From Microbiology and Tumor Biology Center Karolinska Institutet, Stockholm, Sweden

Health hazards associated with dissemination of bacterial strains in waste water recycling

By

Mokhlasur Rahman

Stockholm 2005

All previously published papers were reproduced with permission from the publisher. Published and printed by Karolinska University Press Box 200, SE-171 77 Stockholm, Sweden © Mokhlasur Rahman, 2005 ISBN 91-7140-468-6

This work is dedicated

TO MY FAMILY

ABSTRACT

Treated waste effluents with low levels of chemical and microbiological contents are

used for domestic, industrial, agricultural and aquacultural purposes worldwide, and it is

estimated that one tenth or more of the world’s population consume food produced through

irrigation with wastewater. Treated hospital waste effluents may contain pathogenic and drug

resistant bacteria, which constitutes the most dangerous single risk factor for dissemination of

pathogenic and drug resistant organisms to the environment. This thesis focuses on the

possibility of persistence and transmission of pathogenic and drug resistant bacteria like

Aeromonas and Enterococcus from wastewater to environment and to humans, especially in

relation to a sewage treatment process based on waste water recycling.

In one study (Paper I), we have shown that a prevalent ampicillin and ciprofloxacin

resistant clonal lineage of Enterococcus faecium in Swedish hospital sewage water may be

transmitted from hospital patients to hospital sewage water. Recycling of hospital sewage water

may in the second step disseminate the disease-causing organisms to the environment and in the

third step to the human food chain. In other studies (Papers III, V), we have investigated the

persistence of Aeromonas in a duckweed aquaculture based hospital sewage water treatment

plant, where sewage grown duckweed is used as fish food. In this treatment system, the numbers

of Aeromonas organisms, diversities and resistance patterns to different antibiotics were found to

reasonably decrease through the purification process, with the exception of the fish culture pond

where a high prevalence of resistance was observed. We found that a phenotypically and

genotypically stable clonal lineage of Aeromonas could survive in the treatment system for

years. Both virulent and avirulent clonal lineages of Aeromonas could be transmitted from

hospitalised children suffering from diarrhoea to the fish that will be consumed by human

through recycling of hospital sewage water. It was also shown that transport vehicles such as

duckweed and amoebas (if present) might play a role in this transmission. We were able to

detect a gentamicin resistant (multiresistant) clonal lineage that was transmitted from hospital

sewage water to fish. Furthermore, we found that a transmitted clonal lineage of Aeromonas had

a high biofilm formation ability compared to other Aeromonas clones. As in other bacteria, we

could show that transition between biofilm formation and motility of one strain of this clone is

regulated by the cyclic di-guanosine-monophosphate concentration. We also characterized

Aeromonas from fish with Epizootic Ulcerative Syndrome disease and showed that one

pathogenic clonal group was associated with this fish disease in Bangladesh (Paper II).

To facilitate the monitoring of resistance in different sites of the waste treatment plant,

we developed a user-friendly semi automated MIC determination method, the ScanMIC method,

to screen a large number of isolates in a short time (Papers IV, V). We compared this method

with a reference method and in both cases the ScanMIC method was found to meet the

performance criteria guidelines for automated susceptibility testing.

In conclusion, the results indicate that sewage water recycling in aquaculture constitutes

a potential health hazard through dissemination of pathogenic and drug resistant bacteria to the

environment and/or to human.

TABLE OF CONTENTS

LIST OF PUBLICATIONS............................................................................................ 3

ABBREVIATIONS ........................................................................................................ 4

1 INTRODUCTION .................................................................................................... 5 1.1. Waste water and treatment systems 5

1.1.1 Waste water................................................................................................. 5 1.1.2 Treatment of waste water ............................................................................ 5 1.1.2.1 Activated sludge .......................................................................................... 6 1.1.2.2 Waste stabilization ponds ............................................................................. 6 1.1.2.3 Duckweed based waste treatment system....................................................... 7

1.2. Aquaculture 7 1.2.1 Aquaculture as a source of protein.............................................................. 7 1.2.2 Duckweed as a feed for aquaculture ........................................................... 8

1.3. Transmission and health 8 1.3.1 Transmission of pathogen from waste water to human .............................. 8 1.3.2 Water and/or food borne pathogen.............................................................. 9 1.3.3 Hazard identification for food safety .......................................................... 9 1.3.4 Bacterial typing in epidemiological surveillance...................................... 10 1.3.5 The host-pathogen relationship on waste related infections ..................... 11

1.4. The Bacterial Genus Aeromonas 12 1.4.1 Opportunistic pathogen ............................................................................. 13 1.4.2 Historical aspects of Aeromonas taxonomy.............................................. 13 1.4.3 New Species of Aeromonas ...................................................................... 13 1.4.4 Genospecies .............................................................................................. 14 1.4.5 Identification to the genus level ................................................................ 14 1.4.6 Identification to the species level.............................................................. 14 1.4.7 Ecology of Aeromonas .............................................................................. 16 1.4.7 Pathogenicity of Aeromonas ..................................................................... 17 1.4.8 Putative virulence factors of Aeromonas .................................................. 18 1.4.9. Biofilm formation of Aeromonas ............................................................. 21

1.5. Enterococci 22 1.5.1 Genus description of Enterococcus........................................................... 22 1.5.2 Habitat of Enterococcus ............................................................................ 22 1.5.3 Clinical diseases and nosocomial infections by Enterococcus ................. 22

1.6. Antibiotic resistance 23 1.6.1 Antibiotic susceptibility testing and MIC breakpoint ............................... 23

2 AIMS OF THE STUDY ......................................................................................... 25

3 MATERIALS AND METHODS............................................................................ 27

4 RESULTS AND DISCUSSION.................................................................... 32 4.1 Transmission of Enterococcus faecium from hospital patients to environment 32 4.2 A clonal lineage of Aeromonas is associated with fish diseas 34 4.3 Persistence and transmission of Aeromonas in the sewage treatment plant 36 4.4. General comments on duckweed aquaculture based sewage treatment plant 45 4.5 Usefulness of “ScanMIC” method for antibiotic related hazard identification 48

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4.6 Cyclic-di-GMP signalling in Aeromonas (preliminary data) 50 5 ABSTRACTS OF INCLUDED PAPERS..............................................................54

6 ACKNOWLEDGEMENTS ....................................................................................57

7 REFERENCES.......................................................................................................58

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LIST OF PUBLICATIONS

This thesis is based on the following original papers, which will be referred to in the text by

their Roman numerals:

I. Aina Iversen, Inger Kühn, Mokhlasur Rahman, Anders Franklin, Lars G. Burman,

Barbro Olsson-Liljequist, Erik Torell and Roland Möllby. Evidence for

transmission between humans and the environment of a nosocomical strain of

Enterococcus faecium. Environmental Microbiology 2004; 6: 55-59.

II. Mokhlasur Rahman, Patricia Colque-Navarro, Inger Kühn, Geert Huys, Jean Swings

and Roland Möllby. Identification and characterization of pathogenic

Aeromonas veronii biovar sobria associated with Epizootic Ulcerative Syndrome

in fish in Bangladesh. Applied and Environmental Microbiology 2002; 68: 650-655.

III. Mokhlasur Rahman, Geert Huys, Motiur Rahman, John Albert, Inger Kühn and

Roland Möllby. Persistence and transmission of Aeromonas in a duckweed

aquaculture-based hospital sewage water recycling plant. Submitted to the

Applied and Environmental Microbiology.

IV. Mokhlasur Rahman, Inger Kühn, Motiur Rahman, Barbro Olsson-Liljequist and

Roland Möllby. Evaluation of a scanner assisted colorimetric MIC method for

susceptibility testing of Gram negative fermentative bacteria. Applied and

Environmental Microbiology 2004; 70: 2398-2403.

V. Mokhlasur Rahman, Inger Kühn, Motiur Rahman, Geert Huys, and Roland Möllby.

Influence of a duckweed-aquaculture based hospital sewage water recycling

plant on the antibiotic resistance of Aeromonas spp. as determined by the

ScanMIC method. (Manuscript)

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ABBREVIATIONS

AFLP Amplified Fragment Length Polymorphism

ARE Ampicillin Resistance Enterococci

ASL N-Acyl-L-Homoserine Lactones

c-di-GMP Cyclic di Guanosine Monophosphate

EPS Extracellular Polymeric Substance

EUS Epizootic Ulcerative Syndrome

FAME Fatty Acid Methyl Ester

GDP Gross Domestic Products

GFP Green Fluorescent Protein

HACCP Hazard Analysis Critical Control Point

HSL N-butanoyl-L-Homoserine Lactone

MIC Minimum Inhibitory Concentration

PFGE Pulsed Field Gel Electrophoresis

UPGMA Unweight Pair Group Method

WSP Waste stabilization pond

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INTRODUCTION

Due to growing populations, increased human consumption and urbanization,

freshwater reserves are decreasing in many developing countries (33, 102). To address these

problems, treated sewage water with a low level of chemical and microbiological

contaminations has been used for domestic, industrial, and agricultural purposes (33, 100).

Worldwide it is estimated that 40% of all food is produced with irrigated water and at least

20 million hectars of land in 50 countries are irrigated with raw or partially treated waste

water (33, 59). One tenth or more of the world’s population consume foods produced by

irrigation with wastewater (172). There are several kinds of waste purification systems

available, but most of them are too costly to provide solution for sewage water purification

in growing developing countries (61). In most developing countries only 10% of all

wastewater is subject to purification (79). Duckweed based waste treatment systems are a

relatively new concept of waste purification since sewage grown duckweed could be used as

feed in fish farm. This thesis focuses on the possibility of persistence and transmission of

pathogenic and drug resistant bacteria like Aeromonas and Enterococci from the wastewater

to environment and to humans, especially in relation to a sewage treatment process based on

waste water recycling.

1.1. Waste water and treatment systems 1.1.1 Waste water

Water from households, industries, and run off water comprise the municipal waste

water. Most of the waste water contains a number of organic substances that are more or less

biodegradable (64). In the waste water, microorganisms consume oxygen to use the organic

substance as energy sources and combust them to carbon dioxide and water. As a result, the

reduction of oxygen concentration creates anaerobic conditions in the waste water that may

be toxic to higher forms of life in the natural recipient, which is one of the major problem

with the effluent of untreated waste water (49). Furthermore, excessive nutrient enrichment,

mainly nitrogen and phosphorous contributes to the eutrophication of the natural water. The

consequences of the eutrophication are large algae blooms, sometimes poisonous, and

oxygen depletion in the bottom sediments, which leads to a diminishing bottom fauna (64). It

is thus of great importance to decrease the nutrient loads in order to restore the natural

conditions of water.

1.1.2 Treatment of waste water Treatment of waste water implies reduction of the level of floating and suspended

solids, organic materials such as nitrogen and phosphorus and elimination of the load of

pathogenic microorganisms (81). In a conventional municipal waste treatment plant, the

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sewage is first passed through a series of screens or strainers to remove floating materials

and solids. After this removal, suspended solids are settled in the primary sedimentation. The

wastewater then enters the biological step in the treatment plants. There are two main types

of biological treatment systems: biofilters and activated sludge. Both of them are based on

removal of organic substances with biological removal of nitrogen. The biological unit is

sometimes followed by a second sedimentation and in some cases also chemical treatment

before letting out the effluent into the receiving water.

1.1.2.1 Activated sludge Activated sludge treatment of wastewater has been around for about 100 years in one

form or another, and is hither the most successful mean of achieving pollution control. The

activated sludge separator is composed of two basic compartments: the aerator reactor and

the sludge separator. Aerator reactor is used to mix the microorganisms and sludge that lives

in the plant. The sludge contains microorganisms, inorganic and organic substances. A mass

of activated sludge is kept moving in the water by stirring. The overall goal of the activated-

sludge process is to remove substances that have a demand for oxygen from the system.

Microorganisms play an important role in the waste treatment process and are responsible for

removal of organic substances, such as nitrogen. Bacteria constitute the majority of

microorganisms present in activated sludge. The organic material is oxidised by

heterotrophic bacteria to CO2 and water. Nearly every city in the world uses a form of this

treatment, because it is a relatively simple technology. But these systems are energy

consuming and they require trained personal for operation and maintenance, therefore this

system is too costly for developing countries.

1.1.2.2 Waste stabilization ponds Waste stabilization ponds (WSP) are generally the waste water treatment process of

first choice in developing countries. They are suitable for both large and small populations

(from a few hundreds to hundreds of thousands). WSP have several important advantages for

developing countries: low capital costs, simple operation and maintenance, and high

performance. They can easily be designed to produce high-quality effluents suitable to

irrigate crops, support fish farms, for aquatic vegetable culture or simply to control river

pollution. The principal of the system are it is an entirely natural method of wastewater

treatment (they obtain all their energy directly from the sun) but the disadvantage is that it

requires much more land than conventional processes such as activated sludge. WSP

treatment systems have been used in different countries, such as Turkey, Cyprus, Israel,

Jordan, Egypt, Tunisia, Algeria, Morocco, Portugal, Spain, France, Greece, as well as at

several sites in India. However, land increases in value over time and in an overpopulated

developing country like Bangladesh municipalities cannot afford the land needed for waste

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stabilization ponds. Moreover, in Bangladesh, like in many other developing countries,

planning for domestic waste water reuse is one area that has received special attention.

1.1.2.3 Duckweed based waste treatment system Current mainstream technologies for sewage water treatment are too costly to

provide a satisfactory solution for the growing sewage water problem of developing

countries (61). Cost effective, environmental friendly, macrophyte-based sewage water

treatment systems like the duckweed based waste treatment system is a relatively new

concept and is becoming more common practice in different south East Asian countries (

such as Thailand, Malaysia, Taiwan and Bangladesh) as well as in China and USA (7, 80).

Duckweeds are small aquatic plants belonging to the family Lemnaceae, which may be used

as food for livestock and fish. The use of free-floating duckweed in sewage water treatment

systems has been advocated for, since it is fast growing and easy to harvest, has a low fiber

and a high protein content, and shows a high efficiency in removal of nitrogen and

phosphorous from sewage water (111). The duckweed based waste treatment system uses

sewage water as a nutrient source for duckweed production. In this way waste water is

transferred into high quality protein instead of being disposed in the environment thus

solving the waste purification problem. The growing interest from the potential cost recovery

through the use of duckweed as animal feed mostly for intensively cultured duck, chicken or

aquaculture farms. Thus, the duckweed serves two purposes: it is purifying the wastewater as

well as being used as animal feed.

1.2. Aquaculture 1.2.1 Aquaculture as a source of protein

Aquaculture is the fastest growing sector among the world’s food production sectors.

According to the latest FAO statistics, the contribution of aquaculture to global supplies of

fish, crustaceans and molluscs continues to grow, increasing from 3.9% of total production

by weight in 1970 to 29.9% in 2002. Total world aquaculture production was reported to be

51.4 million tons by volume and 60.0 US$ billion by values in 2002 and countries in Asia

accounted for 91.2% of the production and 82% of these values. The major aquaculture

productions of fish, crustaceans and molluscs continue to come from the freshwater

environment (57.7% by volume and 48.4% by values) (177).

Fish is an important part of the diet for a large proportion of the people living in the

developing world. Many types of fish and aquatic products are available at affordable prices

in developing countries. At present, food fish represents the primary source of animal protein

(contribution more than 25% of the total animal protein supply) for a one billion people

within 58 countries worldwide. Fish are important source of many nutrients, including

protein of very high quality, Vitamin A, vitamin D, vitamin E, iodine and selenium.

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However, affordability is a key factor explaining why aquaculture is making an essential

contribution in the developing world (177). In the overall agriculture based economy of

Bangladesh, aquaculture supplied 80 % of all animal protein produced, and it contributed to

4.7 % of Gross Domestic Products (GDP). The contribution of fisheries in Bangladesh is

very promising and important for creating job opportunities for unemployed people, earning

foreign exchange, alleviating poverty and improving nutritional status of the people.

1.2.2 Duckweed as a feed for aquaculture In developing countries like Bangladesh, in spite of limited resources, aquaculture

provides considerable amounts of high quality protein. Environmental pollution, abused use

of pesticides (about 7000 metric tons per year in agricultural fields, of which 25% are being

washed into open water bodies) and fish diseases are now depleting the fisherie’s resources

in the open water bodies in Bangladesh. In the future, farmed fish will be a more important

source of protein than they are today. Day by day integrated fish farming is getting more

popular in Bangladesh and also throughout Southeast Asia.

The integrated fish farming system are relatively confined units with a high density

fish farming. The fish used to receive supplementary feed and, in most cases, fresh extract of

animals which supports the growth of plankton, the natural food for fish, thus reduced the

cost of feed and fertilizers for fish ponds. But using animal waste in fish culture is not

socially accepted. Thus there is an increased demand of low cost or natural production of

fish food in many developing countries like Bangladesh. In this context, the use of duckweed

as fish feed has attracted much attention, since it can grow on waste water, which does not

cost any investment. The duckweed aquaculture based fish farming system maximizes

production through recycling of waste water, optimum utilization of resources and a

maximum return with minimum cost involved in fish culture.

1.3. Transmission and health 1.3.1 Transmission of pathogen from waste water to human

Protection of public health is the fundamental purpose of waste treatment.

Environmental protection is the second major purpose. Waste water is known to be possible

reservoirs of pathogenic and/or drug resistant bacteria and other pathogenic organisms.

Pathogenic organisms can be transmitted from wastewater to human through direct and

indirect transmission. Direct transmission of pathogens could happen when drinking water or

swimming water is contaminated with waste water. Indirect transmission could happen when

waste water is used for agricultural and aquaculture purposes.

Management and treatment of waste water have largely been insignificant in

preventing pollution from affecting surface water quality on both local and global scales (6).

More recent studies in developing nations have demonstrated that breaking pathogen

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transmission cycles through proper sanitation and sewage management improve health

benefits more than only simple provision of clean drinking water (50) The treatment and

disposal of waste water is becoming more and more important since recent studies have

shown that health problem often are related to inadequate sewage treatment. Due to the lack

of proper treatment of sewage water in developing countries, inadequately treated sewage

water has been used/recycled directly or indirectly in agriculture and aquaculture.

1.3.2 Water and/or food borne pathogen In developing countries with inadequate water supplies, transmission of pathogen

from waste water to human is mainly responsible for food and water borne diseases (faecal

oral transmission). For the past decade, the increase in food born and water borne infections

has become an important public health concern worldwide (133). Besides the classical food

and water borne pathogens such as Clostridium, Salmonella, Shigella, Vibrio a number of so

called new or emerging pathogens have recently been identified as food poisoning organisms

such as E.coli 0157:H7, Camplylobacter, Listeria and Aeromonas (133). The number of

identified food and water borne pathogens since the 1950s has nearly trebled. Several newly

recognized food and water borne pathogens have contributed greatly to the occurrence of

infectious diseases during the last two decades.

Public health authorities have estimated that half the population of the developing

world is suffering from the major microbial diseases associated with water supply and

sanitation (194, 195). Infection by pathogenic bacteria, protozoa, and viruses are the most

prevalent global health risks associated with waste water. However, common organisms in

drinking and waste water that have been identified as posing major threats to human health

include (i) bacteria: enteropathogenic Escherichia coli (notably E. coli 0157:H7), Vibrio

cholerae, Shigella, Campylobacter jejuni, Salmonella, Yersinia enterocolitica, (ii)

protozoans: Giardia lamblia, Cryptosporidium parvum, Entamoeba histolytica, Toxoplasma

gondii, Balantidium coli, and (iii) viruses: Norwalk and Norwalk-like, Rotavirus, Hepatitis A

and E; (26, 53, 68, 99, 130, 137)

1.3.3 Hazard identification for food safety Control and prevention of food and water borne diseases can be difficult. To

increase food safety, it is important to identify pathogens or situations that may lead to food

borne illness and to determine the magnitude of the impact these have on human health

(118). Such information is needed for the effective use of resources, since hazard analysis

critical control point (HACCP) programs can identify the hazards and might play an

important role in reducing food borne pathogens. The term “hazard” refers to a biological

agent, that is, the microorganism and/or its toxins, that has the potential to cause adverse

health effects (118). A systematic examination is necessary to effectively manage of food

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safety. Risk assessment is a process that provides an estimation of the probability and impact

of adverse health effects attributable to potentially contaminated foods (124). Risk

assessment is a science-based investigation consisting of four steps: hazard identification,

exposure assessment, hazard characterization and risk characterization. Thus the hazard

identification is the primary and fundamental step in risk assessment (37).

Hazard Identification What agents are present in the food and capable of causing adverse health effects

Exposure Assessment Consumption, Pathogen in food serving

(occurrence, density), Predictive microbiology (growth/decline), ingested number of

pathogens

Hazard Characterization What is the nature of adverse effect ?

Dose response assessment (non-threshold model, threshold model)

Risk Characterization and Risk Estimation Probability and severity of illness attributable to the

food/pathogen source; e.g. no of illness per year and per 100 K population

1.3.4 Bacterial typing in epidemiological surveillance

Identification and characterization of isolates below the species level is important in

epidemiological studies, e.g. for tracing the source of infections and for identification of

epidemic clones. Bacterial typing has been used for different purposes in epidemiology, but

most reports concern the use of bacterial molecular typing method for investigation of an

outbreaks and route of transmission (18, 44, 67). In the investigation of outbreak, a limited

number of isolates from a limited number of samples/persons are typed to identify which

isolates are clonally related (i.e., directly descended from a common ancestor). In this case

each isolate is compared to each other. This application implies that the same

epidemiological markers are used consistently: markers must be standardized and

reproducible over time. Such typing is called library typing. Any library typing results has a

universal meaning for the epidemiologist.

Molecular epidemiology is based on one postulate. When several isolates are

deemed identical using a molecular typing method with a high discriminatory power, it

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assumed that they are derived from a common ancestor. Thus in a molecular epidemiological

investigation, identification of cases will be with identical strains for which the transmission

can be confirmed by epidemiological links. When a typing method with a high

discriminatory power is tested on a set of epidemiological unrelated isolates, one must

considered the existence of highly prevalent clones in the population of the microorganisms

under study. In this situation, epidemiological unrelated cases might be harbouring a strain of

identical genotype. The predominant clone might have been present for a long period of

time, have had time to spread without significant mutations or might have spread very

rapidly compared to other strains. Several examples of such predominant clones have been

described. For example, epidemiological unrelated S. aureus isolates with similar PFGE

patterns have been found in different geographical regions suggesting that some genotypes

are ubiquitous (65, 165). Another example is the species Legionella pneumophila where

isolates showing similar PFGE patterns have been recovered from many patients and from

different parts of Paris water systems (119). Moreover, this predominate genotype, called the

“Paris strain” has been found between 1998 and 2002 in patients from all over France (15).

Thus molecular typing enables us the tracking of the dissemination of specific clones with or

without epidemiological links (19)

1.3.5 The host-pathogen relationship in waste related infections Waste related microbial water and food borne disease may occur when a susceptible

individual consume water and/or food contaminated by a viable microbial pathogen(s) and/or

microbial toxins . However, not every exposure to a pathogen in water and/or food will result

in infection or illness and not all individuals in a given population are equally susceptible to

all pathogens. Therefore the risk of food and/or water borne disease is a combination of the

likelihood that exposure will result in infection or intoxication and subsequently illness and

the severity of the illness.

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Figure 1 The pathogen-host relationship and possible transmission routes for sewage related

infections. Adapted from Martin Strauss (126)

1.4. The Bacterial Genus Aeromonas

The genus Aeromonas were at first recognized as pathogens of cold blooded animals

(fish, frogs, snakes, lizards) that suffered from Aeromonas associated septicaemia (16, 112,

168,169). In fact, nonmotile psychrophilic Aeromonas (originally named Bacterium

salmonicida but today known as Aeromonas salmonicida) was first identified as the primary

cause of fish furunculosis, a disease leading to severe economical losses in fish farm (174,

175, 197). The first human infection with an Aeromonas strain was reported by Hill and

colleagues in 1954 (78). From that date to until today, clinical microbiologists recovered

various Aeromonas species from a wide range of infection sites. Hundreds of reports,

varying from single case studies to long term monitoring surveys, have revealed a

relationship between high Aeromonas densities in drinking water and diarrhea in humans

(14, 21, 31). Gastrointestinal infections of Aeromonas are generally considered waterborne;

for this reason, Aeromonas has been placed on the United States Environmental Protection

Agency candidate list of emerging pathogens in drinking water. Furthermore, well

documented field studies have changed the traditional belief that Aeromonas associated

gastroenteritis mainly occurs in African, South American and South East Asian countries (8,

9, 104, 114, 129, 143)

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1.4.1 Opportunistic pathogen Several degrees of pathogenicity have been observed among the thousands of

bacterial species described so far. Some of the bacteria have a very high probability of

developing diseases in any susceptible host, for example, Bordetella pertussis that causes

whooping cough, is regarded as a primary pathogen. In contrast, other bacteria, such as

Pseudomonas aeruginosa, enter the human body virtually on a daily basis through

consumption of drinking water and vegetables, yet they only rarely cause diseases in healthy

individuals. However P. aeruginosa strains have been known to produce severe infections

(e.g. meningitis) with high fatality (45, 162) especially in hospitalized patients under

immunosuppressive treatment. Bacteria that show this kind of behaviours are called

opportunistic pathogens. Aeromonas spp. are considered as opportunistic pathogen.

1.4.2 Historical aspects of Aeromonas taxonomy The Aeromonas group of bacteria was first described over 100 years ago by Sanarelli

in 1891(163). But the genus name Aeromonas was first proposed by Kluyver and van Niel in

1936 (109). This group of bacteria was allocated too many different bacterial genera in

different time table such as Bacillus (1891, 1901, 1917), Bacterium (1891), Aerobacter

(1900), Achromobacter (1923), Pseudomonas (1930, 1936, 1954), Proteus (1936) and Vibrio

(1955) (52). However, in the seventh edition of Bergeys Manual Determinative

Bacteriology, Snieszko (1957) citated the genus Aeromonas in the family

Pseudomonadaceae (four species, namely the motile species A. hydrophila, A. punctata, A.

liquefaciens and the nonmotile species A. salmonicida)(176). In the eighth edition of

Bergeys Manual of Determinative Bacteriology (1974), the genus Aeromonas was

considered a member of the Vibrionaceae, with a new taxonomy [Aeromonas hydrophila,

(with the subspecies hydrophila, anaerogenes and proteolitica) and A. punctata and the non-

motile species A. salmonicida (with the subspecies salmonicida, achromogenes and

masoucida], which also included the genera Vibrio, Photobacterium, Plesiomonas (192).

Subsequently, phylogenetic investigations indicate that the genus Aeromonas is not closely

related to Vibrio but rather forms a monophyletic unit in the γ-3 subgroup of the class

proteobacteria (128, 161). These conclusions necessitated the removal of Aeromonas from

the family Vibrionaceae and transfer to a new family, the Aeromonadaceae (38).

1.4.3 New Species of Aeromonas The genus Aeromonas has undergone a number of taxonomic and nomenclature

revisions over the past 15 years. Since 1987, a number of new Aeromonas species have been

identified that include A. veronii, A. schubertii, A janda, A torta, A. allosaccharophila, A.

encheleia, A. bestiarum, and recently A. popoffii (94). Most recently, Aeromonas hydrophila

subsp. Dhakensis, one clonal group of Aeromonas, suspected to cause diarrhoea in patients

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in Bangladesh (85). But the major problem associated with several of the more recently

described Aeromonas species is the extremely limited number of strains. For example, for A.

allosaccharophila, only three strains are known to exist; A. encheleia is based on 4 strains

isolated from European eels; A. popoffii is based on 8 strains isolated from drinking water

(85). In such cases, it is unclear whether the isolates analyzed were in fact separate strains or

whether they simply represented multiple isolates of the same strains.

1.4.4 Genospecies Aeromonas bacteria could be classified into phenotypically defined phenospecies

and into genospecies delineated on the basis of DNA-DNA hybridisation studies (107). Since

the advent of polyphasic molecular approaches in the study of bacterial systematics, the

genus Aeromonas has undergone a number of significant nomenclature changes. Several

approaches have been proposed in order to differentiate between Aeromonas species,

including ribotyping and multilocus enzyme electrophoresis, restriction fragment length

polymorphism (RFLP) (144), amplified fragment length polymorphism (AFLP) (84), the

complete 16S rRNA gene sequences (24), Pulse field gel electrophoresis (PFGE) (23, 181).

Nevertheless, the use of genomic studies based on classical DNA-DNA hybridisation

method is necessary to determine the taxonomic position of Aeromonas species. Aeromonas

genospecies can be defined on the basis of the re-associational kinetics between individual

strains and isotopically labelled DNA extracted from reference strains (93).

1.4.5 Identification to the genus level Identification of Aeromonas to the genus level is relatively simple and

straightforward. Aeromonas species can grow on any standard media such as nutrient agar,

brain heart infusion agar and tryptic soy agar within overnight incubation at 25-30oC for

mesophilic Aeromonas and 22-25o C for psychrophilic Aeromonas (153). Aeromonas genus

can also be easily distinguished from other gram negative, facultative anaerobic genera (i.e.

Vibrio, Plesiomonas, and Enterobacteriaceae) through oxidation and vibriostatic reagent test.

1.4.6 Identification to the species level One of the major difficulties in the identification of Aeromonas strains to species

level concerns the current number of recognized taxa (n=15) and the lack of clear-cut

phenotypic characters useful in distinguishing each species from the others (1). Genospecies

identification of Aeromonas to the species level is unpractical in most clinical laboratories

because of the time required, reagents involved, high instrument cost (2). To facilitate,

routine identification in hospital and clinical laboratories, at least 14 different commercial

systems are available in the USA but the resolving power of most of those system are often

too low to deal with high phenotypic relatedness among Aeromonas taxa (135). For example,

API 20E system can correctly identify only 52% of the Aeromonas strains (98). However,

14

Janda et al proposed that Aeromonas should simple be reported as “Aeromonas species” or

“Aeromonas hydrophila group” or “Aeromonas hydrophila complex”(94). Another

alternative is to continue to biochemically identify strains of the three major species namely

“A. hydrophila, A. caviae, A. sobria”. However, a feasible alternative is to identify

Aeromonas to the species level by a set of reliable biochemical tests. In the early 1990s,

Carnahan et al (31) and Abott et al (1) recommended the use of 20 to 25 selected phenotypic

features for correct allocation of mesophilic Aeromonas strains. However, subsequent

systematic investigations have resulted in nine biochemical key tests as a primary battery to

identify Aeromonas to HG levels (1, 95). The use of these nine phenotypic characters,

however still do not allow differentiation at HG level within A. hydrophila complex (HGs 1

to 3) and A. caviae complex (HGs 4 to 6).

Table 1. Species and DNA hybridization groups currently situated in the genus Aeromonas

HG a, b

(Geno-species)

Species name a, b

(Phenospecies)

Pathogenic status C Main source of isolation b

References b

1 A. hydrophila Major pathogen Humans, animals, water

(152)

2 A. bestiarum Environmental Animal, water, humans

(10)

3 A. hydrophila Environmental Water (52) A. salmonicida Major pathogen Fish (152)

4 A. caviae Major pathogen Humans, animals, water

(152)

5A A. caviae Environment Water (75) 5B A. media Environment Water (75) 6 A. eucreniophila Environment Water (166) 7 A. sobria Environment Fish (152) 8 A. veronii biogroup

sobria Major pathogen Humans, animals,

water (77)

9 A. jandai Minor pathogen Humans (29) 10 A. vronii biogroup

veronii Minor pathogen Humans (77)

11 A. veronii like Environment Human, water (77) 12 A. schubertii Environment Humans, water (76) 13 A .trota Environment Human, water (32) 14 A. allosaccharophila Environment Human, fish (127) 15 A. encheleia Environment Fish (51)

a Genospecies and Phenospecies represent taxa that are delineated on the basis of DNA hybridization data and phenotypic characterization (30) HG, DNA hybridization groups.

b Data adapted from the thesis by Geert Huys (83). C associated with human diseases based upon clinical frequency rather than disease

presentation; data presented from Janda and Abbott (94).

15

1.4.7 Ecology of Aeromonas Aeromonas spp. are ubiquitous organisms which can be readily isolated from a

variety of sources such as water, soil, food, animal and human. Recent progress in the

development of selective media for Aeromonas has increased our knowledge of quantitative

Aeromonas from different sources (148). Despite the new Aeromonas species, most of the

Aeromonas bacteria appear to be environmental species. Only six of the Aeromonas species

have been unquestionably established as human and animal pathogens. Within this five

species, three species accounts for more than 85% of all clinical isolates and they include

HG groups 1, 4 and 8 (94).

Environmental Water: Aeromonas spp are widely distributed in the aquatic

environment. Numerous ecological investigations have showed that Aeromonas occur in

virtually all kinds of aquatic environments throughout the world but in varying

concentrations (13, 98). They can be found in both polluted and unpolluted water, sewage

water, drinking water, and chlorinated and non chlorinated water distribution systems and

even in bottled mineral water. These species were first considered to be absent from the

marine water, but a number of recent reports demonstrated that they are widespread in

marine environments too (136).

Plant and phytoplankton: Aeromonas spp not only have been found in aquatic

water but also in aquatic plants, soil sediments and phytoplankton. Islam et al reported that

Aeromonas sobria could be isolated in plant and phytoplankton’s during hot weather with

the highest isolation rates (47.5%) (90).

Mineral water: Aeromonas was isolated from mineral waters in Spain, which was

published in two papers (134). Gonzales detected this organism in one of three investigated

mineral water sources (60), while Quevedo et al (154) referred that approximately 10% of all

bacteria recovered from mineral water are belonged to Aeromonas spp. In another report,

these organisms were detected in 4.9% mineral water samples and three of these (1.8 %)

with A. hydrophila counts >103 CFU/ml (41). In Taiwan, Aeromonas were isolated from 31

of 88 domestic and 11 of 48 imported mineral water samples (188).

Foods: Aeromonas have been isolated from a wide range of foods including raw

and processed meat, poultry, seafood, raw milk, vegetables and pre-prepared salads (113,

132). The isolation of Aeromonas from vegetables have been shown to represent a potential

risk to consumers health, due to their possible pathogenicity (production of cytotoxin at

refrigerator temperature) and ability to grow at refrigerator’s temperature (132). Organic

vegetables have been considered to represent an increased risk to public health, where

natural fertilizers such as animal manure and sewage water are used and where no chemical

treatment is employed to reduce the microbiological load of the raw products (132).

16

Seasonal abundance: The abundance of Aeromonas in the aquatic ecosystem has

been shown to be seasonally distributed. Islam et al (90) showed a high incident of

Aeromonas sobria during the warmer months (May to July) and the beginning of the winter

in November in a Bangladeshi pond. Different studies also show that the number of

Aeromonas increased with the temperature, with a maximum occurring during the summer

and the lowest incidence during the winter. Monteil reported that Aeromonas were probably

one of the leading causes of bacterial enteritis during summer months, affecting mainly

young children and travellers (138). The correlation between summer seasons, increased

water contamination levels and increased number of gastroenteritis cases may be associated

(54, 138).

1.4.7 Pathogenicity of Aeromonas Cold blooded animals: The pathogenicity of Aeromonas was first established

with cold-blooded animals, A. hydrophila can cause red leg disease in frogs. But recent

studies have shown that turtles, snakes, crocodiles and lizards can also be infected with

Aeromonas with a high rate of mortality (189). Recently, Turutoglu et al described a case

report of skin lesions and septicaemia associated with A. hydrophila in a Nile crocodile

(Crocodylus niloticus) (189). Psychrophilic species Aeromonas salmonicida is known as the

etiological agent of furunculosis in salmon worldwide. Furunculosis has caused enormous

economical losses in commercial salmon and trout fish farms during the last decades. Recent

studies indicate that motile mesophilic Aeromonas play a major role as opportunistic

pathogen in freshwater fishes worldwide. McGarey reported that 50-80% of daily catches of

fish from the St. Jons River system exhibited UDS (Ulcerative Diseases Syndrome) lesions

(131). This disease is frequently occurring not only in different intensive culture fish farms

but also in open water bodies (i.e lakes, rivers) in Indo-Pacific regions (155). Different

studies have shown that Aeromonas is frequently associated with diseases in carp, eels,

milkfish, channel catfish and tilapia (156) .

Warm blooded animals: Aeromonas species have been isolated from a wide

range of mammalians including cats, dogs, horses, rabbits, monkeys, guinea pigs, chicken (4,

57, 146). Recently Andre reported the pure culture of Aeromonas hydrophila from liver and

kidney of a dog suffering from leptospirosis with septicaemia due to this bacteria (11).

Aeromonas infections in human: Aeromonas associated major human infection

could be divided into two major categories. One of them is gastroenteritis that occurs mainly

in young children or aging people but also in travellers (72). Besides of gastroenteritis extra-

intestinal may occur infection in adult individuals (94).

17

Gastroenteritis: The first Aeromonas associated Gastroenteritis was reported by

Ronser et al 1964 (159). From 1964 until today, numerous studies have demonstrated a

strong correlation between high number of Aeromonas in the gastrointestinal tract and

human diarrhoeal diseases. Several case control studies have shown the epidemiological

links between Aeromonas and acute diarrhoea (9, 27, 149). However, none of these

investigations have been able to identify Aeromonas as the true causative agent of

gastroenteritis (94). Thus the single greatest topic “the association of Aeromonas with

gastrointestinal disease” is still unresolved. The fact is that no well documented

epidemiologically linked outbreaks of diarrhoeal disease attributed to Aeromonas have been

reported as yet (94). Even volunteer studies that involve the ingestion of Aeromonas of

various strains showed that only certain strains were associated with colonization in most

individuals when the doses of challenge exceeded 109 CFU (139) .

Extra intestinal infections

Wound infection: Wounds are the second most common infections by Aeromonas

after the gastrointestinal infections (94). Wound infections caused by Aeromonas often

associated with aquatic accidents or occupational accident where the source of Aeromonas

was shown to be water and soil (94).

Septicaemia: Septicaemia is generally considered to be the most life threatening

infection caused by Aeromonas. Aeromonas sepsis usually occurs in immunocompromised

patients but also in patients suffering from malignancy, hepatobiliary diseases and diabetes

(96). Mortality rates for Aeromonas associated septicaemia are usually ranged between 25-

50% (46, 94).

Other infections: Aeromonas species have been shown to be associated with

several other types of infection including meningitis, peritonitis, catheter related infection,

myonecrosis (94, 105, 140, 170).

1.4.8 Putative virulence factors of Aeromonas

Virulence often refers to a measure of the capacity of a microorganism to infect or

damage a host and is often quantified by the size of the dose required to infect (ID50) or to

kill (LD50) 50% of hosts (121). Molecular studies have shown that virulence is a composite

trait and virulence will vary often be selected independently, and selective pressures may

vary between different macro- and microenvironments within a single host or even within a

single tissue (121). Due to the lack of adequate animal models, the significance of the precise

putative virulence in Aeromonas remains to be established. It is well known that some of the

motile Aeromonas possess a number of putative virulence factors. The Table 2 shows the

putative virulence factors described in the literature. Some of these virulence factors were

18

19

found to be distributed in strains isolated from the environment and from the cases of

infections. Thus an association of prevalence of putative virulence factors and infection

could not be established. However, certain species are more frequently isolated from

infections than from environments.

20

Table 2 Putative virulence factors of Aeromonas Virulence factor Gene Prot. Size Effect Ref.

Toxins

Aerolysin aerA 48 kD channel form. (5) HlyA hlyA hemolysis (74) AexT aexT ADP ribosyltransferase (25)

Cytotonic enterotoxin alt cAMP and PGE2 levels* (36) ast 32 kD

(35) Cytolytic enterotoxin act 52 kD haemo- /cytolytic Shiga-like toxin 1 (plasmid encoded) (73)

Enzymes

Acetylcholinesterase 45 kD (151)

Lipase Phospholipase C

Serine protease aspA 70 kD (193) Metalloproteinase asaP1 20 kD caseinolys (190) GCAT (glycerophospholip:cholesterol acetyltransferase) lipase Metallo-gelatinase P2 Aminopeptidases Amonabactin T and P fstA and fsp (150)

Surface proteins/outer membrane proteins/fimbriae S-layer

Mucin binding component 22-95 kD Type IV pili

*: elevated cAMP and prostaglandin (a receptor-mediated activator of adenylate cyclase) levels in CHO cells.

Involved in cell differentiation and apoptosis.

1.4.9. Biofilm formation of Aeromonas In general, biofilms are defined as assemblages of microorganisms associated by

their extracellular products. A typical biofilms are communities of microorganisms attached

to an abiotic or biotic surface (43). Bacteria found in environmental, clinical and industrial

settings often exist as surface associated biofilms (40) and can be beneficial or deleterious.

Biofilms give the bacteria a significant survival advantage, since bacteria in biofilms can be

resistant to antibiotics, chlorination, disinfections (39). Bacteria residing within a biofilms

receive certain degree of shelter since cells in biofilm are known to enmeshed within a

matrix of extracellular polymeric substance (EPS) by the organisms themselves and, EPS

vary not only upon the organism, but also within the same organisms when other

environmental conditions are applied (43, 147).

Biofilms are extremely important in the waste water treatment process since they

remove many kind of key waste water contaminants such as nitrogen and phosphorous

compounds (142). On the other hand biofilms control is now recognised as an important part

of the operation of drinking water plants and distribution systems in developed and

developing countries Aeromonas was identified in 7.7% of biofilm samples in a water

distribution system (34), a finding that may be related to future outbreak of gastroenteritis.

In natural settings biofilms form in aqueous environments, and aquatic environments are also

known as reservoirs of different pathogenic and non-pathogenic bacteria such as V. cholera,

V. parahaemolyticus, Aeromonas spp. In the aquatic environment, these bacterial may form

biofilms on the aquatic flora and fauna, persist for a long time and be are transmitted within

the environment by using the aquatic flora and fauna as a vehicle. Genetic exchange between

community members in environmental biofilms is another key aspects environmental

biofilms harbouring pathogens

Bacteria sense the environmental signal and settle on the surface. Sensing of cell

density (quorum sensing) plays a major role in bacterial biofilm formation (28). Quorum

sensing could be define as accumulation of a low molecular mass signal enables individual

cells to sense when the minimal population unit (’quorum’) has been achieved (for a

concerted population response to be initiated) (180). Quorum sensing in gram-negative

bacteria is mainly based on N-acyl-homoserine lactones (AHLs) and, its co-regulatory

receptors which control the expression of genes (97). AHLs are the mostly studied quorum

sensing signalling molecules in Gram negative bacteria (178). Chemically, AHLs consist of

a homoserine lactone ring, an amide bond and acyl side chain of variable length (to date 4 to

18 carbons in length have been identified). Aeromonas spp are known to produce C4-HSL

(N-butanoylhomoserine lactone) (108, 123, 179) .

21

1.5. Enterococci 1.5.1 Genus description of Enterococcus

Enterococcus are gram positive cocci, arranged single, in pairs or as short chains.

They are facultative anaerobes with a growth range from 10o to 45oC. All Enterococci

hydrolyze esculin in the presence of bile salts, grow in broth containing 6.5% NaCl, produce

leucine aminopeptidase, and most enterococci but not all, and hydrolyze pyrrolidonyl-b-

naphtylamide (PYR). At present there are 23 distinct Enterococcus species based on

molecular characterization techniques such as DNA-DNA reassociation, 16s RNA gene

sequences, and whole cell protein analysis.

1.5.2 Habitat of Enterococcus Enterococci are natural habitants of the human intestinal microflora, and can be

found in faeces of most adults. However, they represent only a minority, i.e. less than 1% of

the microorganisms found in faeces of healthy individuals (167). E. faecalies and E. faecium

are the most commonly detected with E. faecalies as the dominating species but E.

casseliflavus, E. durans and E. avium have also been isolated from small proportions of

human individuals (12, 48).

Enterococci are less frequently isolated from other human sites, but have been found

in vagina, oral and skin specimens. In addition they can be found in soil, on plants, in waters

and in various food products (47). Enterococci have a hardy nature and can endure harsh

environmental conditions, such as 65oC for 10 minutes and a pH from 4.8 to 9.6 (103). They

can survive in nutrient deficient environments and have been recovered from dry surfaces

after weeks and from water after several months (103, 191).

1.5.3 Clinical diseases and nosocomial infections by Enterococcus The most commonly isolated enterococci of clinical significance are E. faecalis and

E. faecium, both of which may be the causative agent of different clinical diseases such as

urinary tract infections, wound infections and serious blood stream infections. E. faecalis is

most frequently encountered in infections acquired in the community, in long term care

facilities and in hospitals (20, 58, 110).

During the last few decades, enterococci have emerged as important nosocomial

pathogens with great ability to acquire antibiotic resistance. Enterococci are in the USA the

third and in Europe the fifth most common clinical isolate causing serious nosocomial

infections such as bacteraemia. They also spread their resistance genes to other bacteria

outside the genus. Transmission of resistant enterococci strains between patients treated in

different settings has also been noted. The hardy nature of Enterococcus makes them able to

survive in the environment for a long time. Increased movements of colonized patients

between different units and hospitals is partly responsible for their spread.

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1.6. Antibiotic resistance

Antibiotic resistance can be divided into intrinsic resistance and acquired resistance.

Intrinsic or inherent resistance can be regarded as a specie’s characteristics and occurs in

most stains of a particular species. The genes of this type of resistance typically reside on the

chromosome. Acquired resistance is the result of mutations in the existing DNA or

acquisition of new DNA from other organisms. Basically antibiotic resistance in bacteria

disseminate in two ways: clonal spread of resistant clonal lineage and horizontal transfer of

resistance genes. The most important vehicles for resistance gene transfer are mobilisable or

self transmissible plasmids in conjugation with transposons, IS-elements and integrons.

Previous studies have shown that class 1 integrons associated resistance gene cassettes are

highly prevalent on conjugative R-plasmids within Aeromonas population in different fish

culture environment from different countries. The occurrence of integrons associated

resistance gene cassettes located on antibiotic resistance plasmids was also isolated from

waste water treatment plant. It has been shown that plasmid mediated horizontal gene

transfer play an important role in waste water purification facilities.

1.6.1 Antibiotic susceptibility testing and MIC breakpoint

In order to find out if an antibiotic exhibits activity against a particular isolate of an

organism, antibiotics susceptibility testing is performed. The susceptibility testing is done by

determining the minimal inhibitory concentration (MIC) of the drug for the isolates of

interest. The MIC results are then interpreted in a breakpoint system and isolates are divided

into susceptible (S), intermediate (I) and resistance (R). Ideally, a breakpoint system should

satisfy both the need to guide therapy and the need to detect biological resistance. From the

clinical point of view this means that if an organism is classified as susceptible to a particular

antibiotic, it can be assumed that the drug will inhibit the organism and therapy will be

successful. However, clinical breakpoints do not take into account whether or not the

organisms harbour resistance mechanisms. To meet the needs of epidemiological monitoring

of emerging resistance, the MIC breakpoint should be able to divide the susceptible wild

type strains from those one that have acquired resistance mechanisms.

Many different methods are available for bacterial susceptibility testing, including

disk diffusion, agar dilution, broth microdilution and antibiotic gradient disks. Broth

microdilution is a convenient and widely used for susceptibility testing of several antibiotics

on a large number of bacterial isolates in a short time.

23

24

AIMS OF THE STUDY

1 To study the prevalence of FMSE1 like PhP clonal group of Enterococci in hospital

sewage water.

2 To characterise the Aeromonas strains isolated from Epizootic Ulcerative Syndrome

in fish in Bangladesh.

3 To study the persistence and transmission of Aeromonas in a duckweed aquaculture

based hospital sewage water recycling plant.

4 To develop a user-friendly semiautomatic susceptibility testing method to screen

large numbers of isolates in a short time.

5 To study the influence of a duckweed aquaculture based hospital sewage water

recycling plant on the antibiotic resistance of Aeromonas.

6 To study the cyclic- di - GMP signalling in Aeromonas.

25

26

MATERIALS AND METHODS

Sample and Isolates

Study I. PhP fingerprinting of 9676 Enterococci from different kinds of sources

(such as untreated urban sewage water, treated urban sewage water, hospital sewage water,

surface water, pig, pig faeces, pig manual, broiler chicken, cattle, hospitalised patients,

outpatients, healthy children) from our databases were used in this study (117). The number

of isolates of Enterococci from the different studies and sample types are described in Table

1 in Paper I.

Study II. Fourteen Aeromonas spp isolated from different diseased fish from several

fish farms, with deep hemorrhagic ulcers in the mid-body and tail regions, were obtained

from a collection of Bangladesh Agricultural University fish diseases laboratory. For

comparison, 26 environmental Aeromonas isolates were randomly chosen from the same

disease ponds, and twelve randomly selected human diarrheal isolates of Aeromonas spp

from an earlier investigation (114) on Bangladeshi children suffering from diarrhoea were

included in the study. The bacteriological methods used in this study are described in Paper

II.

Study III, IV, V. The sampling sites were a model system of duckweed aquaculture-

based hospital sewage (Kumudini hospital and its residential area complex) water treatment

plant located in Tangail, Bangladesh. A total of 1,318 presumptive Aeromonas from a total

of 691 samples from sewage water, duckweed, fish, patients suffering from diarrhea, control

ponds and healthy humans were isolated over a period of three years (Papers III, V).

Furthermore, a total of 1,005 coliform bacterial were also isolated from the same samples

(paper IV). The procedures for samples collection, samples processing and bacteriological

analysis are described in the corresponding papers.

Biochemical Phenotyping with the PhenePlate™ system (study I, II, III, IV, V)

Bacterial isolates were typed through a biochemical phenotyping method,

PhenePlate™ system (PhP; PhPlate Microplate Techniques AB, Stockholm, Sweden)

according to the manufacturer’s instructions. The biochemical reactions of the isolates were

compared pair wise and a similarity matrix consisting of the correlation coefficient between

all possible pairs was constructed. The similarity matrix was clustered according to the

unweight pair group method using average linkages (UPGMA)(173). Isolates with a level of

similarity greater than 0.97 were assigned to the same PhP phenotypes. All data processing,

including optical reading and calculation of correlation coefficient, as well as clustering and

27

printing of dendrograms, was performed with PhP software (PhPlate Microplate Techniques

AB, Stockholm, Sweden).

FAME analysis (Study II, III, IV)

Gas-liquid chromatographic analysis of cellular fatty acid methyl esters (FAMEs)

were prepared and extracted by using the standardized protocol of the Microbial

Identification Systems (MIDI; Microbial ID Inc., Newark, Del.). The extracts were analysed

with a Hewlett-Packard model HP5890 gas chromatograph as described previously (145).

Identification of the FAME fingerprinting, generation of database and unweighted arithmetic

average cluster analysis were performed using the MIDI software package. Aeromonas

isolates were identified by comparing their FAME profiles with the laboratory-based

identification library AER48C (86).

AFLP analysis (Study II, III)

Whole-genome fingerprinting were carried out using amplified fragment length

polymorphism (AFLP) analysis according to the method of Huys and Swings (84). The

AFLP profiles of unknown isolates were compared with the laboratory-based identification

library AEROLIB comprising AFLP profiles generated from a collection of well-

characterized type and reference strains encompassing all currently recognized Aeromonas

taxa (87).

PFGE analysis (Study I)

DNA fingerprinting by pulsed field gel electrophoresis (PFGE) was performed

according to the method described by Torell et al (186) and the resulting DNA band pattern

was interpreted according to criteria proposed by Tenover et al (182).

Cytotoxic and hemolysin activity (study II, III)

Cytotoxic activity of the isolates was tested on the fish cell line EPC (epithelioma

papulosum of carp, Cyprinus carpio ) or Hep- 2 cell lines as described previously (56).

Briefly, confluent monolayers of the cell were grown in 24 well tissue culture plates (Coster,

Corning, NY, USA) in minimal essential medium supplemented with 10 % fetal bovine

serum, 1% (w/v) glutamine and 1% (w/v) penicillin-streptomycin. The cells were incubated

for 6 h at 18°C with 100 µL of sterile culture supernatant (containing 150 mgl-1 protein as

determined by Bio-Rad protein assay) serially two-fold diluted in supplemented MEM. The

cytotoxic activity was measured as rounding up, detachment and loss of viability of the cells

as seen in the light microscope within 6 h. The titer was determined as the highest dilution of

28

supernatant affecting at least 50 % of the cells. Isolates showing a cytotoxic effect at a

dilution of 1/8 (final concentration) or more were regarded as positive.

Hemolysin activity (study II, III)

Hemolytic activity of the isolates was measured on 1% (v/v) human and rabbit

erythrocytes as described earlier (101). Isolates were considered positive for haemolysin

production when the culture supernatant at a final concentration of 1/8 lysed at least 50 % of

the erythrocytes as determined by eye. Known positive (BD2-9) and negative (BD12)

isolates from an earlier investigation were included as controls into both cytotoxin and

haemolysin assay (115).

Hemagglutination (Study II)

Hemagglutination tests were performed on glass slides by mixing a loopful bacteria

with a 3 % (v/v) suspension of erythrocytes from rabbit, human or fish (Labeo rohita) in

PBS. Visible agglutination within five min was considered as positive. Agglutination

inhibition test was performed using a dilution of 1 % (w/v) D-mannose, D-galactose and L-

fucose sugar in PBS (66).

AHCYTOEN genes detection (study II, III)

The AHCYTONE gene, a cytolytic enterotoxin gene (AHCYTOEN) has been

reported as a multivirulence gene involved in lethality in mice, hemolysins, cytotoxicity and

enterotoxicity, were detected by PCR method using the primer AHCF1 ( 5 / – GAG AAG

GTG ACC ACC AAG AAC A–3 / ) and AHCR1 ( 5 / –AAC TGA CAT CGG CCT TGA

ACT C–3 / ) (107). The specificity of the primer combination was corroborated with positive

and negative controls during PCR experiments.

Antimicrobial susceptibility testing

The tested agents and range of concentrations for tested antimicrobial agents were:

ampicillin (2-128 mg /L), tetracycline (1-64 mg/L), chloramphenicol (2- 128 mg/L),

nalidixic acid (2-128 mg/L), cephalothin (1-64 mg/L), erythromycin (2-128 mg/L),

streptomycin (2-128 mg/L), and gentamicin (1-64 mg/L). Preparation of microdilution plates

were performed according to the NCCLS recommendations (141). Furthermore, in the

“ScanMIC” method, a growth indicator (TTC) was also added to the microplates and the

microplates were dried overnight by dry air at 25o C.

Bacteria were cultured over night on blood agar plates. Three colonies were

transferred to 5 ml Mueller Hilton broth (Becton Dickinson, USA) without blood and

29

incubated at 37o C for four hours to reach the exponential phase of growth. From these

cultures, bacterial turbidity was adjusted to 0.5 on the McFarland turbidity standard as

measured by absorbance (0.08-0.1 at 625 nm) in spectrophotometer (Hitachi U-1100),

corresponding to approximately 10 8 CFU/ml. The above adjusted bacterial suspensions were

first diluted 1: 200 in Mueller-Hilton broth, and 100 µl were added to each well (5 X 104

CFU/ well) in the pre prepared microplate (broth microdilution or “ScanMIC” microplate)

followed by incubation for 16 h at 37 oC. For “ScanMIC”, the microplates were scanned

with a reflective flat bed scanner (UMAX-Astra 6450, USA) connected to a Windows based

PC. The images were analysed by an in-house developed software and MICs were

determined by software. For broth microdilution, MIC was defined as the lowest

concentration of an antimicrobial agent at which no growth was detected (100% inhibition)

as turbidity seen visually. The detailed methods are described in paper IV. The “ScanMIC”

method was compared to the reference broth microdilution method, in two different ways. i)

The distribution of differences in MICs results ii) Interpretative categorical results, obtained

according to the NCCLS recommended interpretive standards (SIR=Susceptible,

Intermediate and Resistant) for Enterobacteriaceae (141), were compared for the “ScanMIC”

method versus the reference method. The details of these calculations were described in

paper IV.

Interaction of Acanthamoeba castellanii and Aeromonas

Acanthamoeba castellanii (ATCC 30234) and Aeromonas hydrophila (HG1) strain

(AEW 44) isolated from child suffering with diarrhea and AEW 20 isolated from the

environment were used in this study. A. castellanii was grown without shaking at 30°C in

ATCC medium no. 712 (ATCC). For invasion experiment, invasion positive strains Dublin

strain SH9325 and invasion negative strain TG1 were used as reference strains (183). For

visualization of the bacteria by fluorescence microscopy we transformed the Green

fluorescent protein (GFP), vector pGFPuv (Clonetech, BD Bioscience) to Aeromonas

hydrophila AEW 20 by electropoeration. The Co culture experiments and invasion

experiment were carried out as described previously (3, 183).

Biofilm and c-di-GMP related assays.

One Aeromonas veronii strain (AEW43), isolated from a child suffering from

diarrhoea, was chosen to study the c-di-GMP signalling pathway in this study. The strain

representative a clonal group of Aeromonas (MT7), which showed persistence ability in

different niches, such as environment, human, fish, and was positive for all the tested

virulence factors.

30

The detailed of the construction of the plasmids (GGDEF domain containing protein

AdrA cloned into p LAFR3 and named pWJB9; EAL domain containing protein YhJH

cloned into p LAFR3 and named pRGS3 and pLAFR3 itself as vector control) has been

described by Simm et al (171). The E. coli DH5 α strains carried those plasmids were

transcongugated to the selected Aeromonas strains (AEW43) through the plate mating

methods resulting the AdrA, YhJH and vector expressing Aeromonas.

Isolation and detection of c-di-GMP from Aeromonas were performed as described

by Simm et al (171). For phenotypic assessment, the AdrA, YhJH and vector expressing

Aeromonas was grown on Congo Red (CR) agar plates for 20 h at 37°C as described by

Römling et al (157). Swimming motility was observed using 0.3% LB agar plates inoculated

with a constant cell number from overnight cultures. Biofilm formation was observed in

glass tubes. Overnight cultures of Aeromonas were inoculated in LB medium for 16 h with

shaking 150 r.p.m. After staining the glass tubes with crystal violet, the biofilms were

dissolved in dimethylsulfoxide and the absorbance at 540 nm was measured. In some cases,

a score was assigned on the absorbance values.

Furthermore, to observe the adhesion of constructed strains to duckweed surface,

duckweeds were washed several times with gentamicin. The duckweeds were then incubated

with a bacterial suspension of 107 CFU/ml for 1 hour at room temperature and washed five

times with PBS in order to eliminate non-attached bacteria. For the quantification, the

duckweed and attached bacteria were then lysed in 0.1% Triton in sterile water for 10 min.

The ability of the bacteria to adhere to the duckweed was determined by spreading the Triton

induced cell lysine on nutrient agar plate and counting the number of colonies after

incubation overnight at 37oC.

The quorum sensing molecules, HSL were extracted from AdrA, YhjH and vector

expressing strain as method described by Lynch el al (123). To estimate the bioluminescence

of extracted HSL, overnight culture of Chromobacterium violaceum CV026 or E. coli

JM109 (pSB536) were diluted in LB containing antibiotics to a final optical density at

600nm within the range of 0.005 - 0.006. A 100 µl of the above bacterial culture were added

to a microtiter plate which containing HSL extraction, and incubated for overnight at 30oC

with shaking at 150 r.p.m. Bioluminescence was measured in the luminometer (VICTOR

1420 Multilab counter, Wallac) and relative light units as a function of C4-HSL

concentration was expressed.

31

RESULTS AND DISCUSSION

4.1 Transmission of Enterococcus faecium from hospital patients to environment (Paper I)

During the 1990s a constant increase in the incidence of clinical Ampicillin

Resistant Enterococci (ARE) would be observed in many Swedish hospitals (69-71). Torell

et al showed that 22% of the hospitalized patients and 6% of the outpatients were faecal

carries of ARE (185). Biochemical fingerprinting of these isolates revealed that, 73% of 180

ARE isolates from hospitalised patients and 54% of 39 ARE isolates from outpatients

belonged to the same PhP types (arbitrary named as FMSE1) and almost all isolates of this

type showed high level resistance to ampicillin and ciprofloxacin (186). Thus there was

evidence for a faecal carriage of clonally related ARE isolates among Swedish hospitalised

patients and outpatients nationwide.

The prevalence of Enterococcus faecium FMSE1 (an ampicillin and ciprofloxacin

resistant) strain of was searched for in the PhP typing databases in materials collected

(databases comprising 9,676 isolates of Enterococci) from different sewage waters,

slaughtered animals, pig farms, humans, etc. The FMSE1 type was identified in 1.4% of all

isolates. The FEMS1 type was most commonly found in samples collected from hospital

sewage (50%), followed by surface water (35%), treated urban sewage water (28%),

untreated urban sewage water (17%) and rarely in healthy children (0.8%) and animals (2%)

(Table 1, Paper I). The high prevalence of FMSE1 in different sewage and surface water

samples might be related to a spread of faecal bacteria from hospital patients to sewage and

then to surface water.

The available FMSE1 PhP type isolates were further typed through the PFGE typing

and it was verified that the isolates from hospital sewage and the FMSE1 reference isolates

were genetically closely related (Figure 1, Paper I), whereas the FMSE1 isolate from surface

water that was similar according to PhP typing, showed a different banding pattern, though it

could still be regarded as related. The antibiotic resistance pattern of this isolate also showed

a slightly different profile, while all hospital sewage isolates showed identical profiles

(Figure 1, Paper I). Thus, there is a possible transmission of ampicillin and ciprofloxacin

resistant strains from hospital patients to hospital sewage water, and also possibly to the

surface water. The hospital sewage seems likely the source of resistant bacteria. Thus it

could be possible that ampicillin and ciprofloxacin resistant Enterococcus faecium might be

transmitted to the environment from hospital patients to hospital sewage water and further

through urban sewage waters to treated sewage water and surface water.

32

Drug resistant Enterococcus strains may be carried for months or even years in the

faecal flora of humans (22, 122) and have the potential to spread through colonization of

patients (106, 187). Drug resistant isolates such as FMSE1 like isolates (in Sweden), might

have acquired resistance to ciprofloxacin in hospitals, giving an increased incidence in

hospitalised patients through clonal spread (184, 186). Further on, this same clonal group

transmitted to the environment through hospital sewage water. Current infectious disease

control measures and antibiotic usage routine controls might not prevent this silent spread of

FMSE1 like isolates in the community. But in near future, early detection of new FMSE

isolates with acquired antibiotic resistance might contribute to the control of the clonal

spread in the society.

Table 3 Prevalence of FMSE1-like isolates from PhP Enterococcus database.

% (Positive/number)

FMSE l like

isolates in*

Hospital

sewage**

Surface water Untreated urban

sewage

Treated urban

sewage

Samples 50 (7/14) 35 (13/35) 17 (6/35) 28 (9/32)

Isolates 6.4 (24/374) 3.8 (24 /625) 1.5 (10/677) 1.2 (10/869)

* Samples and isolates analysed for FMSE l like type;

** Samples or isolates were positive within the investigated samples or isolates

In paper I, the samples have been taken from a broad spectrum of sources, and

unfortunately a limited number of isolates were available for further typing. Therefore, we

could not precisely confirm the transmission route(s) of the bacteria from the hospitalised

patient to the environment. Further sampling within a narrow spectrum may permit the

detection of the precise transmission route of the bacteria from hospital to the human

environment. Thus a multidisciplinary project has been chosen, where hospital sewage water

was recycled for production of food, to be consumed by human (Paper III).

33

4.2 A clonal lineage of Aeromonas is associated with fish disease (Epizootic Ulcerative Syndrome) (Paper II)

Epizootic Ulcerative Syndrome, a fish disease, frequently occurs in many fish farms

and open water bodies (lakes, rivers) in Bangladesh as well as in many other countries of the

Indo-Pacific region. The disease generally develops with ulcers on the fish bodies, especially

on the head, middle of the body and dorsal regions (Figure 2). The fish usually die within

one week of infection. The diseases have caused substantial economical losses to fish

farmers and open water fisheries in Bangladesh.

In this study (Paper II), a collection of Aeromonas strains isolated from fish ulcer

scraps were characterized. Biochemical phenotyping with the PhP system revealed that the

isolates belonged to one PhP type. It is shown in the dendrogram that EUS associated

isolates belonged to a PhP clonal group of Aeromonas when 52 Aeromonas isolates from

fish, from environment and from human diarrhoea patients were clustered (figure 1, Paper

II). This EUS associated PhP type was searched for in PhP-AE database, containing a

collection of 1,600 Aeromonas isolates, which comprised Aeromonas isolated from human

diarrhoea and the environment in Bangladesh. But the EUS associated PhP type was not

identical to any type in the database, which indicates that this PhP type might be a unique

clone.

Figure 2 Fish suffering from Epizootic Ulcerative Syndrome (EUS) with dermal ulcer in the middle of the body.

Figure 3 Infected fish are ready to sell in the market from a fish farm; inside infected fish

34

When we compared the virulence properties of the EUS isolates to those of human

diarrhoea and environmental isolates, we found that all the EUS isolates produced all the

virulence factors tested (haemolysin, cytotoxin, the presence of cytolytic enterotoxin genes,

agglutination to different erythrocytes). But some of the human diarrheal and the

environmental isolates also showed the same characteristics. Interestingly, only the EUS

isolates had the ability to agglutinate fish erythrocytes (Table 1, Paper II). In a previous

study by our group we have shown that one phenotypic clonal group of Aeromonas (BD 2,

later assigned a new subspecies Aeromonas hydrophila sub. dhakensis) was associated with

children suffering from diarrhoea and this group was distinguishable from environmental

phenotypes. All of the isolates from that clonal group, except one were positive when tested

for putative virulence factors. Similar to that study, we assumed that the specific EUS

associated clonal group in this study was the causative agent in the fish disease in

Bangladesh.

To test the pathogenicity of the isolates (unpublished data), one representative

isolate of the human diarrhoeal type and one of the EUS associated isolates were subjected to

pathogenicity testing in rainbow trout. The mortality rate was found 100% when challenged

with 104 cells/fish for the diarrhoeal isolate. The infected fishes displayed several ulcerations

with muscle necrosis spreading from the site of injection and the fish kidneys were liquefied.

However, only 67% mortality was found upon challenge with 104 cells/fish for the EUS

associated isolate. The disease signs included haemorrhaging in the muscle around the

injection sites and also the fish kidneys were liquefied. Our results showed that EUS

associated isolates are pathogenic to fish, and human diarrhoeal Aeromonas may also cause

infection in fish. It seems that human diarrhoea associated isolates were more pathogenic to

fish than the fish isolates. Thus if human pathogenic Aeromonas are transmitted to fish

environment, they may increase the risk of infection in fish. But for practical reasons, we

could not detect whether fish associated isolates were pathogenic to human.

The origin of the EUS associated clone in the fish culture environment is not known.

But because of the remarkable survival potential of Aeromonas in the environment, it would

be guess that the EUS associated clone originates from human diarrhoea and was transmitted

to the fish culture pond. In a visit to a fish farm (anonymous) in Bangladesh, we observed

that infected fish was caught for sale in the market indicates that there must be a circulation

of strains between human and fish environment (Figure 3). It is not documented whether

Aeromonas infected fish would be considered as a public health hazard or not. However, fish

culture environment should be aware from contamination of human sewage, since human

pathogenic Aeromonas could increase the rate of fish infection. Our result showed the

evidence for circulation of strains between human and fish in Paper III.

35

36

4.3 Persistence and transmission of Aeromonas in the sewage treatment plant (Papers III, V)

To provide the evidence for possible transmission and/or circulation of bacteria from

hospitalised patients to environment and/or to the human food chain through the sewage

water, we have sampled from a duckweed aquaculture based hospital sewage water treatment

plant and Aeromonas species were chosen as the indicator bacteria (Figure 4). In this studied

treatment plant, the hospital sewage water was first allowed to settle in a settlement pond

and, was then transferred to a lagoon where the duckweed was grown. By growing the

duckweed in the sewage water, organic substances such as, nitrogen and phosphorus loads

were decreased; thus purifying the sewage water. After the duckweed treatment of the

sewage water, treated sewage water effluent was discharged into the nearby river and the

sewage grown duckweed was used as fish food in intensive culture fishponds within the

treatment plant.

4.3.1 Persistence of Aeromonas strains in different samples

For the bacteria, to be able transmit from wastewater to human, the first criteria is survival

and persistence in the treatment system. Among our collection of 1,318 Aeromonas isolates, only 57%

(arbitrarily defined and named to 23 major types) were recovered at several occasions and at several

sites by our testing methods and the remaining isolates occurred sporadically. These findings suggest

that only members of these major types were able to survive and persist during the purification

process (Table 2, Paper III), i.e. were present or possible to detect in different samples. For example,

PhP type MT1 (described as major type in Paper III) was detected in 6 out of 19 Aeromonas positive

hospitalised children suffering from diarrhoea while type MT13 was detected in 4 out of 10

Aeromonas positive human workers. Few major types (MT:s 2, 4 and 11) were found to be distributed

in different sites of the treatment plant, in hospitalised children suffering with diarrhoea and in the

control ponds. Furthermore, some interesting types (MT:s 3, 7 and 8) were detected in the treatment

plants and in the hospitalised children suffering with diarrhoea but never in the environment, i.e. the

control ponds. It is not well understood why certain types where only detected in specific sites but

possibly some strains were adapted to a specific niche e.g. the human gut, the fish pond, while other

strains survive in several niches. For example, some strains were only found in the human samples but

not in the environment, and certain strains survived only in sewage water while others in all niches.

However, our data indicate that phenotypically and genetically stable certain Aeromonas strains

(MT10 type) can survive and persist in the sewage water treatment plant for years (Figure 4, Paper

III). The remarkable survival and persistence of Aeromonas in different environments, such as water

microcosm for 180 days and water distribution systems for years, is documented (116, 134).

37

Hospital

Waste settlement pond

Duckweed lagoon

Aeromonas

Figure 4 Different sampling sites of the duckweed aquaculture based hospital sewage water treatment plant.

Fish pond and fish feed

When the PhP profile of our collected 1318 isolates were compared to a PhP references

database for Aeromonas it was found that A. hydrophila was the dominating species (45 %)

in the human samples while its relative frequency decreased to 11 % in fish (Figure 2, Paper

III). On the other hand, A. veronii was found at the lowest percentage (22 %) in human

samples but this percentage increased throughout the purification process up to 51 % in the

fish. We found both A. hydrophila and A. veronii were common in the control ponds.

4.3.2 Evidence for transmission of Aeromonas from hospitalised patients to human food through sewage water recycling

One of the main concerns of public health in recycling of waste water is that

pathogenic and drug resistant bacteria may persist in the treatment plant and from the plant

possibly disseminate to the environment or spread to the community through the food chain.

To be able to track the transmission of single Aeromonas strains a large number of isolates

were characterized. A series of different identification and typing methods were used to

understand the clonal relationships of the isolates, since identification of Aeromonas species

is not straightforward. This study was based on PhP-AE typing, and it was assumed that

isolates belonging to the same PhP-AE type probably belonged to the same clonal group of

strains. The consequent AFLP analysis of the PhP typed strains not only confirmed the

previous PhP grouping but also the subdivision of the PhP types into multiple genotypes

(Figure 4, Paper III).

4.3.2.1 Transmission of a virulent clonal lineage of Aeromonas bacteria

The PhP clonal groups of bacteria that persisted in different parts of the sewage

treatment plant were subjected to AFLP DNA fingerprinting to confirm the genetic

relatedness. High AFLP fingerprinting homology was found within the isolates belonging to

PhP major types 7 and 11 (Figure 5). One of these clonal groups (MT7) of strains that was

found in children suffering from diarrhoea, but also found in sewage water, duckweed, and

fish. The isolates of this clonal group were positive for the putative virulence factors tested.

On the other hand, the other clonal group (MT11) was found to be distributed in all parts of

the treatment plant samples. But the isolates of this latter type appeared to be avirulent. This

finding shows that both virulent and avirulent clonally related Aeromonas strains were able

to transmit from the hospital sewage water to the fish for human consumption through the

treatment plant.

38

39

4.3.2.2 Transmission of multiresistant clonal lineages of Aeromonas bacteria

Hospital sewage water receives bacteria that were previously exposed to different

antibiotics used for treatment human infections and is known to be a reservoir for many

multiresistant bacteria. Although only 6% of the tested isolates were gentamicin resistant,

these isolates were also resistant to all the eight antibiotics tested. PhP data analysis revealed

that they belonged to two PhP phenotypic groups. Further AFLP analysis of ten isolates

indicated a molecular homogeneity of the isolates thought they could be divided into two

AFLP types (Figure 6). The isolates originated from sewage water, duckweed growing

lagoon, duckweed, fish pond and fish. But these two AFLP types could not be detected

either in the human samples nor in control ponds. Our data revealed that two specific

multiresistant clonal lineages of Aeromonas bacteria persisted in the waste treatment plant

and that the same clonal groups were transmitted from hospital sewage water to cultured fish

for human consumption. Thus through recycle of sewage water virulent and multiresistance

strains of Aeromonas were disseminated to the environment through the sewage water

effluent.

"Imagination is more important than knowledge” Albert Einstein

Isolate no PhP type Origin* Isolation Year Species**

Virulence

107 MT7 Sewage 2001 A. veronii HG8/10 ++ 108

MT7 Lagoon 2001 A. veronii HG8/10 ++

104 MT7 Human 2001 A. veronii HG8/10 ++

103 MT7 Fish 2001 A. veronii HG8/10 ++

56 MT11 F. Pond 2001 A. veronii HG8/10 --

3 MT11 Sewage 2001 A. veronii HG8/10 --

54 MT11 P. Duckweed 2001 A. veronii HG8/10 --

55 MT11 L. Duckweed 2001 A. veronii HG8/10 --

53 MT11 Fish 2001 A. veronii HG8/10 --

40

Figure 5 UPGMA/Pearson correlation clustering of digitalized and normalized AFLP patterns of a selection of gentamicin resistant Aeromonas isolates from different

sites of the sewage water treatment plant.

Origin Amp Tet Ery Chl Nali Cep Stp Gen

Fish 128 32 128 64 128 64 128 64

Duckweed

128 32 128 64 128 64 128 64

Sewage 128 32 128 64 128 64 128 64

Lagoon 128 32 128 64 128 64 128 64

Fish pond 128 32 128 64 128 64 128 64

Lagoon 128 32 128 64 128 64 128 8

Duckweed 128 32 128 64 128 64 128 8

Fish pond 128 32 128 64 128 64 128 8

Fish 128 32 128 64 128 64 128 8

Sewage 128 32 128 64 128 64 128 8

100

959085807570

....AEW-119

....AEW-120

....AEW-116

....AEW-118

....AEW-117

....AEW-112

....AEW-115

....AEW-114

....AEW-113

....AEW-111

Figure 5 AFLP patterns of a selection of Aeromonas isolates from different sites of the sewage water treatment plant with isolation year, species and virulence.

41

4.3.3 Performance of the duckweed based sewage treatment plant

Protection of public health is the fundamental purpose of the waste water treatment

and aim is the reduction and elimination of the load of pathogenic microorganisms.

From the sites sampled at the sewage water treatment plant, the highest diversity

value of Aeromonas was found in the sewage water and the diversity of the populations

appeared to decrease further down the sewage water system from the duckweed lagoon to

the fish. In the treatment plant, the highest number of Aeromonas was found in the duckweed

lagoon and the lowest number in the fish pond. The high number of Aeromonas observed in

the duckweed lagoon was due to the fact that the samples from duckweed contained the

highest numbers of Aeromonas. However, the bacterial load was decreased from the sewage

water to the fish culture pond in the duckweed aquaculture based sewage treatment plant

(Table1 Paper III).

0,00

0,20

0,40

0,60

0,80

1,00

1,20

Sewage Lagoon Duckweed Fishpond Fish Patient Control

Tet Ery Chl Nal Stp Gen

Figure. 7 Relative frequency of resistant bacteria found in different sampling sites. The percentage of

resistant bacteria found in the initial stage of the treatment plant i.e. in sewage water was referred to 1

for all tested antibiotics.

The use of sewage water grown duckweed as fish feed might have an impact on the

occurrence of antimicrobial resistant bacteria in the fish pond. The percentage of resistant

bacteria was found to decrease for all antibiotics throughout the purification process in the

sewage water treatment process, i.e. from the sewage water to the duckweed to be used as

fish feed (Table 2 Paper V). The relative frequencies of resistance are shown to illustrate the

changes within the treatment system (Figure 7). In the sewage water treatment system we

studied here, the duckweed lagoon water is discharged to the river. There is a considerable

decrease of resistance in the lagoon water as compared to the sewage water.

42

In conclusion, duckweed based waste treatment system had a positive effect on both

the number and the resistance pattern of Aeromonas bacteria. Despite the fact that we found

high numbers Aeromonas attached to the duckweed.

4.3.4. Roles as vehicles in transmission

Duckweed as a vehicle for bacterial transmission: In this study, we followed up

the two unexpected results we found. Firstly, in the duckweed lagoon as well as in the fish

pond the duckweed itself appeared to contain higher numbers (>105 cfu/mL) of Aeromonas

(Table1, Paper III). Possibly, the high numbers of Aeromonas found in the duckweed might

be due to attachment and biofilm formation on the duckweed. In the sewage treatment

system studied here, the duckweed grown on sewage water is used as fish feed, which means

that the fish might become colonized with Aeromonas and provide a reservoir for further

transmission to humans. In fact, we were able to isolate specific Aeromonas clones in both

duckweed and fish (MT11 in Figure 4 Paper III), which supports the hypothesis that these

organisms can use duckweed as transmission vehicles. This could make up a disadvantage of

duckweed-based sewage treatment plants if such strains are pathogenic. In previous studies it

has been reported that aquatic animals and plants may function as reservoirs of pathogenic

bacteria, for example Vibrio cholerae may survive longer on duckweed than in water on

which duckweed was floating or in control water (88, 91, 92). Parveen and colleagues (148)

also observed high Aeromonas numbers on aquatic plants and phytoplankton in Bangladesh.

Furthermore, Vibrio cholerae were transmitted as far as from the Philippines to California by

using seaweed as vehicles (198).

Plasmids as a vehicle for resistance transfer: Secondly, although resistance

decreased generally from sewage water to the duckweed, there was an increase in resistance

frequency in the fish pond (Table 2 and Figure 2, Paper V). In our studied sewage water

treatment system the intensive fish culture ponds makes up a relatively confined unit with

little exchange of water. One explanation could be that the antimicrobial drugs, or their

residues together with multiresistant bacteria present in the sewage, enter the fish pond

through the use of duckweed for fish production. Another explanation could be the

horizontal transfer of resistance genes through plasmids, as it is known that vehicles for

resistance gene transfer are mobilisable or self-transmissible plasmids in conjugation with

transposons, IS-elements and integrons. In this study, 24 isolates were investigated for

transfer of resistance and plasmid, 21 of which were shown to contain plasmids and 18 of

these were positive in the conjugative transfer analysis. A single plasmid of 12 Kb, designed

pAEW1, was found in 81 % of the plasmid positive strains including all of the tested isolates

from the fish pond water. The presence of pAEW1 (arbitrary named in this study) was found

43

in isolates of different PhP and AFLP types. A multiresistant clonal group of Aeromonas

caviae also carried that plasmid. Altogether, conjugative transfer analyses in our laboratory

experiments indicate that the horizontal transfer of an R-plasmid might have taken place in

the sewage treatment system. Our results support the hypothesis that plasmid-mediated

spread of resistance in the duckweed aquaculture based sewage water facilities may play a

role in the antibiotic resistance trails. Thus the increased levels of resistant bacteria in the

fish pond may be the results of the introduction of resistant bacteria, a selective pressure

favouring growth of resistant bacteria and/or spread of resistance plasmids within the

Aeromonas population.

Amoeba as a vehicle for Aeromonas transmission (preliminary results):

Free-living amoebas have attracted much interest during the past decades as reservoirs for

different amoeba resistant bacteria (17). The role free-living amoeba plays in the

transmission of both pathogenic and non-pathogenic bacteria have been the subjects of

discussions. To our present knowledge amoeba may increase the transmission rate of

bacteria by acting as an endosymbiont or as a vehicle by carrying a huge number of

microorganisms (62, 63, 125, 18, 19, 123). Like Aeromonas, Acanthamoeba have previously

been isolated from different sewage water systems. To be able to understand the possible

transfer routes of Aeromonas via free-living Acanthamoeba or the utilization of

Acanthamoeba as a natural host, we performed co-culture experiments. We found a mutual

interference of growth between the amoebae and the Aeromonas strains, dependent on the

ratio of bacteria to amoebae cells. But we found that at the high concentrations (107 to 109

CFU ml-1) Aeromonas was able to repress amoeba growth, and within one week the amoeba

cells decreased with 95%. Interestingly, Aeromonas was never able to lyse all amoeba cells,

and even after two weeks of time 100 cell/ml were still left. To investigate the direct

interaction between Aeromonas and A. castellanii, we tagged A. hydrophila (AEW 20) with

GFP and observed the adherence of the bacteria to the amoeba by fluorescent and phase-

contrast microscopy. We observed that the bacteria were always attached in clumps on the

amoeba surfaces, i.e. thus forming a biofilm on the amoebal cell wall (Figure 8 A,

Aeromonas biofilm on Amoeba). These observations show that in a natural setting,

Aeromonas spp may adhere to the amoeba surface and may use amoeba as a vehicle for

transmission. To study the invasion ability of bacteria into Acanthamoeba castellanii, we

carried out gentamicin protection assay, using A. hydrophila (AEW44), A. veronii biovar

sobria (AEW98) and A. caviae (AEW1) at a multiplicity of infection rate of 1:100. We were

not able to recover any intracellular bacteria from the amoeba two hours post infection. But

we recovered a significant number of bacteria 16 hours post infection from the amoebae.

However, we obtained the highest recovery rates of intracellular bacteria after three days of

44

incubation. Fluorescent microscopic observations of the GFP-tagged bacteria indicated

internalization of Aeromonas by amoeba (Figure 5B). Our results suggest that free-living

amoeba may increase the transmission rate of Aeromonas as a vehicle or in endosymbiosis,

wherein they may multiply and later lyse the amoeba in order to disperse. In summary, we

show here that the co-existence of Aeromonas and amoeba in the sewage water increase the

risk of transmission as Aeromonas may use the amoeba as a natural host.

Figure 8 A. Phase-contrast microscopic images of Aeromonas AEW20 adherence to the Acanthamoeba within 15 minutes of co-culture. B-C. Fluorescent microscope images show internalization of Aeromonas AEW 20 by Acanthamoeba castellanii and the increased number of intracellular bacteria with time.

A B C

4.3.5 Biofilm formation of Aeromonas (unpublished data)

Prevalence of biofilm formation of Aeromonas: A total of 92 Aeromonas isolates

were tested for biofilm formation in glass tubes. 25 of them (27%) showed strong biofilm

formation, 41 (44%) showed moderate biofilm formation and the remaining 26 (29%)

isolates did not show any biofilm formation in our setting (Figure 9). Species distribution on

biofilm formation is shown in figure 9. Interestingly, it was found that 100% of the

Aeromonas hydrophilia tested were able to form biofilm either strongly or moderately.

0

10

20

30

40

50

60

"++++ to +++++ "++ to +++ "-- to +

Arbitrary biofilm formation score

Pece

ntag

es

A.hydrophila A.veronii A.caviae

Figure 9 Distribution of species on Aeromonas biofilm formation.

Strong biofilm formation in persistent clonal groups: When the biofilm formation

scores (arbitrary) were analysed in relation to the PhP clonal types, it was found that all

members of PhP MT types 7 and 11 showed strong biofilm formation (Figure 10).

Interestingly, members of these clonal groups were also been shown to be persistent in

different sites of the hospital sewage water treatment plant and to belong to the clonal groups

that are transmitted from the hospitalised patients to the fish to be consumed by human,

through the sewage water treatment system (Paper III). Taken together, our data suggest that

a strong biofilm formation capacity of PhP MT types 7 and 11 clonal groups might have

contributed to the successive persistence and transmission.

0

1

2

3

4

5

6

Arb

itrar

y bi

ofilm

form

atio

n

Fig

Aeromonas.

4.4. Genetreatmen

Wa

organisms f

of microorg

microorgan

according t

removing s

different en

the waste

organisms.

MT7 MT 8 MT 11 MT 3 MT 2

45

ure 10 Biofilm formation capacity of the members of different PhP clonal group of

ral comments on duckweed aquaculture based sewage t plant

ste water treatment plants seldom have been planned to remove pathogenic

rom waste water. Conventional waste water treatment plants reduce the numbers

anisms but the waste water effluents may still contain high numbers of faecal

isms. The removal efficiency of pathogenic and indicator organisms may vary

o the treatment process type, retention time, temperature and the efficiency of

uspended solids. Survival and persistence abilities of different organisms in

vironments may vary and organisms have been demonstrated even multiply in

water treatment system. Thus removal efficiencies may also vary between

Efficient removal of pathogens from waste water is a critical task, since waste

46

water discharge may increase the pathogen contamination in surface water and result in the

increase of food and water borne infections.

The mainstream sewage water treatment, such as activated sludge and waste

stabilization pond systems are too costly for developing countries (61). Duckweed

aquaculture based sewage water treatment systems are a relatively new concept for waste

treatment. The successful implementation of duckweed aquaculture based waste treatment

systems with the benefit of using duckweed for cost recovery far out weighted the problem

with attendant risks of recycling of pathogens. Using the indicator bacteria Aeromonas, we

have shown that virulent and avirulent, and drug resistant bacterium bacteria could be

transmitted from the hospital sewage water to the fish to be consumed by humans through

the recycling of sewage water in a duckweed aquaculture based hospital sewage water

treatment plant in Bangladesh.

Unfortunately, there is no data available on the prevalence of gastroenteritis in

relation to consumption of fish associated with sewage water treatment but there is well-

documented data on shellfish as vectors of infectious agents in USA. Since 1800s, more than

400 infectious disease outbreaks and 14,000 cases have been reported in USA. These

outbreaks are attributed to bacterial and viral agents that are associated either with human

wastes (sewage effluents that have received variable levels of treatment) or to bacterial

pathogens indigenous to coastal marine environments (e.g., Vibrio spp). Before the 1950s,

the most common illness associated with the consumption of shellfish was typhoid fever. In

the mid-1920s there were several outbreaks of typhoid fever (more than 1,500 cases and 150

deaths) in several US cities. After the outbreak, the U.S public health service established

regulations for the sanitary control of shellfish. National shellfish sanitation program appears

to have been effective since then no shellfish associated typhoid cases have been reported in

USA in almost 40 years. The incidence of typhoid fever began to decline for at least two

reasons, i) the technology of the sewage treatment was improved, particularly the removal of

pathogen associated particles, ii) the water quality standard was developed for the shellfish

growing area. These epidemiological results suggest that aquaculture (shellfish) related

infectious diseases could be controlled through proper management of waste water and

aquaculture water.

In our studied duckweed aquaculture based sewage treatment plant, there is a clear

vertical transmission of pathogenic and drug resistant Aeromonas from the sewage water to

the fish through the duckweed. Duckweed itself was also shown to contained high amount of

Aeromonas. These results necessitate a few additional steps to reduce the load of pathogens

47

from duckweed before using it as fish feed. The additional steps could include drying of the

weed and/or washing with the fresh water. Another additional step could be that the water in

the fish culture pond was changed more often. Furthermore, to decreases the gastroenteritis

in relation to consumption of fish associated with sewage water treatment i) Fish should not

be marketed from waste water grown area unless they are shown to be free from any

suspected or dangerous disease producing organisms; ii) Epidemiological studies should be

conducted for all outbreaks (epidemics) that implicate fish as sources, so that the sources of

the fish could be accurately traced.

This investigation serves as a first step of risk assessment, i.e. potential hazard

identification in the duckweed aquaculture based sewage water treatment system. It is

therefore recommended that a quality assurance system based on the HACCP principles

should be introduced to ensure the microbiological safety of the waste recycled food. Thus

our results support that sewage water recycling in aquaculture constitutes a potential health

hazard and further studies are needed for a proper characterization of the risks involved.

Although a detailed assessment of the epidemiological relationship between the

prevalence of enteric disease and the consumption of fish produced in the sewage treatment

system fell outside the scope of the present investigation, this study suggests that it would be

important to perform a community-based study on the prevalence of gastroenteritis in

relation to consumption of fish associated with sewage water treatment.

48

4.5 Usefulness of “ScanMIC” method for antibiotic related hazard identification (Paper IV, V)

Increased bacterial resistance to antimicrobial agents has initiated the monitoring of

the resistance in isolates from humans, foods, animals and environments through national

surveillance and research programs. For antibiotic related hazard identification surveillances

in human food chains, large numbers of isolated are to be investigated. Therefore, a rapid

and effective method is needed to screen large numbers of isolates for antibiotic resistance

patterns in a short time.

In this study, a “ScanMIC” method, i.e. a colorimetric MIC (minimum inhibitory

concentration) method has been developed for susceptibility testing of gram-negative

fermentative bacteria. The method is a slight modification of the NCCLS (National

Committee for Clinical Laboratory Standard, USA) recommended broth microdilution

method, using a redox indicator (TTC) to facilitate the estimation of the bacterial growth

inhibition in a microplate, and a flatbed scanner to capture the microplate image. An in-

house software was developed to transform the microplate image into numerical values

based on the amount of bacterial growth and to generate the MIC values automatically. The

general concept of the ScanMIC method is shown below.

To evaluate the ScanMIC method, we compared the “ScanMIC” method to the

NCCLS recommended broth microdilution method using 197 coliform strains and 288

Aeromonas strains in duplicate against seven and eight antimicrobial agents respectively.

The absolute interpretative categorical agreement was obtained in 92.4 % and 96.7 % of the

assays for coliform and Aeromonas respectively. The percent agreement for MICs

differences (within ± 1 log2 dilution) was obtained in 96% and 97 % for “ScanMIC” versus

broth microdilution for coliform and Aeromonas respectively (Paper IV, Table 1). In both

cases the ScanMIC method was found to meet the performance criteria guideline for

automated susceptibility testing, which has been suggested in the NCCLS recommendations.

The ScanMIC method has been used in this practical study, i.e. epidemiological

surveys of resistance patterns from the hospital sewage water treatment plant, and it was

found to be easy to use for large number of isolates. The main advantage of this method was

the use of a growth indicator, a cheap normal flat bed scanner to capture the microplate

image and the software that directly generated the MIC values from the microplate image.

Besides, the microplate images could be saved and final results sheet with all MIC values

were automatically created. Thus the method could be a useful method for epidemiological

surveys of resistance patterns of large numbers of bacteria in the hospital as well as in the

environment.

49

Schematic presentation of the ScanMIC method

Antibiotic microdilution in microplate with added bacteria

Overnight growth

Scanned the microplate

Software analysis of the scanned microplate; pellet values transferred to numerical values Final result sheet provided by the software

1 2 3 4 5 6 7 8 9 10 11 12A 89 90 84 80 87 84 84 84 81 89 90 94B 86 88 82 67 81 81 83 81 0 82 85 85C 83 90 78 0 80 78 71 81 0 83 90 89D 82 87 82 0 82 78 0 78 0 81 89 94E 80 85 79 0 82 82 0 76 0 83 85 93F 80 0 80 0 82 82 0 80 0 84 93 90G 79 0 81 0 79 75 0 75 0 86 84 90H 76 0 71 0 80 0 0 70 0 88 82 85

Amp Tet Chl Nal Cep Stp Gen Amp Tet Chl Nal CepMIC >128 16 >128 4 >64 128 4 >128 <1 >128 >128 >64

Isolate no Amp Tet Chl Nal Cep Stp Gen Amp Tet Chl Nal Cep 1 256 64 8 8 8 64 2 128 4 16 16 8 2 256 16 4 4 4 16 64 2 2 4 32 4 3 16 2 64 4 2 8 8 32 1 4 16 64 4 4 2 32 4 4 2 4 64 1 8 8 16 5 256 64 4 8 8 64 2 4 64 16 32 1 6 256 16 16 4 4 16 2 4 2 8 32 32 7 16 2 4 4 2 8 2 16 1 32 8 64 8 4 2 8 4 4 2 4 64 1 8 32 2

50

4.6 Cyclic-di-GMP signalling in Aeromonas (preliminary data)

Cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic

guanosine monophosphate (cGMP) are important low molecular weight signalling molecules

in both prokaryotes and eukaryotes (82, 120, 164). In eukaryotes, cGMP is known to be

involved in different mechanisms such as regulation of metabolic processes, cell

differentiation, immunity, and behavioural processes (82, 105, 171). Prokaryotes in general

do not seem to use cGMP for signalling, but recent discoveries suggest that bacteria make

extensive use of a cyclic guanosine compound, cyclic-guanosine–monophosphate (c-di-

GMP) to regulate cell surface associated trials and community behaviours like biofilm

formation in a number of bacterial species such as Yersinia pestis, Pseudomonas aeruginosa,

Salmonella enterica , Vibrio cholera, Gluconacetobacter xylinus (42). It has also previously

been shown that GGDEF and EAL domains are involved in the turnover of cyclic-di-GMP in

pathogenic Salmonella aeruginosa, Pseudomonas aeruginosa and E. coli strains (171). But

nothing is known about the c-di-GMP signalling in Aeromonas spp.

GGDEF and EAL domains protein regulate the cyclic di-GMP levels in

Aeromonas: To elucidate the role of GGDEF and EAL domains in Aeromonas, we

introduced AdrA (GGDEF domain from S. typhimurium) and YhjH (EAL domain from S.

Typhimurium) domains on plasmid carriers to our selected strain. The c-di-GMP

concentration rose to a level of 0.297 nmol mg-1 cells when AdrA was introduced compared

to 0.002 nmol mg-1 cells by vector control strain, and when YhjH was introduced the levels

of c-di-GMP was 0,005 nmol mg-1 cells. Thus our results showed that c-di-GMP level of

Aeromonas are significantly enhanced when the GGDEF domain containing protein AdrA is

expressed. When the EAL domain containing protein YhjH is expressed, c-di-GMP levels

were only slightly enhanced. This phenomenon was also seen in S .typhimurium when YhjH

was expressed. (171).

Phenotypic regulation: GGDEF and/ or EAL domain proteins are known to

regulate the phenotypic trial in different bacterial species such as wrinkled colonies

behaviour for Pseudomonas, radar colonies behaviour for Salmonella, rugose colonies

behaviour for Vibrio (42). In this study, the phenotypic trail was altered when AdrA was

introduced to Aeromonas strains. Figure 11A shows the pink colony of Aeromonas veronii

when AdrA was expressed. Furthermore, when the bacterial colony was stirred with a tooth

pick, it was found that AdrA expressing colonies were highly sticky but neither the YhjH nor

the vector control expressing strain showed such behaviour. A dramatically behaviour

change was observed when swimming motility was tested with the GGDEF and EAL

51

expressing stains. Figure 11B shows that the swimming motility was increased when YhjH

was expressed and the motility was decreased when AdrA was expressed.

AAdrA

B

Dow

nregulated by AdrA

Upr

egul

ated

by

YhJ

H

Vector Y

hjH

Vector Control

Fig 11. A. Phenotyping regulation of Aeromonas when AdrA was expressed; B. Swimming behaviours of Aeromonas was repressed when AdrA was expressed and enhanced when YhjH was expressed.

Adherence on abiotic surface: Biofilm formation on glass test tubes was tested

with the AdrA and YhjH expressing strains. Biofilm formation was found to be increased

when AdrA was expressed and decreased when Yhjh was expressed (Figure 12), as shown

by previous studies on the bacterial species Salmonella typhimurium and Pseudomonas

aeruginosa (171).

CBA

Figure 12. Pattern of adherence of Aeromonas to glass surface; A, Vector control; B, AdrA expressing

strain; YhjH expressing strain.

Adhesion to plant surfaces: c-di-GMP signalling has been suggested to play a

conserved ecological role in bacterial adhesion to plant and animal surfaces with the GGDEF

type regulators (55, 158, 160, 196). In this study, the influence of c-di-GMP on Aeromonas

attachment to duckweed was studied. Figure 13 shows the adherence ability to the duckweed

52

surface. It can easily be seen that there is a considerably higher adherence ability of AdrA

expressing Aeromonas than of the YhjH or vector control strains. Thus our data indicate that

GGDEF type regulatory protein AdrA may have an effect on Aeromonas attachment to the

duckweed surface.

0

10000

20000

30000

40000

AdrA YhjH Vector

CFU

/duc

kwee

d (a

pp. 0

.01g

)

Figure 13 Adherence ability of AdrA, YhjH and Vector control expressing Aeromonas strains to the duckweed surface

Influences on quorum sensing molecules: Quorum sensing is a mechanism for

controlling gene expression in response to an expanding bacterial population. For

identification and analysis of quorum sensing in Aeromonas bacteria i..e, C4 -HSL

bioluminescent sensors have been developed (Figure 14), and are based on the LuxR

regulatory mechanism, which reflects the need to accumulate sufficient pheromones to

trigger the induction of gene expression of LuxR homologues (178, 179)

Biosensor strain

Pheromone producer (AdrA) Vector control

YhJH

Figure 14 Pheromone production of AdrA, YhJH and vector expressing strains against biosensor strain Chromobacterium violaceum CV026 on agar plate

To study the influence of c-di-GMP on quorum sensing molecules, HSL were

extracted from AdrA, YhjH and vector expressing Aeromonas strains. To estimate the

extracted HSL, bioluminescence were measured as relative light units using the detector E.

coli (pSB536) in a microtiter plate counter luminometer. It can easily be seen that AdrA

expressing strains produce considerably higher luminescence that the YhjH and vector

control strains, i.e. they contain higher level of C4-HSL (Table 4). Our results suggest that,

the level of c-di-GMP is related to the level of quorum sensing molecules C4-HSL. Thus c-

di-GMP may influence the quorum sensing of Aeromonas spp.

Table 4. Relative light units were measured as a function of C4-HSL from extracted HSL of AdrA, YhJH and vector expressing strains.

Strains Relative light units

expressed by the sensor

AdrA 31874

YhjH 19486

Vector 18667

Altogether, the AdrA and YhJH expressing strains of Aeromonas were constructed

that contain different levels of c-di-GMP. They showed different morphotypes, motility,

biofilm activities, adhesion abilities to plant surfaces and amounts of quorum sensing

molecules C4-HSL. Thus, c-di-GMP may play an important role in the physiology of

Aeromonas.

53

ABSTRACTS OF INCLUDED PAPERS I. Evidence for transmission between humans and the environment of a

nosocomial strain of Enterococcus faecium. The prevalence of an ampicillin and

ciprofloxacin resistant strain of Enterococcus faecium, named FMSE1, with a characteristic

biochemical fingerprinting pattern was investigated from database comprising 9,676 isolates

of enterococci from 13 different kinds of sources. The FMSE1 type was identified in 1.4% of

all isolates. The FEMS1 type was most commonly found in samples collected from hospital

sewage (50%), followed by surface water (35%), treated urban sewage water (28%),

untreated urban sewage water (17%) and rarely in healthy children (0.8%) and animal origin

(2%). PFGE typing of the isolates showed that they were genetically closely related. This

suggests that a possible transmission route for nosocomial E. faecium from patients in

hospital is through hospital and urban sewage waters and further via treatment plants to

surface water to healthy humans.

II. Identification and Characterization of Pathogenic Aeromonas veronii

Biovar Sobria Associated with Epizootic Ulcerative Syndrome in Fish in

Bangladesh. Epizootic ulcerative syndrome is a fish disease characterized by the presence

of severe, open dermal ulcer on the head, on the middle of the body and on the dorsal regions

of the fish. We identified a pathogenic unique clonal group of Aeromonas veronii biovar

sobria associated with Epizootic Ulcerative Syndrome (EUS) in fish in Bangladesh by the

aid of the Pheneplate biochemical fingerprinting system, fatty acid methyl ester analysis

(FAME) and amplified fragment length polymorphism analysis (AFLP). In this study, the

EUS associated isolates produced haemolysin and cytotoxin, carried the cytolytic enterotoxin

gene, exerted adhesion and invasion ability to fish cell lines and a unique ability to

agglutinate fish erythrocytes. Our results suggest that this clonal group may be considered a

possible causative agent of EUS in fish in Bangladesh.

III. Persistence and Transmission of Aeromonas in a Duckweed

Aquaculture-Based Hospital Sewage Water Recycling Plant. To study the

persistence and transmission of Aeromonas spp. in a duckweed aquaculture-based hospital

sewage water treatment plant in Bangladesh, 1,364 presumptive Aeromonas isolates from a

total of 691 samples were characterised. Aeromonas spp. were found at all sampled sites of

the sewage treatment plant, in 40% of the control environmental samples, in 8.5% of the

faeces from hospitalised children suffering from diarrhoea as well as in 3.5% of the faeces

from healthy control humans. The mean CFU/ml and diversities of Aeromonas species were

found to decrease throughout the purification process indicating that a part of the Aeromonas

population could be eradicated in the sewage treatment system. High numbers of Aeromonas

54

were found associated to the duckweed, which may point to the fact, that aquatic weed may

serve as a reservoir of Aeromonas in the environment. Certain PhP phenotypes from both

duckweed and fish or phenotypes isolated during two subsequent years or from hospitalised

humans suffering from diarrhoea, from different stages of the sewage treatment and from

human consumable fish, were found to be indistinguishable by the AFLP fingerprinting

method, and some of these clonal groups showed virulence properties. Thus, our data

indicate that a few genetically stable Aeromonas clones may have the potential to persist in

the sewage treatment plant for a prolonged period and can possibly be transmitted from the

sewage water to the fish for human consumption via the harvesting of duckweed in the

treatment system.

IV. Evaluation of a scanner assisted colorimetric MIC method for

susceptibility testing of Gram-negative fermentative bacteria. To facilitate the

screening of antibiotic resistance patterns of large numbers of isolates in a short time, we

developed a semi automated colorimetric MIC method, the “ScanMIC” method. The method

used concentration gradients of antibiotics in 96-well microplates, tetrazolium salt as growth

indicator and a flat bed scanner to read the results and an in-house developed software for

image analysis and to calculate and store the MIC values. We compared the “ScanMIC”

method to the NCCLS recommended broth micro dilution method using 197 coliform strains

against seven antibacterial agents and a high agreement (96%) between our “ScanMIC”

method and the NCCLS reference method for MIC determination was obtained. We

conclude that the “ScanMIC” method might become an acceptable and useful method for

epidemiological surveys of resistance patterns of large numbers of coliform bacteria.

V. Influence of a duckweed-aquaculture based hospital sewage water

recycling plant on the antibiotic resistance of Aeromonas spp. as determined by

the ScanMIC method. To understand the influence of duckweed-aquaculture based

hospital sewage water treatment on the resistance patterns of the Aeromonas spp, we

determined the resistance for 288 Aeromonas strains against eight antibacterial agents by the

ScanMIC method as well as with the NCCLS reference method. The ScanMIC method was

again found to be an acceptable (97% agreement) and useful method for susceptibility

determinations in epidemiological surveys on environmental bacteria. The frequencies of

resistant strains decreased during the purification process in the duckweed ponds and even

lower frequencies of resistant bacteria were found attaching to the duckweed used in fish

feed. However, frequencies of resistance were found to increase again in the fish culture

pond. A multiresistant biochemical phenotype was found to persist in all sites of the

treatment plant, and this type was verified by molecular fingerprinting (AFLP) to consist of

one single clone of Aeromonas caviae. Such a clone might spread resistance to the

55

environment, both through the outlet of purified wastewater and through the human

consumption of cultivated fish.

56

ACKNOWLEDGEMENTS

This work has been performed with financial support from Swedish International

Development Agencies (SIDA) Project No 1999-255.

I would like to thank each and everyone who helped me during these years to finish my PhD

works - colleagues, family, and friends. Especially I would like to thank:

My supervisor Roland Möllby, for giving me the freedom and opportunities to carry out my

ideas while still keeping me on track, for teaching me how to write a manuscript, for listening to all

my stupid research ideas, for helping me to write all the project proposals and for giving me a new

name Mokhi (which I did not accept in the beginning !). I have enjoyed this journey with you. I

would also like to thank Gunneli (Roland’s wife) as I have stolen some Roland’s time from her.

My co-supervisor Inger Kuhn, although I still have a problem to writing her name. I must

thank to her for helping me improving the project proposals and manuscripts, and thanks for carefully

listening to all the PhP associated problems.

My unofficial supervisor Ute Römling, for listening to my all ideas, for helping me to

improve my knowledge in molecular biology, for improving my project proposals in the middle of the

night and during weekends and for letting me use her laboratory materials.

The members of Roland Möllby’s group; Patricia Colque (lab mum) for being nice

sometimes !!!, and for encouraging me during my hard times (Mokhi fight fight !!!), Jenny Gabrielson

for helping me with all the computer problem (magic fingers), Aina Iversen for giving me advice

about different things (family, research) and for revising my thesis, Beatrix for being a nice colleague

and helping me revising my thesis, Sara for being a nice colleague, Lena Guldevall for being a lab

partner and all the nice music in the Lab, Maj Ringman for being a nice friend, Magnus for helping

me with all the computer problems. Also thanks to Ruth, Felix, Margarita, Mats (Pling !), Anna Karin,

Hadi Abd, Lena Gezelius, Barbro Olsson-Liljequist and Malin Grape for being to nice with me. Geert

Huys and members of his group for carefully typing the bacteria. The members of Ute’s group: Kader,

Roger, Ulrich, Dinesh, Xiaoda, Devrim, Agaristi for being nice with me and for sharing the lab and

writing space.

Professor Monica Thelestam, Professor Staffan Arvidsson, Professor Gunnar Sandstrom,

Docent Agneta Richter-Dahlfors, Docent Elisabeth Norin, Docent Öjar Melefors and their group

members, MTC administrative people: Eva Pijnenburg, Anna Lögdberg, Leena Norenius, MTC

prefect Mats Wahlgren for helping me with different things in different times.

Especial thanks to Motiur and his lab members, the members of ICDDR,B for laboratory

support and Prism Bangladesh Ltd Staffs and Kumudini Hospital staffs for sampling support.

My family - my parents, my brothers and sisters, my wife Shapla and all my friends for

encouraging me during these years.

57

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