. In
contrast, modern typing methods are based on characterization of the genotype of the organism.
Thus, molecular typing or fingerprinting of Salmonella isolates is an invaluable epidemiological
tool that can be used to track the source of infection and to determine the epidemiological link
between isolates from different sources (Rakesh et al., 2009).
Some molecular typing systems can distinguish among epidemiologically unrelated isolates
based on genetic variation in chromosomal DNA of a bacterial species (Swaminathan and Matar,
1993). Usually, this variability is high, and differentiation of unrelated strains can be
accomplished using a variety of fingerprinting techniques.
The genotypic methods are those methods, which are based on the genetic structure of an
organism and include polymorphisms in DNA restriction patterns based on cleavage of the
chromosome. The digestion of the chromosomal DNA provides variable number of the DNA
fragments, thus revealing genetic variations. Genotyping methods are less subject to natural
variation, though various factors may be responsible for genetic variants such as insertions or
deletions of DNA into the chromosome, the gain or loss of the extra chromosomal DNA, and
random mutations that may create or eliminate restriction sites (Tenover et al., 1997).
There is currently no gold standard typing system available for Salmonella fingerprinting,
however, the combination of different genotyping methods such as plasmid profile analysis,
26
ribotyping, characterization of virulence factors in Salmonella serovars, enterobacterial repetitive
intergenic consensus sequence analysis (ERIC-PCR), random amplified polymorphic DNA
(RAPD) and pulsed field gel electrophoresis methods have been evaluated for more precise
subtyping of Salmonella serovars (Mohand et al., 1999; Lagatolla et al., 1996; Shangkuan and
Lin, 1998).
1.7.4.1 Random amplified polymorphic DNA (RAPD)-PCR
Over the last decade, polymerase chain reaction has become a widespread technique for several
novel genetic assays based on selective amplification of DNA (Bardakci, 2001). The popularity
of PCR is primarily due to its apparent simplicity and high probability of success. Unfortunately,
because of the need for DNA sequence information, PCR assays are limited in their application.
The discovery that PCR with random primers can be used to amplify a set of randomly
distributed loci in any genome facilitated the development of genetic markers for a variety of
purposes (Williams et al., 1900; Welsh and McClelland, 1990).
Random Amplification of Polymorphic DNA (RAPD) is a modification of the PCR in which a
single, short and arbitrary oligonucleotide primer, able to anneal and prime at multiple locations
throughout the genome, can produce a spectrum of amplification products that are characteristics
of the template DNA. No knowledge of the DNA sequence for the targeted gene is required, as
the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes
the method popular for comparing the DNA of biological systems that have not had the attention
of the scientific community, or in a system in which relatively few DNA sequences are compared
(Senthil Kumar and Gurusubramanian, 2011).
The simplicity and applicability of the RAPD technique have captivated the interest of many
scientists. Perhaps the main reason for the success is the gain of a large number of genetic
markers that require small amounts of DNA without the requirement for cloning, sequencing or
any other form of the molecular characterization of the genome of the species in question.
The standard RAPD technology utilises short synthetic oligonucleotides (about 10 bases long) of
random sequences as primers to amplify nanogram amounts of total genomic DNA under low
annealing temperatures by PCR. Amplification products are generally separated on agarose gels
and stained with ethidium bromide (Bardakci, 2001).
27
Welsh and McClelland (1990) independently developed a similar methodology using primers
about 15 nucleotides long and different amplification and electrophoretic conditions from RAPD
and called it the arbitrarily primed polymerase chain reaction (AP-PCR) technique. PCR
amplification with primers shorter than 10 nucleotides known as DNA amplification
fingerprinting (DAF) have also been used to produce more complex DNA fingerprinting profiles
(Caetano-Annoles et al., 1991). Although these approaches are different with respect to the
length of the random primers, amplification conditions and visualization methods, they all differ
from the standard PCR condition in that only a single oligonucleotide of random sequence is
employed and no prior knowledge of the genome subjected to analysis is required.
At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of
random sequence bind several priming sites on the complementary sequences in the template
genomic DNA and produce discrete DNA products if these priming sites are within an
amplifiable distance of each other. The profile of amplified DNA primarily depends on
nucleotide sequence homology between the template DNA and oligonucleotide primer at the end
of each amplified product.
Nucleotide variation between different sets of template DNAs will result in the presence or
absence of bands because of changes in the priming sites. Recently, sequence characterized
amplified regions (SCARs) analysis of RAPD polymorphisms showed that one cause of RAPD
polymorphisms is chromosomal rearrangements such as insertions/deletions. Therefore,
amplification products from the same alleles in a heterozygote differ in length and will be
detected as presence and absence of bands in the RAPD profile (Bardakci and Skibinski, 1999).
RAPD technique has found a wide range of applications in gene mapping (Hemmat et al., 1994),
population genetics (Chalmers et al., 1992; Kambhampati et al., 1992), molecular evolutionary
genetics (Fani et al., 1993; Naish et al., 1995), and plant and animal breeding (Russel et al.,
1993). This is mainly due to the speed, cost and efficiency of the technique to generate large
numbers of markers in a short period compared with previous methods. Therefore, RAPD
technique can be performed in a moderate laboratory for most of its applications. It also has the
advantage that no prior knowledge of the genome under research is necessary.
28
Although the RAPD method is relatively fast, cheap and easy to perform in comparison with
other methods that have been used as DNA markers, the issue of reproducibility has been of
much concern. In fact, ordinary PCR is also sensitive to changes in reaction conditions, but the
RAPD reaction is far more sensitive than conventional PCR because of the length of a single and
arbitrary primer used to amplify anonymous regions of a given genome. This reproducibility
problem is usually the case for bands with lower intensity. The reason for bands with high or
lower intensity is still not known. Perhaps some primers do not perfectly match the priming
sequence, amplification in some cycles might not occur, and therefore bands remain faint. The
chance of these kinds of bands being sensitive to reaction conditions of course would be higher
than those with higher intensity amplified with primers perfectly matching the priming sites. The
most important factor for reproducibility of the RAPD profile has been found to be the result of
inadequately prepared template DNA. Differences between the template DNA concentration of
two individual’s DNA samples result in the loss or gain of some bands (Welsh and McClelland,
1994; Bardacki, 1996).
Since RAPD amplification is directed with a single, arbitrary and short oligonucleotide primer,
DNA from virtually all sources is amenable to amplification. Therefore, DNA from the genome
in question may include contaminant DNA from infections and parasites in the material from
which the DNA has been isolated. Special care is needed in keeping the DNA to be amplified
from other sources of DNA.
1.7.4.2 Salmonella Pathogenicity and virulence genes
The nature of pathogenicity of an organism lies in the virulence genes or virulence factors.
However, these terms are still not strictly defined (Wassenaar and Gastraa, 2001). The possible
virulence factors of Salmonella have been understood with the gain in the knowledge on the
molecular mechanism behind the pathogenicity of Salmonella. Recently, the involvement of
effector proteins in the survival and replication of Salmonella in host cells has been elucidated.
The majority of virulence genes of Salmonella are clustered in a region distributed over the
chromosome, called Salmonella Pathogenicity Islands (SPI) (Groisman and Ochman, 1996;
Marcus et al., 2000). Until recently, five SPIs (SPI 1-5) have been identified on the Salmonella
chromosome at centisome 63, 31, 82, 92 and 25 cs, respectively (Blanc-Portard and Groisman,
1997; Hayward and Koronakis, 2002).
29
Each SPI was responsible in various cellular activities towards the virulence factor of the
organism (Wong et al., 1998; Wood et al., 1998). On completion of genome sequence of
Salmonella Typhi strain CT 18, five more regions were identified and designated as SPI-6, SPI-
7, SPI-8, SPI-9 and SPI-10.
SPI-6 encodes for saf and tcf fimbrial operon and SPI-7 encodes for Vi biosyntheis genes and
also for the IV fimbrial operon (Parkhill et al., 2001; Pickard et al., 2003). The 6.8 kb large SPI-
8 encodes for genes conferring resistance to bacteriocin, SPI-9 for type 1 secretion system,
whereas SPI-10 encode for sef fimbrial operon (Galan et al., 1992; Parkhill et al., 2001). The
flagella mediated bacterial motility accelerates but is not required for Salmonella enteritidis
invasion in differentiated Caco-cells (van Asten et al., 2004).
Salmonella virulence factors were also detected in virulence plasmids in certain Salmonella
serovars namely Salmonella abortusovis, Salmonella cholerasuis, Salmonella dublin, Salmonella
enteritidis, Salmonella gallinarum, Salmonella pullorum and Salmonella typhimurium, although
not all isolates of these serotypes carry the virulence plasmid (Rotgar and Casadesus, 1999). All
plasmids contain the 7.8 kb Salmonella plasmid virulence (spv) locus. This locus harbored five
genes designated spv RABCD and expressions of spv genes which may play a role in the
multiplication of intracellular Salmonella (Chu et al., 2001). The results showed that spvB
together with spvC conferred virulence to Salmonella typhimurium when administered
subcutaneously to mice (Matsui et al., 2001).
Salmonella Typhi CT 18 exhibited a 106 kb large cryptic plasmid with some homology to a
virulence plasmid of Yersinia pestis. However, the majority of Salmonella typhi tested did not
harbor this plasmid. Cryptic plasmid has also been reported for Salmonella paratyphi C,
Salmonella derby, and Salmonella copenhagan, Salmonella durban, Salmonella give and
Salmonella infantis (Rotgar and Casadesus, 1999). Hybridization analysis has shown a few other
serotypes such as Salmonella johannesburg, Salmonella kottbus and Salmonella newport found
to bear the virulence plasmids.
Salmonella produces both endotoxin and exotoxin and virulence due to these toxins are well
documented. The endotoxin, lipid portion (lipid A) of the outer lipopolysaccharide (LPS)
membrane of Salmonella elicits a variety of in vitro and in vivo biological responses. The best
30
studied exotoxin of Salmonella was the heat labile Salmonella enterotoxin (stn) of approximately
29 kDa encoded by stn gene (Prager et al., 1995; Portillo, 2000). A study on 90 kDa heat labile
enterotoxins of Salmonella typhimurium was also reported by Rahman and Sharma (1995). The
role of fimbriae and the flagella of Salmonella have been well identified in the attachment and
movement of the organism but their roles in pathogenesis are still not properly understood
(Folkesson et al., 1999; Edwards et al., 2000; Portillo, 2000).
Characterization of different virulence factors in Salmonella serotypes have been carried out by
amplifying different gene sequences responsible for specific phenotypic properties. The
amplification of invA gene by PCR indicates the presence of invasion gene in Salmonella
serovars. A PCR based study demonstrated that stn gene was present in all Salmonella enterica
serovars, whereas it was absent in Salmonella bongori (Prager et al., 1995). The cumulative
effects of virulence by these genes were found to be responsible for invasion to the epithelial
cells of intestine and thereafter leading to gastrointestinal disorder. PCR assays for several
virulence (inv, him) and functional (iroB, fimY) genes were developed for detection of
Salmonella in the environment, in food or faeces samples (Bej et al., 1994; Baumler et al., 1998;
Yeh et al., 2002; Malorny et al., 2003a). The fliC gene also has been successfully used for
molecular typing studies on Salmonella, based on high variability of the central region (Dauga et
al., 1998).
1.8 Salmonellosis
1.8.1 Reservoirs and epidemiology
The primary reservoir of Salmonella is the intestinal tract of birds and animals, particularly of
poultry and swine. The organisms are excreted in faeces from which they may be transmitted by
insects and other creatures to a large number of places such as water, soils and kitchen surfaces.
There are host adaptations patterns among serovars, namely; highly host adaptive, less host
adaptive and non-host adaptive (Ecuyer et al., 1996). Human host adaptive serovars include
Salmonella typhi (causative agent of typhoid fever); in contrast, the highly host adaptive chicken
pathogens viz., Salmonella pullorum and Salmonella gallinarum are not human pathogen. There
is no report of Salmonella typhi host range extending beyond human beings. Hence, isolation of
Salmonella typhi from food or water must be indication of contamination from human beings.
31
Other Salmonella serovars are found to be host adapted animal pathogens and sources of
zoonotic infections (Ziprin and Hume, 2001).
Salmonella choleraesuis is a pathogen of swine but sometime causes severe systemic infections
in humans (Ziprin, 1994; Wang et al., 1996). Similarly, Salmonella dublin may cause septicemia
in cattle and can be transmitted to humans from milk and milk products (Reher et al., 1995).
Salmonella enteritidis and Salmonella senftenberg are host adapted to chicken and turkey
respectively. Some Salmonella serovars are not host adapted and also tend to be less virulent
than the host-adapted serotypes, but they are found to be responsible for an overwhelming
number (90%) of incidents of human salmonellosis (Webber, 1996; Hunter, 1997).
Typhoid cases are stable with low numbers in developed countries, but non typhoidal
salmonellosis has increased worldwide. Typhoid fever usually causes mortality in 5 to 30% of
typhoid-infected individuals in the developing world. The World Health Organization (WHO)
estimates 16 to 17 million cases occur annually, resulting in about 600,000 deaths. The mortality
rates differ from region to region, but can be as high as 5 to 7% despite the use of appropriate
antibiotic treatment (Scherer and Miller, 2001).
In Nigeria, typhoid fever is among the major widespread diseases affecting both young children
and young adults as a result of many interrelated factors such as inadequate facilities for
processing human wastes and indiscriminate use of antibiotics. Morbidity associated with illness
due to Salmonella continues to be on the increase, in some cases resulting in death (Talabi, 1994;
Akinyemi et al., 2005). A more accurate figure of salmonellosis is difficult to determine because
normally only large outbreaks are investigated whereas sporadic cases are under-reported
(Scherer and Miller, 2001; Parry, 2006). On the other hand, non typhoidal cases account for 1.3
billion cases with 3 million deaths (Hanes, 2003; Hu and Kopecko, 2003).
The infectious dose of Salmonella depends upon the serovar, bacteria strain, growth condition
and host susceptibility. On the other hand, host factors controlling susceptibility to infection
include; the condition of the intestinal tract, age and underlying illnesses or immune deficiencies.
The infectious dose of Salmonella is broad, varying from 1-109 cfu/g. However, single-food-
source outbreaks indicate that as little as 1 to 10 cells can cause salmonellosis with more
32
susceptibility to infection by YOPI (Young, Old, Pregnant and Immunocompromised) groups
(Yousef and Carlstrom, 2003; Bhunia, 2008).
Information about incidence and serovars distribution of Salmonella in domestic animal
populations is essential for understanding the relationships within and among reservoirs of
Salmonella in animals and humans that are ultimately responsible for zoonotic disease
transmission (Gast, 1997). Salmonella infection is usually acquired by the oral route, mainly by
ingesting contaminated food or drink. Salmonella can be transmitted directly from human to
human or from animal to human without the presence of contaminated food or water, but this is
not a common mode of transmission.
1.8.2 Dynamics of Salmonella
Transmission of Salmonella to humans traditionally has been attributed to contaminated animal-
product foods, but epidemiological studies have demonstrated that cases are sporadic and may
more likely involve environmental sources than previously thought. It has been suggested that
contaminated soils, sediments and water as well as wildlife may play a significant role in
Salmonella transmission (Schutze et al., 1998). Moreover, geographic clusters of cases in which
no verifiable food source have been determined, such as those recently caused by S. javiana in
the southeastern US which do not follow the same geographic patterns as cases which have been
linked to a known food source but rather mimic amphibian distribution patterns (Srikantiah,
2004). S. typhi, which is only transmitted from human to human, is most common in developing
nations where access to safe drinking water may be limited and waste disposal and treatment
may be inadequate (Velema et al., 1997).
First, environment contaminated with Salmonella serves as the infection source because
Salmonella can survive in the environment for a long time. Subsequently, Salmonella is
transmitted to vectors such as rats, flies and birds where Salmonella can be shed in their faeces
for weeks and even months. Following the direct transmission, moving animals such as swines,
cows and chickens act as the important risk factor for infection. These animal reservoirs are
infected orally because Salmonella normally originates from the contaminated environment and
also contaminated feed. Human get infected after eating food or drinking water that is
contaminated with Salmonella through animal reservoirs. However, Salmonella typhi and
Salmonella paratyphi A do not have animal reservoir, therefore infection can occur by eating
33
improperly handled food by infected individuals (Newell et al., 2010). Besides, transmission of
Salmonella to the food processing plants and equipments for food preparation are also of great
importance. Once carried by vectors or transferred to food, consumption by human can result in
the risk of salmonellosis. The Salmonella cells can attach to food contact surfaces such as plastic
cutting board which may develop into biofilm once attached and hence cause cross-
contamination. Consequently, Salmonella can enter the food chain at any point from livestock
feed, through food manufacturing, processing and retailing as well as catering and food
preparation in the home (Wong et al., 2002).
Spread of Salmonella may be facilitated in water storage tanks in a building, from wild animal
feces or even from carcasses. Poor sanitation, improper sewage disposal and lack of clean water
system cause the transmission of typhoid fever. In areas where typhoid fever is endemic, water
from lakes or rivers which are used for public consumption and are sometimes contaminated by
raw sewage are the main sources of infection. The consumption of unboiled water during 1997
typhoid outbreak in Dushanbe, Tajikistan caused 2200 cases of illness and 95 deaths (Penteado
and Leitão, 2004; Bordini et al., 2007).
Salmonella contamination of fresh produce could be due to the entry of Salmonella through scar
tissues, entrapment during embryogenesis of produce, natural uptake through root systems and
transfer onto edible plant tissues during slicing. The human health risk is increased further by
Salmonella preference to grow on fresh produce during retail display at ambient temperature. In
2000, cantaloupe from Mexico resulted in a Salmonella Poona outbreak in USA (Penteado and
Leitão, 2004; Bordini et al., 2007). The table below shows the prevalence of predominant
Salmonella serotypes from different sources (Table 1.2).
34
Table 1.2: Prevalence of Salmonella species from different sources.
Country Sample/source Prevalence Predominant
serotypes
Reference
Iran Chicken
Beef
86/190 (45.3%)
38/189 (20.1%)
Thompson Dallal et al., 2010
Brazil Poultry carcass
Poultry viscera
0/127 (0%)
2/73 (2.7%)
Enteritidis Freitas et al., 2010
Spain Pig
Herd
43/804 (5.3%)
22/67 (32.8%)
Anatum,
Typhimurium
Gomez-Laguna et al.,
2010
China Chicken
Pork
Beef
Lamb
276/515
(53.6%)
28/91 (30.8%)
13/78 (16.7%)
16/80 (20%)
Enteritidis,
Typhimurium,
Shubra,
Indiana,
Derby, Djugu
Yang et al., 2010
Morocco Slaughter
house
Sea food
75/105 (71%)
10/105 (9.5%)
Infantis,
Bredeney,
Blokley,
Typhimurium,
Mbandaka,
Branderup II,
Kiambu
Bouchrif et al., 2009
Bangladesh Chick egg
4/80 (5%) Typhimurium Hasan et al., 2009
Republic of
Ireland
Retail pork 13/500 (2.6%) Typhimurium Prendergast et al., 2009
Turkey Chicken part
Minced meat
Ready-to-eat-
salad
Raw vegetable
Raw milk
1/168 (0.6%)
0/45 (0%)
0/100 (0%)
0/78 (0%)
0/25 (0%)
Typhimurium
Infantis
Centinkaya et al., 2008
Iran Raw poultry
Cooked poultry
Raw meat
Cooked meat
Turkey
Quail
Vegetable
24/134 (17.9%)
3/56 (5.4%)
8/101 (7.9%)
2/118 (1.7%)
1/3 (33.3%)
2/5 (40%)
3/38 (7.9%)
Enteritidis,
Baibouknown
Jalali et al., 2008
Lithuania Faeces
Caecum
Dust
Water
28/85 (32.9%)
12/52 (23.1%)
5/34 (14.7%)
1/10 (10%)
Enteritidis,
Typhimurium
Pieskus et al., 2008
Turkey Tulum cheese 6/250 (2.4%) Colak et al., 2007
Vietnam Pork
Beef
Chicken
32/50 (64%)
31/50 (62%)
16/30 (53.3%)
London,
Havana,
Anatum,
Van et al., 2007
35
Shell fish 9/50 (18%) Hadar,
Albany,
Typhimurium
Malaysia Street food
Fried chicken
Kerabu jantung
pisang
Sambal fish
Mix vegetable
12/129 (9.3%)
1/18 (5.6%)
3/5 (60.0%)
6/9 (66.7%)
2/5 (40%)
Biafra,
Braenderup,
Weltevreden
Tunung et al., 2007
Brazil Chicken
abattoir
29/288 (10.1%) Enteritidis,
Typhimurium
Cortez et al., 2006
South India Egg 38/492 (7.7%) Enteritidis Suresh et al., 2006
Jordan Chicken, meat 25/93 (26.9%) Enteritidis,
Typhimurium
Malkawi and Gharaibeh,
2004
Malaysia Selom
Pegaga
Kangkong
Kesum
16/43 (37.2%)
8/26 (30.8%)
8/25 (32%)
8/18 (44.4%)
Weltevreden,
Agona,
Senftenberg,
Albany
Salleh et al., 2003
Albania Chicken meat
sample
30/461 (6.5%) Enteritidis Beli et al., 2001
India Fish
Crustacean
104/730
(14.3%)
48/276 (17.4%)
Weltevreden,
Typhi,
Paratyphi B,
Mgulani,
Typhimurium
Hatha and
Lakshamanaperumalsamy,
1997
Malaysia Retail poultry
Litter
Poultry farm
158/445
(35.5%)
8/40 (20.0%)
2/10 (20.0%)
Enteritidis,
Muenchen,
Kentucky,
Blockley
Rusul et al., 1996
Malaysia Chicken
portion
Chicken liver
Chicken
gizzard
Cooked meat,
chicken
Vegetable
Safay
Prawn
Oriental shrimp
paste
13/33 (39.4%)
6/17 (35.3%)
8/18 (44.4%)
4/28 (14.3%)
14/60 (23.3%)
4/16 (25%)
2/19 (10.5%)
Blockley,
Enteritidis,
Chincol,
Muenchen,
Agona
Arumugaswamy et al.,
1995
(Source: Pui et al., 2011)
36
CHAPTER TWO
MATERIALS AND METHODS
2.1 Materials
2.1.1 Culture Media
Dehydrated bacteriological media and compounded media were used for the isolation and
identification of Salmonella from wastewater.
2.1.1.1 Dehydrated Media
For the isolation and identification of Salmonella from wastewater, the following dehydrated
media were used in this study. All dehydrated media used in this study were prepared according
to manufacturer’s instructions.
Table 2.1: List of Dehydrated Media
S/No Media Source
1 Brain Heart Infusion Agar Difco, USA
2 Buffered Peptone Water Lab M, UK
3 Kligler Iron Agar Oxoid, UK
4 MacConkey Agar Sigma, USA
5 Methyl Red and Voges-Proskauer broth Oxoid, UK
6 Motility GI Medium Difco, USA
7 Nutrient Agar Titan Biotech, India
8 Nutrient Broth Titan Biotech, India
9 Rappaport Vassiliadis broth Oxoid, UK
10 Salmonella-Shigella Agar Oxoid, UK
11 Semisolid Motility Media Difco, USA
12 Simmon’s Citrate Agar Titan Biotech, India
13 Urea Agar Oxoid, UK
37
2.1.1.2 Compounded Media
The following media were compounded in the laboratory.
Sugar fermentation broth
Peptone 10g
Sodium chloride 5g
Sugar 10g
Phenol Red 0.018g
Distilled water (DW) 1 liter
pH 7.4±0.2
The following sugars; glucose, lactose and sucrose were used as required. To 100 ml of the basal
media, 1g of the respective sugar was added, dissolved, dispensed in 4 ml quantities in 100x12
mm tubes containing inverted Durham’s tubes. The media was sterilized at 115oC for 20
minutes. For lactose and sucrose, filter-sterilized solutions were added to the pre-sterilized broth.
2.1.2 Phage Isolation- chemicals, reagents and buffers
All chemicals and buffers were prepared in double distilled water (ddH2O).
1. Phage Buffer (PB, 1L) Final Concentration
1M Tris stock (pH 7.5) 10 mM
1M MgSO4 stock 10 mM
NaCl 68 mM
ddH2O 970 ml
100 mM CaCl2 stock 1 mM
All ingredients were added except CaCl2 into an Erlenmeyer flask. The solution was stirred until
the NaCl was completely dissolved. The flask and its content were autoclaved and then allowed
to cool at 55oC in a water bath. After cooling, CaCl2 was aseptically added and mixed by
swirling. Aliquots were stored in sterile bottles at room temperature.
38
2. 100 mM CaCl2 (100 ml)
CaCl2 1.11g of CaCl2 (anhydrous)
ddH2O to 100 ml
To 90 ml of ddH2O, 1.11g of CaCl2 (anhydrous) was added and stirred until the CaCl2 was
dissolved. The final volume was adjusted to 100 ml by adding ddH2O. The solution was
autoclaved and stored at room temperature.
3. 0.5 M EDTA, pH 8.0
EDTA disodium salt dehydrate 186.1g
ddH2O to 1 L
A 186.1g of EDTA disodium salt dehydrate was weighed and dissolved in 950 ml of ddH2O. The
solution was stirred and NaOH was added to adjust the pH to 8.0. The final volume was adjusted
to 1 L with additional ddH2O and sterilized at 121oC for 15 min.
4. Nutrient Agar
N-Agar base 28g
ddH2O 1L
Twenty-eight grams of N-agar base was dissolved in one liter of ddH2O in a conical flask. The
solution was homogenized by boiling and sterilized at 121oC for 15 min. The sterile content was
aseptically poured into Petri dishes and allowed to harden. Solidified agar plates were inverted
and incubated at 37oC for 8 hours (h) to confirm sterility.
2.1.3 Molecular biology- chemicals, reagents and buffers
Electrophoresis chemical and reagents
1. TAE buffer (50X)
Tris base, 242 gm (Sigma)
Glacial acetic acid, 57.1 ml (Merck)
0.5M EDTA, 100 ml (pH 8) (SRL)
39
All components were weighed and dissolved in 600 ml of ddH2O (Millipore). The final volume
was adjusted to 1 liter with additional ddH2O and sterilized at 115oC for 20 min. The solution
was stored at room temperature and always used as 1X TAE buffer.
2. Agarose (Electrophoresis grade) Melford, UK.
1.5% agarose gel was prepared in 0.5X TAE buffer by heating in a microwave oven for 2 min.
3. Ethidium Bromide (10 mg/ml) (Sigma)
Hundred milligrams (100 mg) of ethidium bromide was weighed and transferred to 10 ml of pre-
sterilized TAE. The mixture was dissolved by stirring with a magnetic stirrer for 3-4 h and then
stored in a dark container at 4oC.
4. Gel Loading Buffer
Bromophenol Blue, 0.25g (Sigma)
Xylone Cyanol, 0.25g (Sigma)
Sucrose, 40g (SRL)
TAE 1X, 100 ml
All components were dissolved in 100 ml of sterile TAE and stored at 4oC.
2.1.4 Enzymes, Oligos and DNA markers
DNA molecular weight markers were used as a reference standard to determine the size of PCR
amplicons. 5X FIREPol® Master Mix (Solis BioDyne, Estonia) was used in different PCR
assays and molecular fingerprinting experiments. All Enzymes, Oligos, Master Mix and
molecular weight markers were stored at -20oC in a deep freezer (Ignis, Italy).
Table 2.2: List of Oligos, DNA markers and Master Mix
S/N Oligos, DNA markers and Master Mix Trade name (Country)
1 5X FIREPol® Master Mix Solis BioDyne (Estonia)
2 100 bp DNA Ladder Fermentas (Germany)
3 1000 bp DNA ladder Fermentas (Germany)
40
2.1.5 Oligonucleotide Primers
The following primers were used in the experiments related to the development of Salmonella
specific PCR, identification of the invA gene and RAPD-PCR studies. All primers were stored at
-20oC in a deep freezer and were prepared as instructed by the manufacturers.
Table 2.3: List of Salmonella specific primers used
S/N Primer sequence (5’----3
’) Brand Reference
1 Salm 16S
F: TGT TGT GGT TAA TAA CCG CA
R: CAC AAA TCC ATC TCT GGA
Biomers.net Lin and Tsen, 1996
2 Mult invA
F: ACA GTG CTC GTT TAC GAC CTG ATT
R: AGA CGA CTG GTA CTG ATC TAT
Biomers.net Chiu and Ou, 1996
3 RAPD-PCR
AAC GCG CAA C (787)
CCC GTC AGC A (RAPD 2)
CCG CAG CCA A (1254)
Biomers.net Smith et al., 2006
2.1.6 Salmonella antisera
The following antisera were used in Salmonella serotyping.
Table 2.4: List of Salmonella antisera used
Antisera Source
Salmonella O Poly A-I and Vi Difco, USA
Individual O antisera (A, B, C1, C2, C3, D1, D2,
E1, E2, E4, F, G, K, and N)
Difco, USA
Salmonella H antisera, Spicer-Edwards 1, 2, 3, 4 Difco, USA
Salmonella H antisera, EN complex Difco, USA
Salmonella H antisera, L complex Difco, USA
Salmonella H antisera, 2, 5, 6, 7 Difco, USA
Salmonella Vi antiserum Difco, USA
41
2.1.7 Major equipment
The following major equipment were used for the different experiments.
Table 2.5: List of Major equipment used
S/N Instrument Purposes/experiments
1 Refrigerated centrifuge 5702 R (Eppendorf, Germany) Pelleting
2 Photogel documentation system (Cjinx science
instrument Co, Ltd)
Gel Imaging
3 Shaker Incubator (Sanyo, Japan) Shaking condition
4 Nanodrop spectrophotometer (Nanodrop Pretoria,
South Africa)
Quantify the concentration and
purity of DNA
5 Eppendorf Vapo protect Thermocycler (Hamburg,
Germany)
Amplify segments of DNA using
varying temperatures
6 Incubator Maintain bacterial growth at a
constant temperature
7 Water bath Maintain samples at a constant
temperature using wet heat
(water)
8 Vortexer Mixing samples by gyration
9 Heat block (Techne, Barloworld, UK) Maintain a constant temperature
using dry heat
10 Inoculation hood Removes vapor and odors
emitted by chemicals by
ventilating them to a designated
area
2.1.8 Wastewater samples
Wastewater samples were collected from four (4) different sites (Imhoff tank inlet, Imhoff outlet,
waste stabilization pond A, waste stabilization pond B) of the wastewater treatment plant of the
University of Nigeria, Nsukka. Wastewater samples were collected from October 2012-March
2013. A total of forty (40) wastewater samples were analyzed for isolation of Salmonella.
42
2.2 Methods
2.2.1 Isolation and identification of Salmonella from wastewater samples
A total of 40 wastewater samples were collected from 4 different sites of the treatment plant,
over a period of six (6) months (October 2012-March 2013). All samples were collected in sterile
bottles and immediately transported to the Microbiology Laboratory, University of Nigeria,
Nsukka (UNN) and examined for Salmonella.
2.2.1.1 Isolation and identification
Salmonella species were isolated using the standard methods for the examination of water and
wastewater described by APHA (2005) and ISO 6579: 2002. Wastewater samples from the
Imhoff tank and Waste Stabilization Ponds (WSPs) were used in this study.
Forty milliliters (40 ml) of wastewater was centrifuged at 2000 rpm for 10 min, and then 10 ml
of the supernatant was pre-enriched in 100 ml buffered peptone water (BPW) in a 250ml
Erlenmeyer flask and incubated at 37oC for 24 h. Pre-enrichment was followed by selective
enrichment in Rappaport Vassiliadisis (RV) broth. 0.1 ml of the pre-enrichment broth was
inoculated into RV broth and incubated at 42oC for 24 h. Subsequently, selectively enriched
samples from RV broth were streaked onto Salmonella-Shigella Agar (SSA) and MacConkey
Agar (Mac). Plates were incubated at 37oC for 24 h.
After incubation, suspected Salmonella on SSA (colorless colonies with or without black
centers) were purified on MacConkey agar by streaking dilution method. Typical Salmonella
colonies (transparent and colorless) on Mac were streaked on Nutrient Agar (NA) and then unto
NA slant for further biochemical identification.
Salmonella spp were identified based on key biochemical reactions on Kligler Iron Agar (KIA),
Urease, indole, lactose, MRVP, Simmon’s Citrate and motility. All Salmonella cultures were
identified based on the typical reactions as shown in Table 2.6.
43
Table 2.6: List of biochemical tests for Salmonella.
S/N Tests/Media Salmonella typical reaction
1 Gram stain Gram negative, short rods
2 Motility Motile*
3 KIA Acid butt and alkaline slant
4 H2S on KIA +ve
5 Urease -ve
6 Lysine Decarboxylase +ve
7 Glucose fermentation Acid and gasv
8 Indole -ve
9 Lactose fermentation -ve
10 Sucrose fermentation -ve
11 MR +ve
12 VP -ve
13 Simmon’s Citrate agar +vev
*some are non-motile v=variations noted
2.2.1.2 Biochemical test
1. Gram stain reaction
A bacterial smear was prepared on a clean microscope slide and heat fixed. The smear was
entirely covered with crystal violet solution and allowed to stand for one minute. The slide was
rinsed with water and flooded with Lugol’s iodine. After one minute, the slide was rinsed with
water. Acetone alcohol was flooded onto the slide and rinsed with water after 15 seconds.
Safranin was added to the slide and washed off after a minute. The slide was gently blotted dry
and viewed under the microscope.
Purple colored cells were interpreted as Gram positive and red/pink colored as Gram negative
bacteria.
2. Citrate Utilization
Simmon’s citrate media (Titan Biotech, India) was inoculated with bacteria and the slants were
incubated at 37oC for 48 h. A change from green to bright blue was a positive indicator that the
44
organism can utilize citrate as its only carbon source, while the presence of a green colour
indicates a negative reaction.
3. Indole test
Aliquots of overnight bacterial cultures were inoculated into test tubes containing sterile tryptone
water. Samples were incubated at 37oC for 48 h. After incubation, 0.5 ml of Kovac’s reagent was
added to the tubes and mixed gently. The presence of a red ring on the surface of the medium
within 10 minutes shows a positive reaction, while a yellow ring on the surface indicates a
negative reaction.
4. Urease test
Urea media (Oxoid, UK) was inoculated with bacteria and the slants were incubated at 37oC
overnight. A change in the color of the media from orange to pink is indicative of a positive
reaction, while a change from orange to yellow shows a negative reaction.
5. Motility test
Motility media (Difco, USA) was inoculated with bacteria and the test tubes were incubated at
37oC for approximately 18 h. Growth along the line of inoculation is considered negative for
motility; while growth throughout the media is considered positive for motility (the bacterium is
motile).
6. Glucose utilization test
Aliquots of overnight bacterial cultures were inoculated into test tubes containing media and
Durham tubes specific for the determination of the ability of bacteria to utilize glucose and
produce gas. Samples were incubated at 37oC overnight. A change from purple to yellow with
bubbles in the Durham’s tube was a positive indicator that the bacteria can utilize glucose with
gas production.
7. Kligler Iron Agar (KIA)
Kligler Iron Agar (Oxoid, UK) was inoculated using a straight wire by stabbing the butt and
streaking the slope. The test tubes were incubated at 37oC overnight. After incubation, the test
tubes were examined for the ability of the organism to ferment glucose and/or lactose (changes
in the color of the butt and slope) with or without production of gas and H2S production from
thiosulphate in an acid environment.
45
8. Methyl Red and Voges Proskauer broth test (MRVP)
MRVP media (Oxoid, UK) was inoculated with bacteria in bijou bottles and incubated at 37oC
for 48 h. After incubation, few drops of methyl red solution were added. The presence of a red
ring on the surface of the medium is considered/shows a positive reaction, while a yellow ring
indicates a negative reaction.
2.2.2 Serotyping of Salmonella isolates
All biochemically typical Salmonella isolates were serotyped based on reaction with somatic
(O), flagellar (H), and capsular (Vi) antisera (Difco, USA). Salmonella O antigens were
identified based on the scheme represented in Table 2.7
Salmonella O and Vi antigens were identified by slide test procedure. A drop of 0.85% saline
was placed on a clean glass slide and a loopful of the test culture was transferred to the saline.
This was then mixed properly to form a uniform suspension. A drop of Salmonella O Poly A-I
and Vi antiserum was dispensed to the suspension on glass slide. The slide containing test
organism and O Poly A-I and Vi antiserum was rotated for 1 minute and observed for visible
agglutination. Rapidly formed +++ (3+) agglutination were considered positive for serotype
testing (75% positivity). The culture found positives for Salmonella O Poly A-I and Vi test were
further tested for individual O antiserum viz. A, B, Cl, C2, C3, D1, D2, El, E2, E4, F, G, K and
N by slide test procedure as described above and the cultures found negative for individual
antiserum were tested for Vi antisera.
46
Table 2.7: Scheme for identification of Salmonella O-antigen
Test Salmonella O poly A-I and Vi
Result +ve -ve
Test Individual O antisera tested
(A,B,C1,C2,C3,D1,D2,E1,E2,E4,F,G,K, and N)
Result +ve -ve
Test Salmonella antiserum Vi
Result + -
Test Heated and retested with Vi
antiserum
Result +ve -ve
Conclusion Confirmation
of H-antigen
Not Salmonella Test
boiled
culture
with
individual
O-antisera
group
Not
Salmonella
Check
for rare
groups
(w,x,y,z)
After the confirmation of the individual Salmonella O antisera, culture were further characterized
for H (phase-I) antisera based on Spicer-Edwards antisera by tube test procedure, whereas, L, EN
and 1 complex antigens were identified, separately. Before the identification of H antigens, test
cultures were consecutively sub-cultured in Motility GI medium (Difco, UK) to increase the
motility of the test organism.
For the identification of H antigens; the test cultures were inoculated in Motility medium (Difco,
USA) by stabbing slightly below the surface in l2 x 150 mm test tubes and incubated at 37oC for
18- 20 h. The organisms that have migrated 50-60 mm to the bottom of the tubes were used for
the test. The cultures from the bottom of the tube were transferred to Brain Heart Infusion broth
(BHI) and incubated at 35°C for 4-6 h. The incubation was followed by preparation of test
culture suspension with equal volume of 0.6% formalized saline. A 0.5 ml of culture suspension
and equal amount of diluted H antisera (1:250) was added into a l2x75 mm test tubes and
incubated in a water bath at 50 °C for l h. Agglutination in tubes were recorded after the
incubation.
47
The identification of phase I, H antigens was followed by identification of phase II antigens after
a phase reversal process. Phase reversal of the identified Phase I of H antigens was carried out in
a semisolid motility medium (Difco, USA) by masking the identified phase I antigens with
antisera. One ml of the 1:10 dilution of antisera (phase I) was added to a 25 ml of semisolid
motility GI medium. It was mixed properly and poured into a sterile Petri dish. After the medium
was solidified, the test organism was inoculated by punching the edge of the semisolid medium
and later incubated at 35-37°C for 24 h. At the end of incubation, culture that migrated to the
opposite side of the inoculation site were transferred to a BHI broth and incubated at 37°C for 4-
6 h. The culture from BHI broth was used for the identification of phase II antigen by tube
method as described above.
The antigenic formula obtained from the Salmonella O, H (phase I and phase II) types were
pooled together and the name of the Salmonella serovar was determined using the Kauffmann-
White Scheme (Popoff and Le Minor, 2001). Salmonella isolates were serotyped at WHO
Collaborating Centre for Reference and Research on Salmonella at Pasteur Institut, Paris, France.
2.2.3 Phage isolation and typing
2.2.3.1 Collection of wastewater samples
Wastewater samples were collected in sterile containers from 4 different sites (Imhoff tank A,
Imhoff tank B, WSP A and WSP B) of the treatment plant and immediately transported to the
Microbiology Lab, UNN.
2.2.3.2 Preparation of phage filtrate
Wastewater samples from the different sites of the treatment plant were spun at 5000 rpm for 5
minutes. The supernatant of each sample was filtered through a 0.45 µm membrane filter
(Merck) to remove bacterial cells and cellular debris. The supernatant (phage filtrate) was capped
properly and stored at 4oC.
2.2.3.3 Direct protocol (plaque assay)
A tenfold serial dilution of the phage filtrate was performed. Subsequently, 0.3 ml of each phage
dilution was transferred into sterile test tubes containing 0.1 ml of an overnight broth culture of
the test organism. The mixture was then allowed to sit at room temperature for 10 minutes.
48
Following, 4 ml of molten Top Agar (TA) was added to the test tubes containing the mixture.
The entire mixture was mixed properly and poured onto appropriately labeled sterile Nutrient
Agar plates (warm). The inoculated plates were swirled gently and left to sit undisturbed for 20
minutes at room temperature. The plates were then inverted and incubated at 37oC for 24 h.
After incubation (24 h, 48 h), plates were examined for the presence of clearing zones (plaques).
2.2.3.4 Enrichment protocol (plaque assay)
Enrichment protocol for phage isolation was performed as described by Carey-Smith et al.
(2006). An aliquot (100 µl) of an overnight broth culture of the test organism(s) was added to
40ml of Tryptone Soy Broth (TSB) and 40 ml of the wastewater sample in a 250 ml Erlenmeyer
flask. The mixture was incubated at 37oC on a 200 rpm thermoshaker for 24 h. After incubation,
the phage-host mixture was centrifuged at 5000 rpm for 5 minutes and filtered through a 0.45 µm
membrane filter. The filtrate (phage filtrate) was aseptically capped and stored at 4oC.
A tenfold serial dilution of the phage filtrate was carried out and 0.3 ml of each phage dilution
was transferred into sterile test tubes containing 0.1 ml of an overnight broth culture of the test
organism. The mixture was then allowed to sit at room temperature for 10 minutes.
Subsequently, 4 ml of molten Top Agar (TA) was added to the test tubes containing the mixture.
The entire mixture was mixed properly and poured onto appropriately labeled sterile Nutrient
Agar plates (warm). The inoculated plates were swirled gently and left to sit undisturbed for 20
minutes at room temperature. The plates were then inverted and incubated at 37oC for 24 h.
After incubation (24 h, 48 h), plates were examined for the presence of clearing zones (plaques).
2.2.3.5 Spot assay
Spot assay was performed as described by HHMI (2011). Putative plaques on plates were labeled
and a grid was drawn at the bottom of sterile Nutrient Agar plates, saving a section for the
negative control.
An aliquot (100 µl) of phage buffer (PB) was transferred into microcentrifuge tubes (one tube for
each potential phage). Using a micropipettor and a tip firmly attached, each putative plaque was
picked and transferred into the tubes containing PB. The solution was then vortexed and a ten-
fold serial dilution of the solution was also carried out.
49
One ml (1 ml) of an overnight broth culture of the test organism and 4 ml of molten TA were
aseptically added to clean microcentrifuge tubes. The TA-Bacteria mixture was then added to the
labeled agar plate, swirled gently and allowed to solidify completely. 10 µl of the putative
plaques samples were added to the corresponding blocks on the grid and allowed to soak into the
agar for a few seconds. The plates were inverted and incubated at 37oC overnight. After
incubation, the plates were examined for the presence of plaques on spotted areas.
2.2.4 Molecular typing of Salmonella serovars
2.2.4.1 Preparation of genomic DNA
Bacterial DNA was extracted by boiling according to the method described by Medici et al.
(2003).
A single colony of each isolate was picked and inoculated in 5 ml of Nutrient Broth and allowed
to grow at 37oC overnight. After incubation, 1 ml of the broth was dispensed into a 1.5 ml
eppendorf tube which was subjected to centrifugation (5702R Eppendorf, Germany) at 10000
rpm for 2 mins, 4oC. Pellet was washed twice with sterile distilled water, following by
centrifugation of cells at 10000 rpm for 5 mins. Crude DNA was extracted from pellet by boiling
in a heat block (Techne, Barloworld, UK) for 10 mins at 100oC in 200 µl of sterile distilled water
and immediately chilled on ice. The boiled content was centrifuged at 10000 rpm for 5 mins,
4oC, and the supernatant was carefully transferred into a new eppendorf tube by gentle aspiration
using a micropipette.
2.2.4.2 PCR assay
2.2.4.3 16S rRNA PCR
Salmonella specific 16S rRNA primer (5’-TGT TGT GGT TAA TAA CCG CA-3
’ and 5
’-CAC
AAA TCC ATC TCT GGA-3’) was used for detection of Salmonella (Lin and Tsen, 1996). A 25
µl of PCR mixture containing 2 µl of template DNA, 4 µl of the PCR Master Mix (Solis
BioDyne, Estonia) (1X PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP , 1 U Taq
Polymerase), 20 pmol of each primer, and ddH20 to the total volume of 25 µl.
DNA amplification was carried out in an Eppendorf Vapo Protect Thermocycler (Hamburg,
Germany) with the following cycling condition; initial denaturation at 94oC for 5 min, followed
by 35 cycles of 94oC for 30s, 56
oC for 1 min and 72
oC for 2 min and a final extension of 10 min
at 72oC was employed. The amplified product was electrophoresed on a 1.5% agarose gel stained
50
with ethidium bromide and a product size (574 bp) was determined with a 100 bp DNA
molecular weight ladder (Fermentas, Germany). Finally, gel image was captured using a
photogel documentation system (Cjinx Science Instrument, USA). For each PCR assay, a
positive control (DNA from S. typhi) and a negative control (sterile distilled water) were
included.
2.2.4.4 invA PCR
Salmonella multiplex-invasion gene (invA) primer (5’-ACA GTG CTC GTT TAC GAC CTG
AAT -3’ and 5
’-AGA CGA CTG GTA CTG ATC TAT -3
’) was used for the identification of
Salmonella invA gene (Chiu and Ou, 1996). A 25 µl of PCR mixture containing 2 µl of template
DNA, 4 µl of the PCR Master Mix (Solis BioDyne, Estonia) (1X PCR buffer, 1.5 mM MgCl2,
0.2 mM of each dNTP , 1 U Taq Polymerase), 20 pmol of each primer, and ddH20 to the total
volume of 25 µl.
DNA amplification was carried out in an Eppendorf Vapo Protect Thermocycler (Hamburg,
Germany) with the following cycling condition; initial denaturation at 95oC for 5 min, followed
by 35 cycles of 94oC for 30s, 56
oC for 30s and 72
oC for 2 min and a final extension of 7 min at
72oC was employed.
PCR amplicons were electrophoresed in a 1.5% agarose gel stained with ethidium bromide
(0.5µg/ml) at 100V. A 100 bp DNA size marker (Fermentas, Germany) was used for calibration
and visualized with a photogel documentation system (Cjinx Science Instrument, USA). A band
product of 244 bp was considered the invA gene (Chiu and Ou, 1996).
2.2.4.5 RAPD-PCR assay
Salmonella spp isolated from wastewater were subjected to RAPD-PCR. Parameters in each
PCR reaction were optimized in order to maximize discriminatory power of the reaction for
typing the Salmonella spp.
A set of three (3) primers were analyzed in this study. The primers include 787: 5’-AAC GCG
CAA C-3’, RAPD 2: 5
’-CCC GTC AGC A-3
’ and 1254: 5
’-CCG CAG CCA A-3
’ (Smith et al.,
2006).
51
The reactions for primers 787 and 1254 were prepared using a 25 µl reaction mixture containing
2 µl template DNA, 4 µl of the PCR Master Mix (Solis BioDyne, Estonia) (1X PCR buffer, 2.5
mM MgCl2, 0.2 mM of each dNTP , 1 U Taq Polymerase), 50 pmol of each primer and ddH20 to
the total volume of 25 µl.
Amplification was carried out in an Eppendorf Vapo Protect thermocycler using the following
cycling parameters; an initial denaturation at 94oC for 5 min and 30 cycles of 94
oC for 1 min,
36oC for 1 min and 72
oC for 2 min. This was followed by a final extension of 72
oC for 5 min.
The reaction for primer RAPD 2 was prepared using a 25 µl reaction mixture containing a 4 µl of
the PCR Master Mix (Solis BioDyne, Estonia) (1X PCR buffer, 2.5 mM MgCl2, 0.2 mM of each
dNTP , 1 U Taq Polymerase), 2 µl template DNA, 60 pmol of each primer and ddH20 to the total
volume of 25 µl.
An initial denaturation was performed at 94oC for 5 min and 40 cycles of 94
oC for 1 min, 40
oC
for 1 min and 72oC for 2 min. This was followed by a final extension of 72
oC for 7 min. PCR
amplicons were electrophoresed in a 1% agarose gel stained with ethidium bromide at 100V and
a 1 kb DNA size marker (Fermentas, Germany) was used for calibration and visualized with a
photogel documentation system.
52
CHAPTER THREE
RESULTS
3.1 Isolation and identification of Salmonella from wastewater
3.1.1 Number and percentage of Salmonella isolated from wastewater
The result demonstrated that 7.5% of wastewater tested was contaminated with Salmonella
during this period. A total of 3 Salmonella isolates were detected and identified, 2 from Imhoff
tank A and 1 from WSP A (Table 3.1).
3.1.2 Identification of Salmonella species from wastewater
The result showed that of 100 Salmonella-like colonies (on SSA and MacConkey agar), only 65
(65%) were found to be negative for urea utilization (Table 3.2). Similarly, out of 65 urea
negative isolates subjected to a set of biochemical reactions, only 12 (18.4%) isolates were found
to be consistent as Salmonella strains. The result further highlighted variation from the standard
pattern in biochemical utilization of citrate (8.3%) and production of H2S on KIA (16.7%) by
different isolates of Salmonella from wastewater.
Identification of Salmonella isolates were carried out with a set of biochemical reactions as
shown in Table 3.3.
53
Table 3.1: Number and percentage of Salmonella isolated from the UNN, wastewater
treatment plant
Source Total
samples
examined
Total
positive
samples
% from
source
% from
positive
samples
% from total
examined
Imhoff Tank A
(inlet)
10 2 20 66.7 5
Imhoff Tank B
(outlet)
10 0 0 0 0
WSP A 10 1 10 33.3 2.5
WSP B 10 0 0 0 0
Total 40 3 - 100 7.5
54
Table 3.2: Analysis of suspected Salmonella isolates for Urea utilization
Imhoff
Tank A
Urea test Imhoff
Tank B
Urea test WSP A Urea test WSP B Urea test
A1 + B1 + C1 - D1 -
A2 + B2 - C2 - D2 -
A3 - B3 - C3 - D3 -
A4 - B4 + C4 + D4 +
A5 - B5 + C5 + D5 -
A6 - B6 - C6 + D6 +
A7 - B7 - C7 - D7 -
A8 - B8 - C8 - D8 -
A9 - B9 - C9 - D9 +
A10 + B10 + C10 - D10 -
A11 - B11 - C11 - D11 -
A12 - B12 - C12 - D12 +
A13 - B13 + C13 - D13 -
A14 + B14 - C14 - D14 -
A15 - B15 - C15 - D15 -
A16 - B16 - C16 + D16 +
A17 - B17 + C17 + D17 +
A18 + B18 - C18 + D18 -
A19 + B19 - C19 - D19 -
A20 - B20 + C20 - D20 +
A21 - B21 + C21 - D21 +
A22 - B22 + C22 + D22 +
A23 + B23 - C23 + D23 +
A24 - B24 - C24 + D24 -
A25 - B25 - C25 - D25 -
55
Table 3.3: Confirmatory Tests of Salmonella isolates (n=12)
S/N Test/Medium Reaction Typical Result (%)
1 Gram Reaction Negative 12 (100)
2 Motility Motile 12 (100)
3 KIA
H2S on KIA
Acid butt, Alkaline slant
Positive
12 (100)
12 (83.33)
4 Urease Negative 65 (100)
5 Indole Negative 12 (100)
6 Glucose Acid and gas 12 (91.7)
7 Lactose Negative 12 (100)
8 Sucrose Negative 12 (100)
9 Dulcitol Positive 12 (91.7)
10 Malonate Negative 12 (100)
11 Simmon’s Citrate Positive 12 (91.7)
12 MR Positive 12 (100)
13 VP Negative 12 (100)
14 Serology Positive 12 (25)
56
3.1.3 Serotyping of Salmonella isolates from wastewater
Serotyping of Salmonella isolates from wastewater revealed that 3 (25%) out of 12 isolates were
identified as Salmonella enterica belonging to subspecies 1, serogroup B. A total of 3 Salmonella
isolates were identified as Salmonella enterica serovar Limete (Table 3.4). The result further
demonstrated that 2 of the isolates could not be serotyped and were identified as rough strains.
3.1.4 Phage isolation and typing
The result of phage isolation revealed that no phage was isolated in this study. Plaque assay also
showed that none of our Salmonella isolates from wastewater could be phage typed (Tables 3.5-
3.10). Plaque assay was performed in duplicates. S. typhimurium LT2 was used as reference
culture, while phage buffer was used as negative control.
57
Table 3.4: Serotyping of Salmonella isolates from wastewater
Sample code Detected O-
antigen
Detected H-
antigen
Complete antigenic
identity (O and H)
Serotype
identification
(S. enterica)
A1 - - - -
A5 O:4, O:12[27] b:1,5 1,4,12,[27]: b.1,5 S. limete
A6 O:4, O:12[27] b:1,5 1,4,12,[27]: b.1,5 S. limete
A12 - - - -
A17 - - - -
B18 - - - -
B24 - - - -
B28 - - - -
C1 - - - -
C8 O:4, O:12[27] b:1,5 1,4,12,[27]: b.1,5 S. limete
C28 - - - -
D11 - - - -
58
Table 3.5: Plaque assay for Imhoff Tank (I)
PHAGE
DILUTIONS
ISOLATES
A4 A5 A6 A12 A17 B18 B24 B28 C1 C8 C28 D11 Neg
control
Pos
control
10-1
(+) - - - - - - - - - - - - +
10-2
- - - - (+) - (+) - - - - - - +
10-3
- - - - - - - - - - - - - +
10-4
- - - (+) - - - - - - - - - +
10-5
- - - - - - - - - - - - - +
10-6
- - - - - - - - - - - - - +
(+) Putative plaques, - no plaques, + clear plaques
59
Table 3.6: Plaque assay for Imhoff Tank (II)
PHAGE
DILUTIONS
ISOLATES
A4 A5 A6 A12 A17 B18 B24 B28 C1 C8 C28 D11 Neg
control
Pos
control
10-1
- - - - - - - - - - - - - +
10-2
(+) - - - (+) - (+) - - - - - - +
10-3
- - - - - - - - - - - - - +
10-4
- - - (+) - - - - - - - - - +
10-5
- - - - - - - (+) - (+) - - - +
10-6
- - - - - - - - - - - - - +
(+) Putative plaques, - no plaques, + clear plaques
60
Table 3.7: Plaque assay for WSP (I)
PHAGE
DILUTIONS
ISOLATES
A4 A5 A6 A12 A17 B18 B24 B28 C1 C8 C28 D11 Neg
control
Pos
control
10-1
- - - - - - - - - - - - - +
10-2
- - - - (+) - - - - - - - - +
10-3
- - - - - - - - - - - - - +
10-4
- - - (+) - - - - - - - - - +
10-5
- - - - - - - (+) - (+) - - - +
10-6
- - - - - - - - - - - - - +
(+) Putative plaques, - no plaques, + clear plaques
61
Table 3.8: Plaque assay for WSP (II)
PHAGE
DILUTIONS
ISOLATES
A4 A5 A6 A12 A17 B18 B24 B28 C1 C8 C28 D11 Neg
control
Pos
control
10-1
- - (+) - - - - (+) - - - - - +
10-2
- - - - - - - - - - - - - +
10-3
- - - - - - - - - - - - - +
10-4
- - - - - - - - - - - - - +
10-5
- - - - - - - - - - - - - +
10-6
- - - - - - - - - - - - - +
(+) Putative plaques, - no plaques, + clear plaques
62
Table 3.9: Spot test for Imhoff tank
PHAGE
DILUTIONS
ISOLATES
A4 A5 A6 A12 A17 B18 B24 B28 C1 C8 C28 D11 Neg
control
Pos
control
10-1
- - - - - - - - - - - - - +
10-2
- - - - - - - - - - - - - +
10-3
- - - - - - - - - - - - - +
10-4
- - - - - - - - - - - - - +
10-5
- - - - - - - - - - - - - +
10-6
- - - - - - - - - - - - - +
(+) Putative plaques, - no plaques, + clear plaques
63
Table 3.10: Spot test for WSP
PHAGE
DILUTIONS
ISOLATES
A4 A5 A6 A12 A17 B18 B24 B28 C1 C8 C28 D11 Neg
control
Pos
control
10-1
- - - - - - - - - - - - - +
10-2
- - - - - - - - - - - - - +
10-3
- - - - - - - - - - - - - +
10-4
- - - - - - - - - - - - - +
10-5
- - - - - - - - - - - - - +
10-6
- - - - - - - - - - - - - +
(+) Putative plaques, - no plaques, + clear plaques
64
3.2 Molecular identification of Salmonella from wastewater
3.2.1 Salmonella specific PCR (16S rRNA) assay
The specificity and sensitivity of the 16S rRNA PCR assay revealed that 3 (25%) of the 12
Standard Microbiological test (SMT) confirmed isolates from wastewater produced desired
amplification of 574 bp fragment, whereas no amplicons were observed for non-Salmonella
serovars (Figure 3.1)
3.2.2 Detection of invA virulence gene of Salmonella isolates
Figure 3.2 reveals the amplification of a 244bp fragment of the invA gene on an agarose gel. The
result of the invA PCR assay revealed that 2 out of 3 of the S. limete isolates were found to
harbor the invA gene corresponding to 244 bp. An exception was observed in the case of one S.
limete which did not show the presence of the invA gene. All non-Salmonella serovars gave
negative result.
3.2.3 RAPD-PCR of Salmonella isolates from wastewater
The discriminatory power of the RAPD-PCR assay was tested by considering the number of
profiles (RAPD binding patterns) generated using a set of 3 primers.
The result of the RAPD-PCR revealed that primers 787 and RAPD2 produced four (4) RAPD
binding patterns on the basis of shared amplified products. Primer 1254 did not produce any
discriminatory pattern amongst the Salmonella isolates; hence no typing was possible (Figure
3.3).
65
Figure 3.1: Salmonella-specific PCR (16S rRNA) of isolates from wastewater.
Lane M: 100 bp DNA, Lane 1: Negative Control, Lane 2: Positive control (S. typhi), Lane 3-14:
samples, Lane 9, 10 and 13 reveals amplification of 574 bp fragments of 16S rRNA gene.
66
Figure 3.2: Agarose gel electrophoresis showing amplification of 244 bp fragment of invA gene
in Lane 12 and 13.
Lane M: PCR Marker, Lane 1: Positive Control, Lane 2: Negative Control, Lane 3-14: samples.
244
67
Figure 3.3: Representative RAPD-PCR of Salmonella isolates from wastewater using a set of 3
primers (1254, 787, RAPD 2).
Lane M: 1kb Marker, Lane 3-5 (1254), Lane 8-10 (787), Lane 13-15 (RAPD 2).
68
CHAPTER FOUR
4.0 DISCUSSION AND CONCLUSION
4.1 Discussion
Salmonella species are the etiological agents of a wide range of diseases. They are commonly
found in wastewater and are frequently transmitted through food and water.
Traditional cultural methods have been the most common conventional technique employed to
type Salmonella species, despite several disadvantages. Molecular techniques for typing
microorganisms have advanced significantly and a number of techniques have been standardized
and applied to type Salmonella species. RAPD-PCR has been successfully applied to molecular
fingerprinting and the resulting genetic fingerprints can be of epidemiologic value (Hejazi et al.,
1997; Shangkuan and Lin 1998; Singh et al., 2006).
There are many instances of Salmonella spp isolated from wastewater and polluted water
reported in literature (Kinde et al., 1997; Howard et al., 2004; Bitton, 2005; Oliver et al., 2005;
AWWA, 2006). It is important to recognize that the prevalence and distribution of Salmonella
serovars varies from region to region (Dominguez et al., 2002; Uyttendaele et al., 1998) and
isolation rates depend upon the country where the study was carried out, the sampling plan and
the detection limit of the methodology (Uyttendaele et al., 1998). The composition and
conditions in the wastewater treatment plant (WWTP) are not static and it should not be assumed
that similar conditions exist in the wastewater of that region at a different time or wastewater
from other areas.
The result of this study revealed a low occurrence (4.1%) of Salmonella spp in wastewater
collected from the University of Nigeria, Nsukka wastewater treatment plant (WWTP). This is in
consonance with the work done by El Hussein et al. (2012) in Khartoum State, Sudan, who
reported an occurrence of 11.09% of Salmonella species in wastewater. Conversely, a report by
Howard et al. (2004) showed that municipal wastewater having undergone an activated sludge
process continued to bear Salmonella; the treated water yielded an MPN of 45/100 ml. Also El
Taweel (1994) found that Salmonella spp were detected in raw wastewater samples at oxidation
69
pond in Mit-Mzah treatment plant in Dakahlia governorate, Egypt, in numbers ranging from 102-
105 cfu/100 ml.
The low occurrence of Salmonella spp observed in this study could be attributed to either a few
organisms entering the system and/or failure of the bacteria to survive the movement to the
treatment plant, as well as the treatment processes employed in the WWTP. Since our interest
was in the number that survived the primary and secondary treatment, we did not examine the
wastewater at any point prior to its reaching the treatment plant.
The Standard microbiological tests (SMT) used in this study was characterized by good
analytical parameters which allowed the detection of low numbers of potentially stressed cells
but it is also important to note that the method used for isolation of Salmonella in this study
relied on the ability of the organisms to be successfully cultured on media plates. Therefore, the
low occurrence of Salmonella maybe due to the adoption of a viable but non culturable (VBNC)
state, the integration of the pathogen into an existing biofilm and internalization of the pathogen
into a variety of protozoan host (Jones and Bradshaw, 1996; Barker and Bloomfield, 2000;
Solano et al., 2002; Stepanovic et al., 2003).
Cho and Kim (1999) demonstrated the ability of several subspecies of Salmonella enterica to
enter a VBNC state after lengthy exposure to oligotrophic fresh and seawater under ambient
temperature. Also, the ability of Salmonella to become internalized, and to survive and replicate
in Amoeba was evaluated using 3 separate serovars of S. enterica and 5 different isolates of
axenic Acanthamoeba spp (Tezcan-Merdol et al., 2004).
The presence of substances such as industrial wastes, solvents, soaps, detergents and
disinfectants in wastewater which may be antagonistic and deleterious to the survival of
Salmonella could also be responsible for the low concentration of Salmonella observed in the
wastewater. Espigares et al. (2006) demonstrated that all strains of Salmonella tested were
susceptible to 2%, 20,000 µg/ml concentration of glutaraldehyde and 0.26% peracetic acid.
In a similar study, Oliver et al. (2005) reported that when sodium hypochlorite (free chlorine)
solution was added to provide a final concentration of 1 mg/l of free chlorine in wastewater,
0.39% of the treated S. typhimurium cells responded to the viability assay (as opposed to
70
culturability) after 60 minutes of chlorination, indicating that only a small portion of the cells
were able to resist this treatment.
The complex ecosystem in the WWTP created by the participation of bacteriophages, bacteria,
protozoa, algae and other organisms may play a role in the low concentration of Salmonella
present in wastewater. The competition that exist between Salmonella spp and other organisms
for nutrients and biological space, and the activities of antagonistic microorganisms and/or their
products may inhibit or suppress the growth of Salmonella in wastewater. Furthermore, water
dilution factor and the distance travelled to the WWTP may make it difficult to accumulate large
numbers of Salmonella spp in wastewater.
It is difficult to assess the effect that any single factor may have on the survival or recovery of
Salmonella in wastewater. Each factor probably contributes to the possible outcomes and each
factor may vary with region and time.
The result of the biochemical tests highlighted that 12 (18.4%) out of 65 non-lactose fermenting
(urease negative) isolates recovered from wastewater were found to be consistently Salmonella
spp and were in agreement with Bergey’s Manual of Systematic Bacteriology (Brenner and
Farmer III, 2005).
The biochemical tests carried out for different Salmonella strains from wastewater revealed that
there was less strain variation in Salmonella isolates. Variation in biochemical reactions have
been reported to be very low in Salmonella at serovars level (Rakesh et al., 2009), however,
biochemical tests showed variations at Salmonella subspecies. The presence of slightly diverse
Salmonella biochemical patterns were observed for lactose and citrate utilization and similar
variations were reported by Brenner and Farmer III (2005).
The analysis of Salmonella spp by serotyping revealed that of the 12 isolates confirmed
biochemically as Salmonella spp, 3 (25%) were identified as Salmonella limete belonging to
subspecies 1 (serogroup B) according to the Kauffman-White Scheme (Popoff and Le Minor,
2001). The result also showed that 2 isolates could not be serotyped and were identified as rough
strains. This is in tandem with a study by Rakesh et al. (2009), who reported that some of the
71
Salmonella serovars isolated from seafood could not be serotyped and were identified as rough
strains, lacking O-antigen.
S. limete was the only serovar found in this study. However, this serovar was considered an
unsuccessful pathogen because it was rarely associated with human illness (Boqvist et al., 2003).
It is important to recognize that serotypes isolated from humans do not always coincide with
serotypes isolated from wastewater. The existence of different serotypes proceeding from
wastewater and from humans is a common finding, as wastewater can often contain strains of
animal origin (Usera et al., 2001; Espigares et al., 2006). Some researchers have reported
isolating S. limete from different sources. Paterson and Cook (1955) reported isolating S. limete
from guinea pigs. In another study, Tejedor-Junco et al. (2010) reported the presence of S. limete
in water samples from 3 different farms in Gran Canaria, Spain. Furthermore, S. limete has been
recovered from human faeces and frogs in Sudan and Sweden respectively (Boqvist et al., 2003;
El Hussein et al., 2012). Thus, this reveals the broad host spectrum of this pathogen in both
humans and animals and suggests a strong possibility that contamination of the wastewater may
be of either human and/or animal origin.
Salmonella typhi is regarded worldwide as a significant pathogenic serovar with certain phage
types being associated with serious human illness (Quintaes et al., 2002; Smith et al., 2006).
However, no S. typhi was isolated in this study. The reason could be that the RV broth (Oxoid,
UK) used as a selective enrichment is suitable for isolating Salmonella serotypes other than S.
typhi and S. paratyphi A from environmental samples with high sensitivity and specificity
(Vassiliadis, 1983).
Phage typing results revealed that no phage was isolated, obviously no typing was possible.
Phages are highly host or species specific and phage typing has been successfully employed in
differentiating Salmonella spp especially S. enteriditis and S. typhimurium (Ward et al., 1987,
Anderson et al., 1977). One of the reasons for this observation maybe the failure of the virus to
attach to bacteria due to the lack or absence of suitable receptors or as a result of mutation on the
phage or bacteria which can alter the way receptors are arranged and prevent adsorption
(Engelkirk and Burton, 2006).
72
Kim et al., (2008) also found phage resistance with some of the Listeria monocytogenes used in
their study. In addition to the aforementioned factors; abortive infection, restriction system and
prevention of phage DNA transfer into the host were cited as reasons for conferring phage
resistance.
The specificity and sensitivity of PCR assay (16S) to detect Salmonella spp recovered from
wastewater was investigated. 3 (25%) of the 12 SMT confirmed isolates produced positive
amplifications of 574 bp fragment of the 16S rRNA gene specific for Salmonella spp, while non
Salmonella serovars were negative (100%). This result is similar to those obtained by Lin and
Tsen (1996). These investigators reported that 16S PCR technique using Salm 16S primer was
able to identify all the examined Salmonella serovars, while all non-Salmonella serovars gave
negative results. Conversely, El Hussein et al. (2012) reported that although primer set 16S
rRNA generated target size amplicons with all Salmonella isolates, similar amplicons were
produced from the DNA of some non-Salmonella strains including Proteus spp, Shigella
dysenteriae, Citrobacter freundii and Pseudomonas aeruginosa.
PCR assay was also used to detect Salmonella spp targeting the invA virulence gene. The result
of the invA PCR assay revealed that 2 of the 3 Salmonella isolates previously identified by 16S
PCR were found to harbor the invA gene corresponding to 244 bp, thus indicating the possession
of the invA gene of Salmonella, while all non Salmonella serovars gave negative results. A weak
band was observed for invA gene in one of the Salmonella spp recovered.
The invasion (invA) gene is present in Salmonella Pathogenicity Island (SPI) and found to be
responsible for invasion in the gut epithelial tissue of humans and animals (Astan and Dijk,
2005). Chiu and Ou (1996) reported that all Salmonella carry the invA gene which is not carried
by any other bacterial species. An exception was observed in the case of one of the isolates
previously identified as Salmonella by the 16S PCR assay. Thus, this suggests the possible
genetic variation of invA.
Rahn et al. (1992) showed that 2 Salmonella serovars did not harbor the invA gene. However,
further studies demonstrated that it was due to the natural deletion of the invA gene in the
centisome 63 Pathogenicity Island of environmental isolates (Ginocchio et al., 1997). Finally,
this study revealed that the invasion gene (invA) was present in Salmonella serovars isolated
73
from wastewater, thus highlighting the virulent nature of Salmonella serovars associated with
wastewater.
The discriminatory power of the RAPD-PCR was tested by considering the number of profiles
(RAPD binding patterns) generated using a set of 3 primers. The analysis of the RAPD-PCR
revealed that primers 787 and RAPD 2 were found useful in typing Salmonella isolates and 4
RAPD patterns were observed among the isolates on the basis of shared amplified product.
Primer 1254 did not produce any discriminatory pattern amongst the Salmonella isolates and
obviously no typing was possible. This result is in consonance with studies conducted in Lagos,
Nigeria by Smith et al. (2011) and Akinyemi et al. (2014). These investigators demonstrated that
RAPD-PCR using primer 1254 did not discriminate among the Salmonella isolates. The
implication of this is that RAPD Primer 1254 may not be useful for typing Salmonella in our
locality. However, this study is in contrast to the report of Quintanes et al. (2004) which
recorded highest discriminatory power amongst clinical Salmonella isolates using Primers 784
and 1254 in Brazil.
In another study, Hejazi et al. (1997) and Sandra et al. (1999) demonstrated that RAPD primer
1254 provided high polymorphic profiles among Serratia marcescens and Lactobacillus
delbrueckii strains respectively. Also Singh et al. (2006) revealed the usefulness of RAPD-PCR
assay for typing Indian strains of M. tuberculosis using 7 random decamer primers and the
heterogeneity in the M. tuberculosis strains in population studies.
Standardization of PCR mixtures and conditions are very important for reproducibility of RAPD-
PCR results. We found that it was necessary to perform RAPD-PCR in duplicates to obtain valid
result. It is important to note that to interpret the DNA fragment patterns generated by RAPD-
PCR, we should understand that the occurrence of random genetic events, including point
mutation and insertion and deletions of DNA, can alter the RAPD fingerprinting pattern
(Tenover et al., 1997). For this reason, we presumed the differentiating bands in the profiles
could be due to one or more genetic events. Our findings show that RAPD-PCR yields reliable
and reproducible results under precise assay conditions.
74
4.2 Conclusion
Results from this study revealed a low occurrence of Salmonella species in the University of
Nigeria, Nsukka wastewater treatment plant. It also revealed the sensitivity and rapidity of
molecular typing methods over the conventional methods in the detection, identification and
characterization of Salmonella species in wastewater.
Detailed strain identification is essential for the successful epidemiological investigation of
Salmonella outbreaks. For an appropriate risk assessment to be made, the type of Salmonella
species present and its relative numbers need to be determined. This is particularly important
regarding wastewater treatment and the reuse of wastewater. Thus, determination of the numbers
of different Salmonella species in a wastewater sample is imperative. Also, the efficient
enumeration of microbial pathogens in a wastewater sample pre and post treatment can allow an
effective assessment of the treatment process. The ideal detection method would be rapid,
sensitive, highly accurate, easy to perform, able to be run in high numbers and inexpensive.
The introduction of nucleic acid-based methods, such as the polymerase chain reaction (PCR) for
pathogen detection research, has resolved some of the problems encountered using conventional
methods. Molecular typing or fingerprinting of Salmonella isolates is an invaluable
epidemiological tool that can be used to track the source of infection and to determine the
epidemiological link between isolates from different sources. Hence, the combination of
traditional and molecular typing methods may be the best approach to characterize the
Salmonella isolates.
Wastewater management or the lack of it has a direct impact on the biological diversity of
aquatic ecosystems, disrupting the fundamental integrity of our life support systems, on which a
wide range of sectors from urban development to food production and industry depend. It is
essential that wastewater management is considered as part of an integrated, ecosystem-based
management that operates across sectors and borders.
75
REFERENCES
Ackerman, H. W. (1998). Tailed bacteriophages: the Caudovirales. Adv. Virus. Res. 51:135-201.
Akinyemi, K. O., Iwalokun, B. A., Agboyinu, J. A., Ogunyemi, O. and Fasure, A. K. (2014).
Emergence of Third Generation Cephalosporin Resistance and Typing by Randomly
Amplified Polymorphic DNA (RAPD) among Clinical Salmonella Isolates from Lagos,
Nigeria. British Microbiology Research Journal 4(6):669-677.
Akinyemi, K. O., Smith, S. I., Oyefolu, A. O., Coker, A. O. (2005). Multidrug resistance in
Salmonella enterica serovar Typhi isolated from patients with typhoid fever
complications in Lagos, Nigeria. Public Health 119:321-327.
Alexandrino, M., Grohmann, E. and Szewzyk, U. (2004). Optimization of PCR based methods
for rapid detection of Campylobacter jejuni, Campylobacter coli and Yersinia
enterocolitica serovar 0:3 in wastewater samples. Water Research 38:1340-1356.
American Public Health Association (2005). Standards Methods for the Examination of Water
and Wastewater. American Public Health Association, 21st (edn). Washington DC.
American Water Works Association (2006). Manual of Water Supply Practices-M56:
Fundamentals and Control of Nitrification in Chlorinated Drinking Water Distribution
Systems. Denver, CO.
Anderson, E. S., Ward, L. R., De Saxe, M. J. and De Sa, J. D. H. (1977). Bacteriophage-typing
designations of Salmonella typhimurium. Journal of Hygiene, Cambridge 78:297-300.
Anderson, R. C. and Ziprin, R. L. (2001). Bacteriology of Salmonella. In Foodborne Disease
Handbook, Bacterial Pathogens. Hui, Y. H., Pierson, M. D. and Gorham, J. R. (Eds).
Marcel Dekker Inc, New York; pp: 247-263.
Andrews, W. H. and Hammack, T. S. (2001). Salmonella, chapter 5. In Bacteriological
Analytical Manual, U.S Food and Drug Administration, 8th
(edn), Revision A, AOAC
International, Gaithersburg, Md.
Appelgren, B. (2004). Water in Agriculture. UNESCO, New Delhi.
Arumugaswamy, R. K., Rusul, G., Abdul Hamid, S. N. and Cheah, C. T. (1995). Prevalence of
Salmonella in raw and cooked foods in Malaysia. Food Microbiology 12:3-8.
Asten, A. J. A. M. and Dijk, J. E. (2005). Distribution of classic virulence factors among
Salmonella spp. FEMS Immunol. Med. Microbiol. 44:251-259.
76
Aurelie, T., Thierry, B., Barbara, P. and Fabienne, P. (2005). Detection of Salmonella in
environmental water and sediment by a nested-multiplex polymerase chain reaction
assay. Research in Microbiology 156:541-553.
Bardakci, F. (1996). Applications of the random amplified polymorphic DNA (RAPD) technique
in tilapia: species, subspecies and sex identification. Ph.D. Thesis, University of Wales,
Swansea.
Bardakci, F. (2001). Random Amplified Polymorphic DNA (RAPD) Markers.
Turk. J. Biol. 25:185-196.
Bardakci, F. and Skibinski, D. O. F. (1999). A polymorphic SCAR-RAPD marker between
species of tilapia. Animal Genetics 30:78-79.
Barker, J. and Bloomfield, S. F. (2000). Survival of Salmonella in bathrooms and toilets in
domestic homes following Salmonellosis. Journal of Applied Microbiology 89:137-144.
Barry, M. A., Dower, W. J. and Johnston, S. A. (1996). Toward cell-targeting gene therapy
vectors: selection of cell-binding peptides from random peptide presenting phage
libraries. Nat. Med. 2(3):299-305.
Baumler, A. J., Tsolis, R. M., Ficht, T. A. and Adams, L. G. (1998). Evolution of host
adaptation in Salmonella enterica. Infect. Immun. 66:4579-4587.
Bej, A., Mahbubani, M. H., Boyce, M. J. and Atlas, R. M. (1994). Detection of Salmonella spp.
in oysters by PCR. Applied Environmental Microbiology 60:368-373.
Beli, E., Duraku, E. and Telo, A. (2001). Salmonella serotypes isolated from chicken meat in
Albania. International Journal of Food Microbiology 71:263-266.
Bell, C. and Kyriakides, A. (2002). Salmonella. In Foodborne Pathogens: Hazards, Risk
Analysis and Control. Blackburn, C. W. and McClure, P. J (Eds). CRC Press; pp:307-35.
Bhunia, A. K. (2008). Food borne Microbial Pathogens: Mechanisms and Pathogenesis.
Springer Science and Business Media, LLC, United States of America.
Bitton, G. (2005). Wastewater Microbiology, 3rd
(edn). Wiley-Less, Hoboken, NJ, pp:746.
Blanc-Potard, A. B. and Groisman, E. A. (1997). The Salmonella selC locus contains a
pathogenicity island mediating intramicrophage survival. EMBO J 16:5376-5385.
Boqvist, S., Hansson, I., Nord Bjerselius, U., Hamilton, C., Wahlström, H., Noll, B., Tysen, E.
and Engvall, A. (2003). Salmonella isolated from animals and feed production in Sweden
between 1993 and 1997. Acta vet. Scand. 44:181-197.
77
Bordini, M. E. B., Asturiano Ristori, C., Jakabi, M. and Gelli, D. S. (2007). Incidence,
internalization and behavior of Salmonella in mangoes, var. Tommy Atkins. Food
Control 18(8):1002-1007.
Bouchrif, B., Paglietti, B., Murgia, M., Piana, A., Cohen, N., Ennaji, M., Rubino, S. and
Timinouni, M. (2009). Prevalence and antibiotic-resistance of Salmonella isolated from
food in Morocco. Journal of Infection in Developing Countries 28(3):35-40.
Brenner, D. J. and Farmer III, J. J. (2005). Enterobacteriaceae. In Bergey’s Manual of
Systematic Bacteriology, 2nd
(edn), Brenner, D. J., Kreig, N. R. and Staley, J. T. (Eds).
Springer, New York, USA, pp:587-878.
Brenner, F. W., Villar, R. G., Angulo, F. J., Tauxe, R. and Swaminathan, B. (2000). Salmonella
Nomenclature. Journal of Clinical Microbiology 38(7):2465-2467.
Butler, D. and Davies, J. W. (2011). Urban Drainage 3rd
(edn). Spon Press.
ISBN-13:9780415455268.
Caetano-Annoles, G., Bassam, B. J and Gresshoff, P. M. (1991). DNA amplification
fingerprinting using very short arbitrary oligonucleotide primers.
Biotechnology 9:553-557.
Carey-Smith, G., Billington, C., Cornelius, A. J., Hudson, J. A. and Heinemann, J. A. (2006).
Isolation and Characterization of bacteriophages infecting Salmonella spp. FEMS
Microbiology Letters 258:182-186.
Centers for Disease Control and Prevention (2005). Salmonella Annual Summary 2004.
Retrieved on 16th
September, 2013 from
http://www.cdc.gov/ncidod/dbmd/phlisdata/salmonella.html
Cetinkaya, F., Cibik, R., Soyutemiz, G. E., Ozakin, C., Kayali, R. and Levent, B. (2008).
Shigella and Salmonella contamination in various foodstuffs in Turkey. Food Control
19:1059-1063.
Chalmers, K. J., Waugh, R., Sprent, J. I., Simons, A. J. and Powell, W. (1992). Detection of
genetic variation between and within populations of Gliricidia sepium and G. maculate
using RAPD markers. Heredity 69:465-472.
Chiu, C. H. and Ou, J. T. (1996). Rapid identification of Salmonella serovars in feces by specific
detection of invA and spvC, by an enrichment broth culture multiplex PCR combination
assay. Journal of Clinical Microbiology 34:2619-2622.
Cho, J. C. and Kim, S. J. (1999). Viable but non-culturable state of a green fluorescence protein-
tagged environmental isolate of Salmonella typhi in groundwater and pond water. FEMS
Microbiology Letters 170(1):257-264.
78
Chu, C., Chiu, C. H., Wu, W. Y., Chu, C. H., Liu, T. P. and Ou, J. T. (2001). Large drug
resistance virulence plasmids of clinical isolates of Salmonella enterica serovar
Choleraesuis. Antimicrobiol. Agents Chemother. 45:2299-2303.
Clark, J. R., March, J. B. (2006). Bacteriophages and biotechnology: vaccines, gene therapy and
antibacterials. Trends in Biotechnology 24(5):212-218.
Colak, H., Hampikyan, H., Bingol, E. B. and Ulusoy, B. (2007). Prevalence of L.monocytogenes
and Salmonella spp. in Tulum cheese. Food Control 18:576-579.
Corbitt, A. J., Bennion, N. and Forsythe, S. J. (2000). Adenylate kinase amplification of ATP
bioluminescence for hygiene monitoring in the food and beverage industry. Lett. Appl.
Microbiol. 30(6):443-447.
Cortez, A. L. L., Carvalho, A. C. F. B., Ikuno, A. A., Burger, K. P. and Vidal-Martins, A. M. C.
(2006). Identification of Salmonella spp. isolates from chicken abattoirs by multiplex
PCR. Research in Veterinary Science 81(3):340-344.
Crosa, J., Brenner, D., Ewing, W. and Falkow, S. (1973). Molecular relationships among the
Salmonellae. Journal of Bacteriology 115(1):307-315.
D’Aoust, J. and Maurer, J. (2007). Salmonella species. In Food microbiology: Fundamentals
and Frontiers, Doyle, M. P. and Beuchat, L. R. (Eds). ASM Press, Washington DC;
pp:187-219.
D’Aoust, J. Y., Maurer, J. and Bailey, J. S. (2001). Salmonella species. In Food Microbiology:
Fundamentals and Frontiers, Doyle, M. P., Beuchat, L. R. and Montville, T. J. (Eds).
ASM Press, Washington, DC, pp:141-177.
Dallal, M. M. S., Doyle, M. P., Rezadehbashi, M., Dabiri, H., Sanaei, M., Modarresi, S.,
Bakhtiari, R., Sharifiy, K., Taremi, M., Zali, M. R. and Sharifi-Yazdi, M. K. (2010).
Prevalence and antimicrobial resistance profiles of Salmonella serotypes, Campylobacter
and Yersinia spp. isolated from retail chicken and beef, Tehran, Iran. Food Control
21:388-392.
Dauga, C., Zabrovsakaia, A. and Grimont, P. A. D. (1998). Restriction fragment length
polymorphism analysis of some flagellin genes of Salmonella enterica. Journal of
Clinical Microbiology 36:2835-2843.
de la Torre, E., Tello, M., Mateu, E. M. and Torre, E. (2005). Use of biochemical kinetic data to
determine strain relatedness among Salmonella enterica subsp. enterica isolates. Journal
of Clinical Microbiology 43:5419-5424.
79
del Cerro, A., Soto, S. M., Landeras, E., Gonzales-Hevia, M. A., Guijarro, J. A. and Mendoza,
M. C. (2002). PCR-based procedures in detection and DNA fingerprinting of Salmonella
from samples of animal origin. Food Microbiology 19:567-575.
Department for International Development (2008). Water and Sanitation Policy - Water: An
increasingly precious resource Sanitation, a matter of dignity. Retrieved on February,
2012 from http://www.dfid.gov.uk/Documents/publications/water-sanitation-policy
08.pdf.
Dick, N., Ritter, V., Kircher, S., Warns, P. and Sturm, K. (2005). Evaluation of BBL
CHROMagar Salmonella: AOAC Performance Tested Method.
Annual AOAC Meeting, 2005.
Dominguez, C., Gómez, I. and Zumalacarregui, J. (2002). Prevalence of Salmonella and
Campylobacter in retail chicken meat in Spain. International Journal of Food
Microbiology 72:165-168. ISSN 0168-1605.
Duguid, J. P., Anderson, E. S., Alferdsson, G. A. R., Barker, D. C. and Old, D. C. (1975). A new
biotyping scheme for Salmonella typhimurium and its phylogenetic significance.
Journal of Medical Microbiology 8:149-166.
Ecuyer, P. B., Diego, J., Murphy, D. T. E., Jones, M., Sahm, D. F. and Fraser, V. J. (1996).
Nosocomial outbreak of gastroenteritidis due to Salmonella Senftenberg. Clin. Infect.
Dis. 23:734-742.
Edwards, R. A., Schifferli, D. M. and Miloy, S. R. (2000). A role of Salmonella fimbriae in
intraperitoneal infections. Proc. Natl. Acad. Sci. USA. 97:1258-1262.
El Hussein, A. A., Mohy-Eldin, H. S., Nor Elmadiena, M. M. and Siddig, M. A. M. (2012).
Prevalence, Detection and Antimicrobial Resistance Pattern of Salmonella in Sudan.
Research Journal of Microbiology 5(10):966-973.
El-Lathy, M. A., El-Taweel, G. E., El-Sonosy, W. M., Samham, F. A. and Moussa, T. A. A.
(2009). Determination of pathogenic bacteria in wastewater using conventional and PCR
techniques. Environmental Biotechnolgy 5(2):73-80.
El-Taweel, G. E. (1994).Use of some biological and chemical indicators for evaluating the
wastewater during treatments. Ph.D. thesis, Faculty of Agriculture, Cairo University.
Engelkirk, P. G. and Burton, G. R. (2006). Burton's Microbiology for the Health Sciences, 8th
(edn). Lippincott Williams and Wilkins.
Espigares, E., Bueno, A., Espigares, M. and Galvez, R. (2006). Isolation of Salmonella serotypes
in wastewater and effluent: Effect of treatment and potential risk. International Journal
of Hygiene and Environmental Health 209:103-107.
80
Ewing, W. H. (1972). The nomenclature of Salmonella, its usage and definitions for the three
species. Can. J. Microbiol. 18:1629-1637.
Ewing, W. H. (1986). Differentiation of Enterobacteriaceae by biochemical reaction. In Edwards
and Ewing’s identification of Enterobactriaceae, 4th
(edn). Elsevier Science Publishing
Co. Inc., New York.
Ezaki, T., Kawamura, Y. and Yabuuchi, E. (2000). Recognition of nomenclatural standing of
Salmonella typhi (Approved Lists 1980), Salmonella enteritidis (Approved Lists 1980)
and Salmonella typhimurium (Approved Lists 1980), and conservation of the specific
epithets enteritidis and typhimurium. Request for an opinion. Int. J. Syst. Evol.
Microbiol. 50:945-947.
Fani, R., Damiani, G., Di Serio, C., Gallari, E., Grifoni, A. and Bazzicalupa, M. (1993). Use of
random amplified polymorphic DNA (RAPD) for generating specific DNA probes for
microorganisms. Molecular Ecology 2:243-250.
Fluit, A. C. (2005). Towards more virulent and antibiotic-resistant Salmonella.
FEMS Immunology and Medical Microbiology 43:1-11.
Folkesson, A., Advani, A., Sukupolvi, S., Pfeifer, J. D., Normark, S. and Lofdahl, S. (1999).
Multiple insertions of fimbrial operons correlate with the evolution of Salmonella
serovars responsible for human disease. Molecular Microbiology 69:612-622.
Freitas, C. G. D., Santana, A. P., Silva, P. H. C. D., Goncalves, V. S. P., Barros, M. D. A. F.,
Torres, F. A. G., Murata, L. S. and Perecmanis, S. (2010). PCR multiplex for detection of
Salmonella Enteritidis, Typhi and Typhimurium and occurrence in poultry meat.
International Journal of Food Microbiology 39:15-22.
Fricker, E. J. and Fricker, C. R. (1995). Detection of Legionella spp. using a commercially
available polymerase chain reaction test. Water Science Technology 31:407-408.
Funatsu, T., Taniyama, T., Tajima, T., Tadakuma, H. and Namiki, H. (2002). Rapid and sensitive
detection method of a bacterium by using a GFP reporter phage. Microbiol. Immunol.
46(6):365-369.
Galan, J . E., Ginocchio, C. and Costean, P. (1992). Molecular and Functional Characterization
of the Salmonella invasion gene invA: homology of invA to members of a new protein
family. J. Bacteriol. 174:4338-4349.
Gast, R. K. (1997). Salmonella infections. In Diseases of Poultry, 10th
(edn). Calnek, B. W.
(Eds). Iowa State University Press, America; pp:81-122.
81
Ginocchio, C. C., Rahn, K., Clarke, R. C. and Galan, J. E. (1997). Naturally occurring deletions
in the centisome 63 pathogenicity island of environmental isolates of Salmonella spp.
Infect. Immun 65:1267-1272.
Gomez-Laguna, J., Hernandez, M., Creus, E., Echeita, A., Otal, J., Herrera-Leon, S. and Astorga,
R. J. (2010). Prevalence and antimicrobial susceptibility of Salmonella infections in free
range pigs. The Veterinary Journal 190:176-178.
Goodridge, L., Chen, J. and Griffiths, M. (1999). Development and characterization of a
fluorescent-bacteriophage assay for detection of Escherichia coli O157:H7. Applied
Environmental Microbiology 65:1397-1404.
Gray, J. T. and Fedorka-Cray, P. J. (2002). Salmonella. In Foodborne Diseases, 2nd
(edn),
Cliver, D. O. and Riemann, H. P. (Eds). Academic Press, San Diego; pp:55-68.
Grimont, P. A. D. and Weill, F. X. (2007). Antigenic Formulae of the Salmonella serovars,
Ninth Edition. WHO Collaborating Centre for Reference and Research on Salmonella.
Institut, Pasteur, Paris, France.
Groisman, E. A. and Ochman, H. (1996). Pathogenicity islands: bacterial evolution in quantum
leaps. Cell 87:791-794.
Guibourdenche, M., Roggentin, P., Mikoleit, M., Fields, P. I., Bockemuhl, J., Grimont, P. A. D.
and Weill, F. X. (2010). Supplement 2003 – 2007 (No. 47) of the White-Kauffmann
LeMinor scheme. Research in Microbiology 161:26-29. ISSN 0923-2508.
Hammer, M. J. and Hammer Jr, M. J. (2011). Water and Wastewater Technology, 7th
(edn).
Prentice-Hall. ISBN-13:978-0135114049.
Hanes, D. (2003). Nontyphoid Salmonella. In International Handbook of Foodborne Pathogens,
Henegariu, O., Heerema, N. A., Dloughy, S. R., Vance, G. H. and Vogt, P. H. (Eds).,
Marcel Dekker, Inc, New York; pp:137-149.
Hasan, M. N., Ara, N., Mamun, S. A., Rahman, M. M. and Rahman, M. H. (2009). Prevalence of
Salmonella spp in chicken eggs from Khulna city. Journal of Innovation and
Development Strategy 3(3):1-6.
Hatha, A. A. M. and Lakshamanaperumalsamy, P. (1997). Prevalence of Salmonella in fish and
crustaceans from markets in Coimbatore, South India. Food Microbiology 14:111-116.
Hayward, R. D. and Koronakis, V. (2002). Direct modulation of the host cell cytoskeleton by
Salmonella actin-binding proteins. Trends. Cell Biol. 1(12):1520.
Hejazi, A., Keane, C. T. and Falkiner, F. R. (1997). The use of RAPD-PCR as a typing method
for Serratia marcescens. Journal of Medical Microbiology 32:1935-1938.
82
Hemmat, M., Weeden, N. F., Manganaris, A. G. and Lawson, D. M. (1994). Molecular marker
linkage map for apple. Journal of Heredity 85:4-11.
Hennes, K. P., Suttle, C. A. and Chan, A. M. (1995). Fluorescently labeled virus probes show
that natural virus populations can control the structure of marine microbial communities.
Applied Environmental Microbiology 61(10):3623-3627.
Herikstad, H., Motarjemi, Y. and Tauxe, R. V. (2002). Salmonella surveillance: a global survey
of public health serotyping. Epidemiol. Infect. 129:1-8.
Hoszowski, A. and Wasyl, D. (2001). Typing of Salmonella enterica subsp. enterica serovar
Mbandaka isolates. Vet. Microbiol. 80:139-148.
Howard Hughes Medical Institute (2011). Natural Genomics Research Initiative in Phage: Phage
resource guide. Tuajuanda, C. J., Lucia, P. B., Kevin, W. B., Razi, K. and Melvina, F. L
(Eds). Chevy Chase, Maryland, USA.
Howard, I., Espigares, E., Lardelli, P., Martın, J. L. and Espigares, M. (2004). Evaluation of
microbiological and physicochemical indicators for wastewater treatment.
Environ. Toxicol. 19:241–249.
Hu, L. and Kopecko, D. J. (2003). Typhoid Salmonella. In International Handbook of
Foodborne Pathogens, Millotis, M. D. and Bier, J. W. (Eds). Marcel Dekker, Inc. New
York; pp:151-165. ISBN 0-8247-0685-4.
Hunter, P. R. (1997). Typhoid and Paratyphoid Fevers and Other Salmonella Infections. John
Wiley and Sons Ltd, West Sussex; pp:116-123.
Inal, J. M. (2003). Phage therapy: a reappraisal of bacteriophages as antibiotics.
Arch. Immunol. Ther. Exp. 51(4):237-244.
International Organization for Standardization (2002). ISO 6579: Microbiology – General
guidance on methods for the detection of Salmonella, 4th
(edn). Geneva, Switzerland.
Jalali, M., Abedi, D., Pourbakhsh, S. A. and Ghoukasin, K. (2008). Prevalence of Salmonella
spp. in raw and cooked foods in Isfahan-Iran. Journal of Food Safety 28:442-452.
Jones, K. and Bradshaw, S. B. (1996). Biofilm formation by the Enterobacteriaceae: a
comparison between Salmonella enteritidis, Escherichia coli and a nitrogen fixing strain
of Klebsiella pneumoniae. Journal of Applied Bacteriology 80(4):458-464.
Kambhampati, S., Blavk, W. C. and Rai, K. S. (1992). Random Amplified Polymorphic DNA of
mosquito species and populations (Diptera: Culicidae): techniques, statistical analysis,
and applications. Journal of Medical Entomology 29:939-945.
83
Keller, R., Passamani, F., Vaz, L., Cassini, S. T. and Goncalves, R. F. (2003). Inactivation of
Salmonella spp from Secondary and Tertiary Effluents by UV Irradiation. Water Science
Technology 47(3):147-150.
Kim, J. W., Siletzky, R. M. and Kathariou, S. (2008). Host ranges of Listeria-specific
bacteriophages from the turkey processing plant environment in the United States.
Applied and Environmental Microbiology 74:6623-6630.
Kinde, H., Adelson, M., Ardans, A., Little, E. H., Willoughby, D., Berchtold, D., Read, D. H.,
Breitmeyer, R., Kerr, D. Tarbell, R. and Hughes, E. (1997). Prevalence of Salmonella in
municipal sewage treatment plant effluents in Southern California. Avian Diseases
41(2):392-398.
Kodikara, C. P., Crew, H. H., Stewart, G. S. (1991). Near on-line detection of enteric bacteria
using lux recombinant bacteriophage. FEMS Microbiology Letters 67(3):261-265.
Lagatolla, C., Dolzani, L., Tonin, E., Lavenia, A., Dimichele M., Tommansin, T. and Monti
Bragadin, C. (1996). PCR- ribotyping and Characterizing Salmonella isolates of different
serotypes. Journal of Clinical Microbiology 34:2440-2443. ISSN 0095-1137.
Le Minor, L. (1991). The genus Salmonella. In The Prokaryotes, Balows, A., Truper, H. G.,
Dworkin, C. M., Harder, W. and Scheilfer, K. H. (Eds). Springer-Varlag, New York;
pp:2760-2774.
Lin, C. K. and Tsen, H. Y. (1996). Use of two 16S DNA targeted oligonucleotides as PCR
primers for the specific detection of Salmonella in foods. Journal of Applied
Bacteriology 80(6):659-666. ISSN 0021-8847.
Malkawi, H. I. and Gharaibeh, R. (2004). Rapid and simultaneous identification of two
Salmonella enterica serotypes, Enteritidis and Typhimurium from chicken and meat
products by multiplex PCR. Biotechnology 3(1):44-48.
Malorny, B., Hoorfar, J., Bunge, C. and Helmuth, R. (2003a). Multicenter validation of the
analytical accuracy of Salmonella PCR: towards an international standard. Applied and
Environmental Microbiology 69:290-296.
Marcus, S. L., Brumell, J. H., Pfeifer, C. G. and Finlay, B. B. (2000). Salmonella pathogenicity
islands: big virulence in small packages. Microbes Infect 2:145-156.
Matsui, H., Bacot, C. M., Garlington, W. A., Doyle, T. J., Roberts, S. and Gulig, P. A. (2001).
Virulence plasmid-borne spvB and spvC genes can replace the 90 kilobase plasmid in
conferring virulence to Salmonella enterica serovar Typhimurium in subcutaneously
inoculated mice. Journal of Bacteriology 183:4652-4658.
84
Medici, D. D., Croci, L., Delibato, E., Pasquale, S., Filetici, E. and Toti, L. (2003). Evaluation of
DNA extraction methods for use in combination with SYBR Green I Real-Time PCR to
detect Salmonella enterica serotype Enteritidis in poultry. Applied and Environmental
Microbiology 69:3456-3461.
Metcalf and Eddy, Inc. (2002). Wastewater Engineering- Treatment and Reuse, 4th
(edn).
McGraw-Hill. ISBN-13:9780070418783.
Michael, R. T. and David, B. (2011). Introduction to Wastewater Treatment. Ventus Publishing
APS. ISBN 978-87-7681-843-2.
Millennium Ecosystem Assessment (2005). Millennium Ecosystem Assessment: Concepts of
Ecosystem Value and Valuation Approaches. Island Press, Washington, DC.
Moganedi, K. L. M., Goyvaerts, E. M. A., Venter, S. N. and Sibara, M. M. (2007). Optimization
of PCR-invA primers for the detection of Salmonella in drinking and surface waters
following a pre-cultivation step. Water. SA. 33(2):195-202. ISSN 0378-4738.
Mohand, R. V., Brahimi, N. M., Moustaoui, N., Mdaghri, N. E., Amarouch, H., Grimont, F.,
Bingen, E. and Benbachir, M. (1999). Characterization of extended spectrum β-lactamase
producing Salmonella typhymirium by phenotypic and genotypic typing methods.
Journal of Clinical Microbiology 37:3769-3773. ISSN 0095-1137.
Montville, T. J. and Matthews, K. R. (2008). Food Microbiology: An Introduction, 2nd
(edn).
United States of America: ASM Press, Washington, DC; pp:428.
Mullis, K. and Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase catalyzed
chain reaction. Methods. Enzymol. 155:335-350.
Naish, K. A., Warren, M., Bardakci, F., Skibinski, D. O. F., Carvalho, G. R. and Mair, G. C.
(1995). Multilocus DNA fingerprinting and RAPD reveal similar genetic relationships
between strains of Oreochromis niloticus (Pisces: Cichlidae).
Molecular Ecology 4:271-274.
Narasimhan, T. N. (2008). Water, Law, Science. Journal of Hydrology 349(1-2):125-138.
Negm, M. S., Nagy, N. M., Abdel-Rasik, M. H. (1995). Sewage characteristics and per capita
loads in Ismailia City. Second Middle East Conference, Cairo Egyptian Wastewater
Management.
Newell, D. G., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H., Giessen, J.
V. D. and Kruse, H. (2010). Food-borne diseases: the challenges of 20 years ago still
persist while new ones continue to emerge. International Journal of Food Microbiology
139:3-15.
85
Oliver, J. D., Dagher, M. and Linden, K. (2005). Induction of Escherichia coli and Salmonella
Typhimurium into the viable but non culturable state following chlorination of
wastewater. Journal of Water Health 3(3):249-257.
Parkhill, J., Dougan, G., James, K. D., Thomson, N. R., Pickard, D., Wain, J., Churcher, C.,
Mungall, K. L., Bentley, S. D., Holden, M. T., Sebaihai, M., Baker, S., Basham, D.,
Brooks, K., Chillingworth, T., Connerton, P., Cronin, A., Davis, P., Davies, R. M.,
Dowd, L.,White, N., Farrar, J., Feltwell, T., Hamlin, N., Haque, A., Hein, T. T., Holryod,
S., Jagels, K., Krogh, A., Larsen, T. S., Leather, S., Moule, S., O'Gaora, P., Parry, C.,
Quail, M., Rutherford, K., Sommonds, M., Skelton, J., Stevens, K.,Whitehead, S. and
Barrel, B. G. (2001). Complete genome sequence of a multiple drug resistance
Salmonella enterica serovar Typhi CT18. Nature 413:848-852.
Parry, C. M. (2006). Epidemiological and clinical aspects of human typhoid fever. In Salmonella
Infections: Clinical, Immunological and Molecular Aspects, Matroeni, P. and Maskell, D.
(ed). Cambridge University Press, New York; pp:1-18.
Paterson, J. S. and Cook, R. (1955). Naturally occurring Salmonella limete in guinea pigs.
J. Pathol. Bacteriol. 70(1):242-245.
Pathmanathan, S. G., Cardona-Castro, N., Sanchez-Jimenez, M. M., Correa-Ochoa, M. M.,
Puthucheary, S. D. and Thong, K. L. (2003). Simple and rapid detection of Salmonella
strains by direct PCR amplification of the hilA gene. Journal of Medicinal Microbiology
52:773-776.
Penteado, A. L. and Leitão, M. F. F. (2004). Growth of Salmonella Enteritidis in melon,
watermelon and papaya pulp stored at different times and temperatures. Food Control
15(5):369-373.
Petrenko, V. A., Vodyanoy, V. J. (2003). Phage display for detection of biological threat agents.
J. Microbiol. Methods. 53(2):253-262.
Pickard, D., Wain, J., Baker, S., Line, A., Chohan, S., Fookes, M., Barron, A., Gaora, P. O.,
Chabalgoity, J. A., Thanky, N., Scholes, C., Thomson, N., Quail, M., Parkhill, J. and
Dougan, G. (2003). Composition, acquisition, and distribution of the Vi
exopolysaccharide-encoding Salmonella enterica Pathogenicity Island SPI-7.
Journal of Bacteriology l85:5055-5065.
Pieskus, J., Kazeniauskas, E., Butrimaite-Ambrozeviciene, C., Stanevicius, Z. and Mauricas, M.
(2008). Salmonella incidence in broilers and laying hens with the different housing
systems. The Journal of Poultry Science 45:227-231.
Pimentel, D. and Pimentel, M. (2008). Food, Energy, and Society. CRC Press, Boca Raton,
Florida, USA.
86
Popoff, M. Y. and Le Minor, L. (1997). Antigenic formula of the Salmonella serovars. WHO
Collaborating Centre for and Research on Salmonella. Institut Pasteur, Paris, France.
Popoff, M. Y. and Le Minor, L. (2001). Antigenic Formulas of the Salmonella Serovars, 8th
revision. WHO Collaborating Center for Reference and Research on Salmonella, Pasteur
Institute, Paris, France. Retrieved from: www.prise-pcp.org/.../cd/.../antigenic-formulas
salmonella-serovars.pdf
Popoff, M. Y. and Le Minor, L. (2005). Salmonella. In Bergey’s Manual of Systematic
Bacteriology, 2nd (edn), Brenner, D. J., Kreig, N. R. and Staley, J. T. (Eds). Springer,
New York, USA; pp:764-799.
Popoff, M. Y., Bockemuhl, J. and Gheesling, L. L. (2004). Supplement 2002 (no.46) to the
Kauffmann -White scheme. Res. Microbiol. 155:568-570.
Portillo, F. G. (2000). Molecular and cellular biology of Salmonella pathogenesis. In Microbial
Foodborne Diseases: Mechanisms of Pathogenesis and Toxin Synthesis, Cary, J. W. and
Bhatnagar, D. (Eds). Technomic Publishing Company, Inc, United States of America;
pp:3-34.
Prager, R., Fruth, A. and Tschape, H. (1995). Salmonella enterotoxin (stn) gene is prevalent
among strains of Salmonella enterica, but not among Salmonella bongori and other
Enterobacteriaceae. FEMS Immuno. Med. Microbiol. 12:47-50.
Prendergast, D. M., Duggan, S. J., Gonzales-Barron, U., Fanning, S., Butler, F., Cormican, M.
and Duffy, G. (2009). Prevalence, numbers and characterizations of Salmonella spp. on
Irish retail pork. International Journal of Food Microbiology 131(2-3):233-239.
Pui, C. F., Wong, W. C., Chai, L. C., Tunung, R., Jeyaletchumi, P., Noor Hidayah, M. S., Ubong,
A., Farinazleen, M. G., Cheah, Y. K. and Son, R. (2011). Review Article Salmonella: A
foodborne pathogen. International Food Research Journal 18:465-473.
Qasim, S. R. (1998). Wastewater Treatment Plants: Planning, Design and Operation, 2nd
(edn).
CRC Press. ISBN-13:978-1566766883.
Quintaes, B. R., Leal, N. C., Reis, E. M. F., Fonseca, E. L. and Hofero, E. (2002). Conventional
and molecular typing of Salmonella typhi strains from Brazil. Rev. Inst. Med. Trop. S
Paulo 44:315-319.
Quintanes, B. R., Leal, N. C., Reis, E. M. and Hofer, E. (2004). Optimization of randomly
amplified polymorphic DNA-polymerase chain reaction for molecular typing of
Salmonella enterica serovar typhi. Rev. Soc. Bras Med. Trop. 37(2):143-47.
Raetz, C. R. and Whitfield, C. (2002). Lipopolysaccharide endotoxins.
Annual Review of Biochemistry 71:635-700.
87
Rahman, H. and Sharma, V. D. (1995). Immunogencity and antigenic relationship of Salmonella
enterotoxin with other enterotoxins. Zbl. Bakt. 282:416-422.
Rahn, K., De Grandis, S. A., Clarke, R. C., McEwen, S. A., Galan, J. E., Ginocchio, C., Curtiss
Ill, R. and Gyles, C. L. (1992). Amplification of an invA gene sequence of Salmonella
Typhimurium by polymerase chain reaction as a specific method of detection of
Salmonella. Mol. Cell. Probes. 6:271-279.
Rakesh, K., Surendran, P. K. and Nirmala, T. (2009). Biochemical and Molecular investigations
on Salmonella serovars from seafood. Unpublished doctoral dissertation, Cochin
University of Science and Technology, India.
Raschid-Sally, L. and Jayakody, P. (2008). Drivers and characteristics of wastewater agriculture
in developing countries: Results from a global assessment, Colombo, Sri Lanka. IWMI
Research Report 127, International Water Management Institute, Colombo; pp:35.
Reeves, M. W., Evins, G. M., Heiba, A. A., Plikaytis, B. D. and Farmer III, J. J. (1989). Clonal
nature of Salmonella typhi and its genetic relatedness to other salmonellae as shown by
multilocus enzyme electrophoresis and proposal of Salmonella bongori.
comb. nov. J. Clin. Microbiol. 27:313-320.
Reher, S., Van Schil, P., Rutsaert, R. and DeGeest, R. (1995). Inflammatory aneurysm of the
abdominal aorta infected by Salmonella Dublin. Eur. J. Endovasc. Surg. 9:498 -500.
Rotgar, R. and Casadesus, J. (1999). The virulence plasmid of Salmonella.
Int. Microbiol. 2:177-184.
Russel, J. R., Horsein, F., Johnson, E., Waugh, R. and Powell, W. (1993). Genetic differentiation
of cocoa (Theobroma cacao L.) populations revealed by RAPD analysis. Molecular
Ecology 2:89-97.
Rusul, G., Khair, J., Radu, S., Cheah, C. T. and Yassin, R. M. (1996). Prevalence of Salmonella
in broilers at retail outlets, processing plants and farms in Malaysia. International Journal
of Food Microbiology 33:183-194.
Salleh, N. A., Rusul, G., Hassan, Z., Reezal, A., Isa, S. H., Nishibuchi, M. and Radu, S. (2003).
Incidence of Salmonella spp. in raw vegetables in Selangor, Malaysia. Food Control
14:475-479.
Sandra, T., Giacomo, Z. and Franco, D. (1999). Use of PCR-Based methods for Rapid
Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp.
lactis. Applied and Environmental Microbiology 65(10):4351-4356.
88
Scherer, C. A. and Miller, S. I. (2001). Molecular pathogenesis of Salmonellae. In Principles of
Bacterial Pathogenesis Principles of Bacterial Pathogenesis, Groisman, E. A. (Ed).
Academic Press, United States of America; pp:265-316.
Schutze, G., Kirby, R., Flick, E., Stefanova, R., Eisenach, K. and Cave, M. (1998).
Epidemiology and Molecular Identification of Salmonella Infections in Children.
Archives of Pediatrics and Adolescent Medicine 152(7):659-664.
Senthil Kumar, N. and Gurusubramanian, G. (2011). Random amplified polymorphic DNA
(RAPD) markers and its applications. Sci. Vis.11(3):116-124.
Shangkuan, Y. H., and Lin, H. C. (1998). Application of random amplified polymorphic DNA
analysis to differentiate strains of Salmonella typhi and other Salmonella species.
Journal of Applied Microbiology 85:693-702.
Simon, T. (1999). PCR and the detection of microbial pathogens in water and wastewater. Wat.
Res. 33(17):3545-3556.
Singh, J. P. N., Verma Rishendra, R., Chaudhuri, P. (2006). Random Amplified Polymorphic
DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India. Journal of
Veterinary Science 7(2):181-187.
Smith, S. I., Fowora, M. A., Goodluck, H. A., Nwaokorie, F. O., Aboaba, O. O. and Opere, B.
(2011). Molecular typing of Salmonella spp. isolated from food handlers and animals in
Nigeria. Int. J. Mol. Epidemiol. Genet. 2(1):73-77.
Smith, S. I., Odunukwe, N. N., Niemogha, M. T., Omonigbehin, E. A., Bamidele, M., Akinsinde,
K. A., Goodluck, H., Fesobi, T., Brai, B. I. C. and Bakare, B. (2006). Typhoid fever in
Nigeria: Analysis of phenotypic and genotypic methods of Salmonella typhi
identification. Res. J. Biotech. India. 1:17-21.
Solano, C., Garcia, B., Valle, J., Berasain, C., Ghigo, J. M., Gamazo, C. and Lasa, I. (2002).
Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose.
Molecular Microbiology 43(3):793-808.
Srikantiah, P. (2004). Salmonella enterica serotype Javiana infections associated with amphibian
contact, Mississippi, 2001. Epidemiology and Infection 132(2):273-281.
Stepanovic, S., Cirkovic, I., Mijac, V. and Svabic-vlahovic, M. (2003a). Influence of the
incubation temperature, atmosphere and dynamic conditions on biofilm formation by
Salmonella spp. Journal of Food Microbiology 20:339-343.
Straub, T. M., Chandler, D. P. (2003). Towards a unified system for detection of waterborne
pathogens. Journal of Microbiological Methods 53:185-197.
89
Straub, T. M., Pepper, C. P. and Gerba, C. P. (1995b). Removal of PCR inhibiting substances in
sewage sludge amended soil. Water Science Technology 31:311-316.
Su, L. H. and Chiu, C. H. (2007). Salmonella: Clinical Importance and Evolution of
Nomenclature. Chang Gung Med. J. 30(3):210-218.
Sulakvelidze, A. Kutter, E. (2005). Bacteriophage therapy in humans. In Bacteriophages:
Biology and Applications. Kutter, E., Sulakvelidze, A. CRC Press; pp:381-436.
Suresh, T., Hatha, A. A. M., Sreenivasan, D., Sangeetha, N. and Lashamanaperumalsamy, P.
(2006). Prevalence and antimicrobial resistance of Salmonella enteritidis and other
Salmonella in the eggs and egg-storing trays from retails markets of Coimbatore, South
India. Food Microbiology 23:294-299.
Swaminathan, B. and Matar, G. M. (1993). Molecular typing methods: definition, applications,
and advantages. In Diagnostic Molecular Microbiology: Principles and Applications,
Persing, D. H., Smith, T. F., Tenover, F. C., White, T. J. (Eds). American Society for
Microbiology, Washington, DC; pp:26-50.
Talabi, H. A. (1994). Medical aspects of typhoid fever in Nigeria.
Nig. Post grad. Med. J. 1:51-56.
Tebbutt, T. H. Y. (1998). Principles of Water Quality Control, 5th
(edn). Butterworth
Heinemann. ISBN-13:978-0 750636582.
Tejedor-Junco, M., Gonzalez, M., Rodriguez, N. and Gutierrez, C. (2010). Prevalence, serotypes
and antimicrobial resistance patterns of Salmonella isolates from apparently healthy
camels in Canary Islands (Spain). Journal of Camelid Science 3:44-48.
Tenover, F. C., Arbeit, R. D. and Goering, R. V. (1997). How to select and interpret molecular
strain typing methods for epidemiological studies of bacterial infections: A review for
healthcare epidemiologists. Infect. Control Hosp. Epidemiol. 18:426-439.
Tezcan-Merdol, D., Ljungstrom, M., Winiecka-Krusnell, J., Linder, E., Engstrand, L. and Rhen,
M. (2004). Uptake and replication of Salmonella enterica in Acanthamoeba rhysodes.
Applied Environmental Microbiology 70(6):3706-3714.
Tindall, B. J., Grimont, P. A., Garrity, G. M. and Euzeby, J. P. (2005). Nomenclature and
taxonomy of the genus Salmonella. Int. J. Syst. Evol. Microbiol. 55:521-524.
Tsai, L. Y., Palmer, C. L. and Sangeermano, L. R. (1993) Detection of Escherichia coli in
sewage and sludge by polymerase chain reaction. Applied and Environmental
Microbiology 59:353-357.
90
Tunung, R., Chai, L. C., Usha, M. R., Lee, H. Y., Fatimah, A. B., Farinazleen, M. G. and Son, R.
(2007). Characterization of Salmonella enterica isolated from street food and clinical
samples in Malaysia. ASEAN Food Journal 14(3):161-173.
United Nations Department of Economic and Social Affairs (2009). Millennium development
goals report 2009. United Nations Department of Economic and Social Affairs, New
York.
United Nations Development Programme (2006). Human Development Report, 2006. Beyond
scarcity: Power, poverty and the global water crisis. Retrieved on February, 2013 from
http://hdr.undp.org/en/media/ HDR06-complete.pdf.
United Nations Environment Programme/United National-Habitat (2010). Sick water? The
central role of wastewater management in sustainable development: A Rapid Response
Assessment. Corocovan, E., Nellemann, C., Baker, E., Bos, R., Osborn, D. and Savelli,
H. (Eds). ISBN: 978-82-7707-075-5.
United Nations Water (2008). Tackling a global crisis: International Year of Sanitation 2008.
Retrieved from http://www.wsscc.org/fileadmin/files/pdf/publication/IYS_2008.
Usera, M. A., Aladuena, A., Dıaz, R., De La Fuente, M., Cerdan, P., Gutie´rrez, R. and Echeita,
A. (2001). Ana´ lisis de las cepas de Salmonella spp aisladas de muestras de origen no
humano en Espana en el ano. Bol. Epidemiol. Semanal. 9:281-292.
Uyttendaele, M. R., Debevere, J. M., Lips, R. M. and Neyts, K. D. (1998). Prevalence of
Salmonella in poultry carcass and their products in Belgium. International Journal of
Food Microbiology 40:1-8. ISSN 0168-1605.
van Asten, F. J. A. M., Hendriks, H. G. C. J. M., Koninkx, J. F. J. G. and van Dijk, J. E. (2004).
Flagella mediated bacterial motility accelerates but is not required for Salmonella
serotype enteritidis invasion of differentiated Caco2 cells. FEMS Microbiology Letters
185:175-179.
Van, T. T. H., Moutafis, G., Istivan, T., Tran, L. T. and Coloe, P. J. (2007). Detection of
Salmonella spp. in retail raw food samples from Vietnam and characterization of their
antibiotic resistance. Applied and Environmental Microbiology 73(21):6885-6890.
Vassiliadis, P. (1983). The Rappaport-Vassiliadis (RV) enrichment medium for the isolation of
Salmonellas: An overview. Journal of Applied Bacteriology 54(1):69-76.
Velema, J., van Wijnen, G., Bult, P. and van Naerssen, T. (1997). Typhoid fever in Ujung
Pandang, Indonesia–high risk groups and high-risk behaviours. Tropical Medicine and
International Health 2(11):1088-1094.
91
Viessman, W. and Hammer, M. J. (1998). Water Supply and Pollution Control 6th
(edn).
Prentice-Hall. ISBN-13:978-0321014603.
Wang, J., Liu, Y., Muh-Yong, Y., Wang, J., Chen, Y., Wann, S. and Ching, D. (1996). Mycotic
aneurysm due to non-typhi Salmonella: Report of 16 cases. Clin. Infect. Dis
pp:232743-747.
Ward, L. R., De Sa, J. D. and Rowe, B. (1987). A phage-typing scheme for Salmonella
enteritidis. Epidemiology and Infection 99(2):291-294. ISSN 1469-4409.
Wassenaar, T. M. and Gaastra, W. (2001). Bacterial virulence: can we draw the line? FEMS
Microbiology Letters 201:1-7.
Way, J. S., Josephson, K. L., Pillai, S. D., Abbaszadegan, M., Gerba, C. P. and Pepper, I. L.
(1993). Specific detection of Salmonella spp by multiplex polymerase chain reaction.
Applied and Environmental Microbiology 59:1473-1479.
Webber, R. (1996). Typhoid. In Communicable Diseases Epidemiology and Control. CAB
International, Wallingford. pp:115-123.
Welsh, J. and McClelland, M. (1990). Fingerprint genomes using PCR with arbitrary primers.
Nucleic Acids Res 18:7213-7218.
Welsh, J. and McClelland, M. (1994). Fingerprinting using arbitrarily primed PCR: applications
to genetic mapping, population biology, epidemiology and detection of differentially
expressed RNAs. In The Polymerase Chain Reaction, Mullis, K. B., Ferre, F. and Gibbs,
R. A. (Eds.). Brikhauser, Boston, pp:295-303.
Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V. (1990). DNA
polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic
Acids Res 18:6531-6535.
Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and
Environmental Microbiology 63:3741-3751.
Wong, D. M. A., Hald, T., van der Wolf, P. J. and Swanenburg, M. (2002). Epidemiology and
control measures for Salmonella in pigs and pork. Livestock Production Science
76(3):215-222.
Wong, K. K., McClella, M., Stillwell, L. C., Sisk, E. C., Thurston, S. J. and Saffer, J. D. (1998).
Identification of sequence analysis of a 27-kilobase chromosomal fragment containing a
Salmonella pathogenicity island located at 92 minutes on the chromosome map of
Salmonella enterica serovar Typhimurium. Infect. Immun. 66:3365-3371.
92
Wood, M. W., Jones, M. A., Watson, P. R., Hedges, S., Wallis, T. S. and Galyov, E. E. (1998).
Identification of a pathogenicity island required for Salmonella enteropathogenicity.
Molecular Microbiology 29:883-891.
World Health Organization (2008). The Global Burden of Disease: 2004 Update. World Health
Organization, Geneva.
World Health Organization/United Nations International Children’s Emergency Fund (2010).
Report of the WHO/UNICEF Joint Monitoring Programme on Water Supply and
Sanitation. United Nations Children’s Fund and the World Health Organization, New
York and Geneva.
Yang, B., Qu, D., Zhang, X., Shen, J., Cui, S., Shi, Y., Xi, M., Sheng, M., Zhi, S. and Meng, J.
(2010). Prevalence and characterization of Salmonella serovars in retail meats of
marketplace in Shaanxi, China. International Journal of Food Microbiology
141(1-2):63-72.
Yeh, K. S., Chen, T. H., Liao, C. W., Chang, C. S. and Lo, H. C. (2002). PCR amplification of
the Salmonella typhimurium fimY gene sequence to detect the Salmonella species.
International Journal of Food Microbiology 78:227-234.
Younis, M., Soleiman, H. A., Abou Elmagd, K. (2003). Microbiological and chemical evaluation
of bentonite as a new technique for sewage water treatment, Aswan city, Egypt. 7th
International Water Technology Conference, Egypt, 1-3 April.
Yousef, A. E. and Carlstrom, C. (2003). Salmonella. In Food Microbiology: A Laboratory
Manual, Yousef, A. E. and Carstrom, C. (Eds). John Wiley and Sons, Inc, New Jersey;
pp:167-205.
Ziemer, C. J. and Steadham, S. R. (2003). Evaluation of the Specificity of Salmonella PCR
primers using various Intestinal bacteria species. Letters in Applied Microbiology
pp:263-269. ISSN 0266-8254.
Ziprin, R. L. (1994). Salmonella. In Foodbome Disease Handbook, Diseases Caused by
Bacteria, Hui, Y. H., Gorham, J. R., Murell, K. D. and Cliver, D. O. (Eds) Marcel
Dekker, New York; pp:2760-2774.
Ziprin, R. L. and Hume, M. H. (2001). Human Salmonellosis. In Foodbome Disease Handbook:
General Medical Aspects, Hui, Y. H., Pierson, M. D., Gorham, J. R. (Eds), Marcel
Dekker, New York; pp:285-321.
![Solid Minerals Development in Parts of Southwest Nigeria ... · Fig. 1. Production and reserve of mineral commodities in Africa as at 2010 [1] 1.1 The Geology of Nigeria The Nigeria](https://img.pdfslide.us/doc/110x75/5fa8ced6055b1d45771b7421/solid-minerals-development-in-parts-of-southwest-nigeria-fig-1-production.jpg)
