Identifying the Microbial load in Open Area

Cover page

Identifying the Microbial load in Open Area

ID No. : MBO-04905369

Session: ………….

A Research Report Submitted To The Department of Microbiology,

Stamford University Bangladesh in the partial fulfillment of the

Degree of B.Sc. (Hons.) in Microbiology.

Department of Microbiology

Stamford University Bangladesh

Date, Month, 2011

CONTENTS

Heading Page

ABSTRACT…………………………………………………………………………………….7

INTRODUCTION ……………………………………………………………………….8-42

MATERIALS AND METHODS ……………………………………………………….. 42-44

RESULTS ……………………………………………………………………………………44-49

DISCUSSION …………………………………………………………………………………..49-52

REFERENCES …………………………………………………………………………………53-55

Acknowledgement……………………………………………………………56

Appendices ……………………………………………………………………57

TABLES OF CONTENTS

LIST OF TABLES

ABBREVIATIONS

PCD programmed cell death

CFU colony forming unit

kb kilo base pair(s)

kDa kilo dalton

OD optical density

SDS sodium dodecyl sulphate

PAGE polyacrylamide gel electrophoresis

RT-PCR reverse transcriptase-plymerase chain reaction

VBNC viable but non-culturable

DAPI 4, 6-diaminido-2-phenylindole, dihydrochloride

CTC 5-cyano-2, 3-ditolyl tetrazolium chloride

ROS reactive oxygen species

SOD superoxide dismutase

1.Abstract

To identify the different risks and to establish exposure in surface area, Microbiology of open air

samples from a series of indoor & outdoor environment Like University area, Fish & vegetable Market,

supermarket and Restaurant area should be characterized; and the different microorganisms must be

identified and characterized. This research title, identification and characterization of bacteria air

pathogens present in the air that could constitutes health risk to the inhabitants of these Open

area place. A total of three bacterial pathogens were identified from the five samples collected from five

zones for surface microbial growth count, include of Staphylococcus spp., Escherichia coli, and

Klebsiella spp., had the highest percentage of concern followed by 100% Staphylococcus spp.,

Escherichia coli had 20%, while Klebsiella spp. had 40%.Considering the fact that some of the

isolated pathogenic bacteria are associated with gastrointestinal tract infection(Salmonella spp.,

Shigella spp., Escherichia coli, Bacillus spp., and Staphylococcus spp.) This could be through ingestion

of food or water contaminated by these pathogens and also respiratory tract infection (Pseudomonas

Spp.) constitute a great concern to health practitioners in developing countries because these are

pathogens that are mostly resistant to the commonly available antibiotics used in the treatment

of infection associated with these pathogens.

2. Introduction

2.1 Background

For the last 20 years the open area air quality has becomes a challenging matter (Teresa Sotoet

al., 2009). Over past few years people are giving more attention to the severe health effect, which is

caused by the open area air pollution (Bergen et al., 2005). Because open area air pollution is increasing

regularly (Ayotunde S et al., 2012). According to The United States Environmental Protection Agency

(USEPA), the pollutant level of open area is 100 times higher than indoor area (Badri et al., 2014).

Microorganisms are easily transportable from one area to another area, as a results microbe can infect

any human or animals easily (Fang et al., 2014). In most cases Pathogenic microbes are causing disease

& infections via skin, food, water, breathing and other interpersonal contact (Cordeiro et al., 2007).

Many respiratory disease of human like allergies, asthma, and pathogenic infections of the

respiratory tract are caused by airborne particles; these particles are working as infectious agents

(Bowers et al., 2011). Transportation of this infectious agent happens very swiftly from one host to

another host (Filho et al., 2007). Bangladesh heavily depends on the use of cleaning & disinfectant

bioaerosols to destroy microorganisms; but not only Bangladesh, every developed & under developed

countries are using Fungicides & Bactericides to destroy the pathogenic microbes (Polymenakou et al.,

2012). In last 10 years the fungicide & bactericides importance had been observed due to their harmful

effect on human health (Ayotunde S et al., 2008).

Many studies have suggested that, the widespread contamination of fungi & bacteria depend on

seasonal environment & climatic factors, such as temperature, air humidity, and time of day, wind speed

and direction, presence of human activity, and type of ventilation in closed spaces (Valen et al., 2010).

Additionally the real impact of fungal & bacterial infection may be further understood by the

establishment of a reliable method. This will also detect the fungal & bacterial particles in the open air

(Ayotunde S et al., 2012). Most of these bacteria & fungus are getting shed from human skin surfaces

(Bates et al., 2013). It is not surprising to find hundreds of thousands of bacteria per grams of

dust in floor (Badri et al., 2014). Additionally among the Enteric bacteria, Klebsiella spp., E.coli,

Staphylococcus spp. are commonly found in open area, specially Staphylococcus aureus bacteria in

outdoor area (Eze et al., 2010). In open area environment spores of molds & bacteria are generally

airborne; and gradually become widespread (Pavan et al., 2014). Many microbes are also released by

Human, animals, open dustbin, plant, soil, wastebasket etc. into open area (Yassin et al., 2010). If we

study the negative impact of airborne microbes, then we could take some precautionary step to prevent

our health & economic loss.

Fish & vegetable market, Supermarket and Restaurant area are complex environments that

require ventilation for comfort of public and control of hazardous wastes (Fanget et al., 2014).

Some unique technique like passive sedimentation is highly recommended in research as a

microbiological alert (Badri et al., 2014). Automated techniques are also useful for quantitative analyses

in a research (Ayotunde S et al., 2012). To manage & control environmental health risks, such as air

pathogens, we have to research continuously base on the effect and impacts of air pathogens on

human & animal health.( Lozano et al., 2009). Looking at the high population in Bangladesh, the present

study aims to isolate air-borne and surface bacteria from areas where foods are purchased and

consumed in Dhaka City.

2.2 Literature Review

2.2.1 Airborne Organisms

Earth’s atmosphere is known to team with airborne microorganisms, though the high light

intensities, extreme temperature variations, low concentrations of organic matter, and a scarcity

of water, make the atmosphere as unsuitable environment habitat for microbial growth.

Biological material may contribute about 20%, 22% and 10% to the total airborne particulate by

volume in remote continental, populated continental and remote maritime environments

respectively (MOULI et al., 2005). Most of them originate from natural sources such as soil, lakes,

animals and humans. Moreover, agricultural practices, healthcare units and industrial operations

such as sewage treatment, animal rendering, fermentation processes, and food processing plants

also emit viable microorganisms into the air. Exposure to microbial pollution is now an almost

inescapable feature of urban living throughout the world, which associated with a wide range of

adverse health effects including contagious infectious diseases, acute toxic effects, allergies and

cancer.Inhalation is of the predominant route of exposure resulting in adverse health effects.

Other types of exposure, namely ingestion and skin contact may also be present besides

inhalation. Although the relationship is still poorly defined, increased mortality and morbidity

believed to be caused by urban air microbial pollution are of great concern. Among the

microorganisms present in the atmosphere, bacteria are often the highest in number, despite their

high death rate due to environmental factors producing stress of various kinds, of the major being

dehydration stress. Though, most of the bacteria or bacterial agents are not very potent allergens

with the exception of spore forming actinomycetes; bacterial cell wall components, such as

endotoxin (most prevalent in gram negative bacteria) and peptidoglycens (most prevalent in

grampositive bacteria) are crucial agents with important pro-inflamatory properties that may

induce respiratory symptoms.

It is important to note that geography and climate play an important role indetermining the

outdoor air microbial concentrations because the transport of microbes is primarily governed by

hydrodynamic and kinetic factors, while their fate is dependenton their specific chemical makeup

and the meteorological factors to which they areexposed. The most significant environmental

factors influencing the viability ofmicroorganisms are temperature, relative humidity (RH), and

wind velocity. Also,the additional influences are exerted through oxygen, air ions, solar

irradiance, andopen-air factors. The monitoring of outdoor airborne microorganisms isnecessary

to evaluate the risk on human health and to study its evolution, and theinterest in microbial

characterization has increased over the last few decades. Mostof these studies were carried

through airborne fungi. But up to now there are thelimitations of the data available from

monitoring for the bacteria found in the atmosphere.

Air pollution is one of the most severe of our modern world. This pollution has now reached on

an advance level those poses probablethreat to the health & safety of the population ( Bhatia et

al., 2010).

The increase in population, transportation and industrial development has resulted in the release

of all kinds of pollutants into the environment. These pollutants can cause short-term as well as

long-term effects to the human beings. Air pollutants are grouped into Particulate and Gaseous

pollutants. Particulate matter can be major cause of respiratory allergies and pathogenic

infections of the respiratory tract. Allergic sensitization of the intrathorasic airways (asthma) or

lung parenchyma (hypersensitive pneumonitic) may be a major health problem and this is of

concern in the long term for exposure to in haled aeroallergens(Dutkiewicz.J). Bacterial cells and

cellular fragments, fungal spores and by-products of microbial metabolism, present as particulate

liquid or volatile organic compounds may be components of aeroallergens (Stetzenbach.L.D).

Inhalation of non-infectious microorganisms and constituents can cause inflammation of

respiratory system, while antigens and allergens may activate the immune system and cause

allergic and immunotoxic effects. Particulate matter of bacteria and fungi are mainly non-

infectious, but may exert adverse effects on respiratory tract of the exposed person causing

allergic rhinitis. Diseases like asthma or cystic fibrosis develop a chronic allergic reaction with

cough. The lining bronchi and alveoli become inflamed severely reducing the capacity to inhale.

Fungus like Alternaria, Aspergillus and Cladosporium cause such diseases. Allergic

Bronchopulmonary Aspergillosis (ABPA) mostly caused due to Aspergillus is a life threatening

diseaseImmunotoxic disease caused by inflammation of small air way and the alveoli of the

fungus due to mycotoxins and endotoxins produced by fungus and bacteria. The symptoms of

this disease resemble those of influenza with high temperature shivering, nasal congestion,

irritation of throat, headache and cough. Developed countries have agencies like ACGIH

(American conference of governmental Industrial Hygienists), OSHA (Occupational Safety

Health Administration) which give health alerts periodically. Official statistics regarding

occupational diseases in most of the industries exist in these countries. For an efficient diagnosis

of the allergy and its effective treatment it is very important to know about the prevalence,

seasonal and annual variation of aeroallergens of the area. A study has therefore been undertaken

to evaluate the bacterial and fungal population in the air in some selected area of the Dhaka city

and identify the microbial presence (Reddy et al., 2011).

Presentdaysmany different components are found in atmosphere, which enhance & promote the

survival of microorganisms in the air (Aliabadi et al., 2011). Air pollution by microorganisms is

a growing concern to human health throughout the world. Various alergic & infectious diseases

in man are caused by airborne fungi. The presence of high concentrations of airborne

microorganisms within the indoor & outdoor area increasing concern with many acute disease,

infactions, & alergies. These incidence indicate the degrre of hygine &sanitation of the indoor &

outdoor air environment of different places in our Dhaka city. Also indicate that the degree of

effectiveness in our food chain, in our regular respiratory activity, in perishable food

contamination like fruits , vegetables, & fish contaminaton etc. In our research, Some defferent

area in Dhaka city is detected as the microbial contaminating area. Describing this contaminating

area like Vegetable market, Fish Market, Restaurant, University area, & Supermarket area.

Markets are characterized by high human activity site, responsible for generation of higher

quantity of bioaerosols. Exposure to these bioaerosols often associated with a wide range of

adverse health effects including contagious infectious diseases, acute toxic effects, allergies and

cancer. In the present study, the current atmospheric load of bioaerosols and the influence of

meteorological factors on thesebioaerosols. Gram negative bacilli may present in highercount

among all bacterial groups and Aspergillus sp. prevailed among fungi. Both bacteria and fungi

showedsignificant seasonal variations. Presence of microorganisms in the air is ubiquitous but

their proportion varies according to the environmental conditions and locations. Markets are

characterized as a human activity enriched site and also a highly trafficked site. People in these

areas are actively engaged in various activities, responsible for generation of higher quantity of

bioaerosols. Bioaerosols are airborne particles that are living (bacteria, viruses and fungi) or

originated from living organisms .Spore-forming bacteria and fungi are able to survive in

bioaerosols and stay viable for a long time in the air but the situation becomes worse when they

are able to multiply in these aerosols. It is generally known that bioaerosols present in the air can

affect human health, causing mainly respiratory and related diseases transmitted via respiratory

route, allergic and toxic reactions (Naruka et al., 2014). The presence of different type of

microbes in different market areamay cause many human diseases. The presences of microbes in

market area have been discussed in below;

2.2.2 in vegetable market

The occurrence of pathogens in the internal parts of vegetables is usually associated with

irrigation water or contaminated soil & could pose risk to the air environment of market as the

internalized pathogens are unaffected by external washing. Microbial risk is the most important

form of risk associated with food & recent epidemiological information has shown an internet

outbreak with several foods including vegetables (Donkor et al., 2010).The microbial load of

food measure the extent of food measure the extent of food contamination, the likelihood of the

presence of pathogens & the keeping quality of the food. Microbial contamination of vegetable is

usually associated with the external surface of vegetables. However internalization of microbes

including pathogenic forms in vegetables have been documented (Bernstein et al., 2007).

Microorganisms may enter vegetables through damage to the natural structure, such as punctures

and cuts, which can occur during maturation, harvesting or processing (Donkor et al., 2010). The

occurrence of pathogens in the internal parts of vegetables could pose risk to consumers as the

internalized pathogens are unaffected by external washing. The hygienic safety of vegetables is

threatened by various factors including poor quality irrigation water, such as water could result

in internal 7 external contaminations of vegetables. Pipe water, ground water, surface water, &

human waste water are commonly used for irrigation. Pipe & ground water are generally having

good microbial quality, unless ground water contaminated with surface runoff. Human waste

water is usually having very poor microbial quality & requires extensive treatment before it can

be used safely to irrigate crops.

However it is unclear whether this is limited to external contamination of vegetables from

handling & other sources, or their internal parts could also poses substantial risk to air

environment.

Considering for the nutritional value and health benefits of fresh products like fruits and

vegetables become more and more significantly influence the human diets. Fruits and vegetables

are highly perishable food. Many food contaminating microbes are involved in these food

perishing activities. Microorganisms frequently detected and tend to dominate the bacterial

population in fresh fruits and vegetables include Enterobacter spp. and other coliforms,

Salmonella, Shigella, Bacillus, and Lactobacillus. Enteric pathogens such as Escherichia coli

and Salmonella are among the greatest concerns during food-related outbreaks (Buck et al.,

2003). And recently even an outbreak associated with raw cantaloupes has been linked to

Listeria monocytogenes strain (Hassan et al., 2016). The disease caused by these bacteria is

usually mild and has a short duration; however the frequent of the occurrences of the cases are

quite often. Outbreaks of human infection associated with the consumption of raw fruit and

vegetables often occur in developing countries and have become more frequent in developed

countries over the past decade (Beuchat et al., 2002). Although, the majority of microorganisms

associated with fresh fruits and vegetables are non-pathogenic, the present of these

microorganisms indicates the low quality and microbial safety of the products. Thus, instead of

obtaining the nutritional and health benefits, the health of consumers can be adversely affected

by consumption of microbiologically unsafe fruit and vegetables (Hassan et al., 2016).

2.2.3 in Fish market

Fish and seafood constitute an important food component for a large section of world population

(Wafaa et al., 2011). They come after meat and poultry as staple animal protein foods where fish

forms a cheap source of protein (Wafaa et al., 2011). Sea foods have traditionally being a

popular part of the diet in many parts of the world and in some countries constituted the main

supply of animalprotein. Also Fish & seafood are very swiftly perishable food & may cause

appear new pathogenic microbes in Fish market air. When frozen seafood products are

consumed raw, there is the likelihood of endangering the health of the consumer especially when

the micro-organism present includes pathogenic ones. Bacteria may be found on the skin,

chitinous shell, gills as well as the intestinal tracts of fish or shellfish (ICMSF, 1998). The

microbiological flora in the intestines of sea foods such as finfish, shellfish and cephalopods is

quite different being psychotrophic in nature and to some extent believes to be a reflection of

general contamination in the aquatic environment. In filter feeding bivalve molluscan shellfish

(oyster) and accumulation and concentration of bacteria and viruses from the environment is

generally taking place (Adebayo-Tayo et al., 2012a). However, some sea foods are processed in

a modern fish industry which is technologically advanced and complicated industry in line with

any other sea foods industry and with the same risk of products being contaminated with

pathogenic microorganisms (Adebayo-Tayo et al., 2012a). Several studies have demonstrated

many bacteria species encountered in different fish which are potentially pathogenic under

certain conditions as reported for Pseudomonas angulluseptica and Streptococcus sp. (Emikpe et

al., 2011). It has been estimated that there are more than 80 million cases per annum of sea food

borne illnesses on antibiotic resistance in the United States of America and that the cost of these

illnesses is the order of many billions of dollars per year (Adebayo-Tayo et al., 2012a). a report

by the US National Research Council Committee (FND/NRC) estimated that one-fourth of the

world food supply is lost through microbial activity alone (EEC, 1992) (Adedeji OB et al.,

2012). Many fish contaminating Bacteria can be spread through the air environment. This may

cause many diseases to human. Human infections caused by pathogens transmitted from fish or

the aquatic environment are quite common and depend on the season, patients’ contact with fish

and related environment, dietary habits and the immune system status of the exposed individual.

They are often bacterial species facultatively pathogenic for both fish and human beings and may

be isolated from fish without apparent symptoms of the disease. The infection source may be fish

kept for both for food and as a hobby.

Human infections and intoxications with the following bacteria have been recorded:

Mycobacterium spp., Streptococcus iniae, Photobacterium damselae, Vibrio alginolyticus, V.

vulnificus,V. parahaemolyticus, V. cholerae, Erysipelothrix rhusiopathiae, Escherichia coli,

Aeromonas spp., Salmonella spp., Staphylococcusaureus, Listeria monocytogenes, Clostridium

botulinum, C. perfringens, Campylobacter jejuni, Del�ia acidovorans,Edwardsiella tarda,

Legionella pneumophila, and Plesiomonas shigelloides. Fish tissue histamine intoxications of

people have frequently been described. The purpose of the present paper was to elaborate an

overview of significant bacterial causative agents of human diseases transmitted from fish used

as food or by handling them.Human infections caused by pathogens transmitted from fish or the

aquatic environment are quite common depending on the season, patients’ contact with fish and

related environment, dietary habits and the immune system status of the exposed individual.

They are often bacterial species facultatively pathogenic for both fish and man and may be

isolated from fish without apparent symptoms of disease. The infection source may be fish kept

either for food or as a hobby (Acha and Szyfres, 2003). Thorough anamnesis and microbiological

examination are the prerequisites for correct diagnosis. However, quantification of the

occurrence of these diseases is difficult because many cases, typically gastrointestinal illness, go

unreported; the symptoms usually do not last long and are self-limiting in healthy people

(Novotny et al., 2004).

2.2.4. Supermarket area

Food-borne pathogens take a serious toll on public health. It is estimated in the United States

alone those approximately 14 million incidents of food related illness occur in super market area.

A recognized source for food-borne pathogens is fecal contamination of water used for irrigation,

or for processing, of fresh produce. While many agricultural products are cooked prior to eating,

many Southeast Asian cultures also consume uncooked produce either directly or as fresh

condiments to other dishes, such as soups. Surveys of agricultural produce, meats, and shellfish

have been conducted finding relatively high microbial loads in Southeast Asia indicating that

contamination of water for agriculture and aquaculture, compounded by poor food handling

during distribution, can have a negative impact on public health. Some have also investigated

risk assessment models based on consumption of fresh produce. While these survey studies have

been conducted, actual sampling data is lacking for many developing countries making attempts

to accurately develop risk assessment studies problematic. Some approaches to assess microbial

risk associated with drinking water based on theoretical values for developing countries have

been attempted.

Yet, the additional variables of the transfer of waterbornepathogens to produce through irrigation

and washing of producecan make such theoretical values difficult to calculate.

Other approaches involving direct sampling of produce forpathogens to evaluate microbial risk

assessment have been employed; however, sufficient comprehensive survey dataof produce in

Southeast Asia is lacking.Additionally, studies for the impact of enteric viruses onproduce in the

Asia are also limited. Asmicrobial contaminationof agricultural products is of concern,

additionalsurvey data would be a key to assessing their impact on food safety. A comprehensive

survey for the presence and enumerationof bacterial pathogens, in addition to establishingthe

quantification of somatic bacteriophages as an indicatorfor viral pathogens, would be ideal for

determining microbialcontaminant loads on fresh produce used in salad preparationthrough

culture and molecular methods. This studyinvestigates the prevalence of thermotolerant

Escherichia coli,Salmonella spp. and somatic bacteriophages in fresh produceconsumed

uncooked, namely, bell pepper, cabbage, carrot,lettuce, and tomato, found in both open air

markets andsupermarkets in south Asia.This information would be acornerstone to determining

the risk associatedwithmicrobialcontamination of freshly consumed foods for populations inthe

south Asia and safer vending practices.This study is thefirst in the country to address microbial

contamination offresh produce that may directly or indirectly benefit farmers,consumers,

merchants, vendors, and policy makers towardsfood quality and safety (Vital et al., 2014).

A voluntary recall by one manufacturer in Trinidad in2003 was due to contamination of L.

monocytogenes, butother organisms were detected in finished meat productsat the plant,

including E. coli, Salmonella spp., Campylobacterspp., and unacceptable levels of aerobic

bacteria. Additionally, Hosein et al. 2003 detected L. monocytogenesand E. coli in deli meat

samples (1.4% and2.9%, respectively) collected from local supermarkets onthe island. The

occurrence of pathogenic microorganismsin RTE meats in Trinidad indicates the need for

improvedquality assurance by local producers in order to reduceconsumers’ risks of exposure to

infectious foodborneagents (Syne et al., 2013).

2.2.5 Restaurant area

Restaurant foods can be described as the status of food being ready for immediate consumption

at the point of scale, it could be raw or cooked, and can be consumed without further treatment.

There is an increase in the consumption of ready-to-eat food because of a change in social

patterns, which characterized by increased mobility, large numbers of itinerary workers and less

family centered activities. Thus, good manufacturing practices of foods taken outside the home

such as good sanitation or sanitary measure and proper food handling have been transferred from

individuals/families to the food preparing area, which rarely enforces such practice..

Unfortunately, increasing numbers of local foodborne diseases continue to be implicated with

food service institutions that prepare and sell in Dhaka city food restaurant.Particularly inpost-

cooking areas restaurant area, serves as one of the most important routes for contamination of

meats. The pre cooked meat contaminating agent could be E. coli, Salmonella spp.,

CampylobacterSpp, Listeria monocytogenes.,The microbial load and the presence of the bacterial

pathogens in foods are a good indication of the food quality and the potential health risk they

pose to consumers (Rosmini et al. 2002). Listeria monocytogenes, Escherichia coli O157:H7 and

Salmonella spp. are among the most dangerous food borne bacterial pathogens in terms of

human health and disease (Hoseinet al. 2008).

Need Steps to improve local surveillance of quality and safety of restaurant foods are needed to

help ensure public health. The benefits in cost and convenience derived from restaurant foods

should always be coupled with safety assurance. Street vended and restaurant food can contribute

to food security of those involved in its production, particularly, suppliers of raw produce, food

processors, and consumers.

Food contamination is the introduction or occurrence of a contaminant (any biological or

chemical agent, foreign matter or other substance not intentionally added to food which may

compromise food safety or suitability) in food or a food environment. Food is prone to

contamination at every stage in the food chain, i.e. the various stages from primary production of

food to when it is ready for consumption. The consumption of food contaminated by micro-

organisms will result in food-borne illnesses. These are usually either infectious or toxic in

nature, caused by agents that enter the body through ingestion of food. The main food-borne

pathogens that can contaminate food include, but are not limited to, Salmonella spp,

Staphylococcus aureus, Vibro cholerae or other Vibro spp, Clostridium botulinum, Klebsiella

pneumoniae, Escherichia coli, Pseudomonas spp, while the intestinal parasites include Giardia

lamblia, Entamoeba hystolytica, Ascaris lumbricoides, Trichuris trichiuria and Hookworm spp (

Ankita et al., 2012). Food-borne illnesses are major international health problems and important

causes of reduced economic growth. They not only significantly affect people’s health and

wellbeing, but also have economic consequences for individuals, families, communities, the food

industry and the national economy. Up to one-third of the population of developed countries are

affected by food-borne illnesses each year. The problem is even more widespread in developing

countries, where the poor are most susceptible to illness and death. Food- and water-borne

diarrheal diseases are the leading causes of illness and death in developing countries, killing an

estimated 2.2 million people annually, most of them are children. Although diarrhea is the most

common symptom of food-borne illness, other serious consequences include kidney and liver

failure, brain and neural disorders, and death. Food-borne illnesses have continued to form a

significant part of the morbidity and mortality of every country, and have been on the increase in

recent times. The massive production of restaurants in Dhaka since the beginning of this decade

has resulted in a resultant increase in Restaurant food consumption. The contamination of these

fast foods would lead to a high incidence of food-borne illnesses in the city. Thus, the aim of this

study was to identify factors affecting food contamination and to determine the prevalence of

food contamination in the food available. Food contamination is the results of eating organisms

or toxins in contaminated food. Most cases of food contamination are from common bacteria like

Staphylococcus and/or E. coli. The microbial contamination is a universal problem due to the

presence of microbial propagation in the environment. The different types of microbes like –

thermophilic, thermoduric and pshychrophilic bacteria are present in the environment either in

active condition or in dormant condition. When microbes enter food from soil, water, air, etc.

they may either cause it to spoil or make it dangerous to eat or enhance it to another form i.e. still

acceptable as food. On the basis of spoilage they can be categorized as perishable, semi

perishable and/or non-perishable food.

One can try to keep the contamination to a minimum by cleaning the hand, processing

equipments and utensils. Water and air is used for cleaning and food ingredients. Microbes of air,

water and soil can easily contaminate the processing area, food and processed food. The

possibility of contamination from atmosphere, soil or water is high. Food contamination can

affect one person or it can occur as an outbreak in a group of people who all eat the same

contaminated food. Even though food contamination is common in India, Nepal, Bangladesh,

Pakistan and it affects approx. 60-80 million people worldwide each year and results in

approximately 06-08 million deaths( Ankita et al., 2012). Intake of spoiled or contaminated food

cause food-borne illness and food poisoning to consumers. In developing countries most of the

fatal diseases are due to consumption of contaminated food. We live in a microbial world and

there are several opportunities for food to be contaminated during the processing of foods.

Freshly eaten vegetables constitute an ordinary part of the diet of many people. Food-borne

parasitic infections may be associated with consumption of these contaminated fresh items.

Fruits and vegetables, particularly those eaten raw and without peeling, have been demonstrated

to be the vehicle for transmission of a range of parasites. Improperly washed vegetables

represent a major risk factor and mode of parasite transmission. Contamination could be due to

several factors including the use of untreated wastewater and water contaminated with sewage

for irrigation as well as handling vegetables with contaminated hands. Most people are not aware

of the danger to their health posed by parasites nor do they know from where and how parasites

are acquired.

The food borne illness such as diarrhea, vomiting, fever, sore throat with fever and jaundice has

been reported time to time may be caused by bacterial contamination. An outbreak of food-borne

illness occurs when a group of people consume the same contaminated food. It may be a group

that ate meal together somewhere or it may be a group of people who do not know each other at

all but who all buy and eat the same contaminated food items from food stalls or a restaurant. For

an outbreak to occur something must have happened to contaminate a batch of food that was

eaten by a group of people. A food-borne outbreak is an indication of malfunctioning of food

safety system. The full investigation of such outbreaks requires a team with multiple talents,

including the epidemiologists, microbiologists, food sanitarians, veterinarians and factory

process engineers.

The cross contamination of such foods with pathogenic microorganisms could occur during the

processing of food. The food we eat carries some form of microbial association. Microorganisms

affecting food comes from natural micro flora or are introduced by manufacturing steps ranging

from harvesting, processing storage and distribution. The Salmonella Spp, Pseudomonas Spp,

Staphylococcus Spp etc are food conaminatingbacterium; they are facultative anaerobe growing

better in the presence of air. Their presence in food indicates human contact such as poor

personal hygiene and poor manufacturing practices of the food Restaurant. They produce

enterotoxins that can withstand high temperature, which on ingestion can cause vomiting, and

diarrhea. They also can withstand high sodium chloride concentration [24]. Although death from

Staphylococci food poisoning is rare. It can cause death in small children and the

immunocompromised. The presence of Staphylococcus auerus, a pathogenic organism of public

health concern and significance in these products might have contaminated the stored products

from source because of handling by retailers. Improper handling and improper hygiene might

lead to the contamination of food and this might eventually affects the health of the customers

(khater et al., 2013).

2.2.6 University area

The control of indoor air is related to the quality for internalenvironments. The air of

representative areas within the different of the University area of Dhaka city was analyzed

todetermine the level of microbial types most frequently found in suspension.Samples were

collected by an impaction method and viablecounts (bacteria and fungi) determined both in

absence orpresence of human activities to evaluate the human contribution tothe microbial

contamination. Also, a comparison between indoorand outdoor microbial population densities

showed that the bacterialconcentration was higher inside than in the outside. Most

bacteriaidentified were in different university area in world gram-positive cocci belonging to

Micrococcus,Staphylococcus and Streptococcus species, whereas Cladosporiumwas the

predominant genus isolated among fungi. The microbialconcentration of indoor air was within a

range which indicates alow-intermediate level of contamination according to the

guidelinesestablished in 1993 by the European Community Commission. Although the air does

not represent a true ecosystemor aeroplancton whereby the microorganismscan grow and

reproduce, it does contains microbial forms maintained in suspension coming fromthe soil,

water, plants or animals, including men(Atlas & Bartha 2002). Air movements favor

themaintenance of microorganisms in the aerial mediawhile their deposition is barely affected

bygravity due to their small size.

Factors as temperature,humidity, light and nutrient availability aredeterminants of microbial

survival and abundance.Although pathogenic species are ratherscarce in the air, some relevant

microorganisms travel by aerial transmission and are involved inserious processes causing

pneumonia and otherdiseases. Aerial fungi are much more importantthan bacteria as agents for

allergic diseases. Manyfungal species of Penicillium, Aspergillus, Alternariaand Cladosporium

have been shown to triggerrhinitis, asthma and dermatitis.Respiration by men is not limited to

open atmosphericmedia(Soto et al 2009). The daily intake of air bybreathing in closed

environments greatly outnumbersthe ingestion of other elements like water ornutrients (Cardona

et al., 2003). These and other argumentsjustify the recent concern about the

microbiologicalquality of the indoor air. The term indoorair usually applies to the air of non-

industrialinterior environments, like colleges, hospitals, offices,restaurants, homes and similar

partiallyclosed settings. The indoor air quality (IAQ)began to be taken into consideration during

thepast 60´s when the problems on biological contaminationcame into the focus of attention

forearlier environmental researchers. Since air is an important vehicle for thedissemination of

infectious agents and allergiccomponents developing potential undesirable effectson human

beings, the control of the microbialcharge became an important key to define theenvironmental

quality of ambient media surroundingwide human populations which are largely exposedto

indoor air during their daily activities.The IAQ of a particular building depends onseveral

parameters, such as the quality of the outdoorair, the design of both the ventilation and

airconditioning systems (including their operativeand maintenance conditions), the

compartments inwhich are divided, as well as the endogenoussources of contamination and their

magnitude(Vargas & Gallego et al., 2005). The most frequent defectsin quality derive from

inadequate ventilation,pollution generated inside the building and contaminationfrom exogenous

origin. When morethan 20% of the occupants of a building complainabout the quality of the air

or show clear symptoms of drowsiness, fatigue, nausea, cough,asthma or related respiratory

disorders, the phenomenonis known as the “sick building syndrome”.In general, predominant

bacteria in indoor air are gram positive and usually not dangerousfor human health but, because

they often derivefrom the skin and the respiratory tract of occupants,high viable counts are used

as markers ofcrowded conditions and poor ventilation.Recently, we published data on the

microbiological quality of the outdoor air in open urban areasof the city of Murcia, Spain (Soto

et al. 2009). Inthis work we complement those results by studyingthe microbial charge of the

indoor air in a universityinstitution of the same city.

The activity of people and equipment within the indoor environments is thought to be the

principal factor contributing to the buildup and spread of airborne microbial contamination.

Particular activities like talking, sneezing, coughing, walking and washing can generate airborne

biological particulate matter. Food stuffs, house plants and flower pots, house dust, textiles,

carpets, wood material and furniture stuffing, occasionally release various fungal spores into the

air. Moreover, the environmental factors mainly include temperature, humidity, air exchange

rate, air movement, building structures and location, poor design, ventilation system as well as

interior or redesign which enhance microorganism’s growth and multiplication in the indoor

atmosphere. A review made by WHO on the number of epidemiological studies showed that,

there is sufficient evidence for an association between indoor dampness-related factors and a

wide range of effects on respiratory health, including asthma development, asthma exacerbation,

current asthma, respiratory infections, upper respiratory tract symptoms, cough, and wheeze.

Thus microbiological air quality is an important principle that must be taken into account when

indoor workplaces are designed to provide a safe environment. This study provides information

on the current concentration of microorganisms and describes bacterial and fungal loads for

different libraries of Stamford University Bangladesh. Moreover, coexistence of bacteria and

fungi were established to see the impact of environmental factors on their multiplication and

growth in the indoor air of the libraries.

The microbial density was analyzedin several areas in the presence and absenceof users to assess

for the contamination introducedby the human activity and to determinethe microbiological

status of this environmentwhich had not been studied previously in Bangladesh.

2.2.7 Transmission of airborne pathogens

An airborne particle that is living or originate from living organisms, is one of the most

important indoor air contaminants, and they contribute to about 5–34% of indoor air pollution

(Srikanth et al., 2008). Potential health risk of exposure to airborne bacteria can occur in Market

places and university area at any time, especially to those residents like patients, infants and old

people who are physiologically sensitive to them (Fanget al., 2014). It was reported that children

on a per-bodyweight basis tent to inhale relatively more air than adults and elderly persons were

more likely to have weak body defense systems, and people with compromised immunity (e.g.,

pregnant women, post-operative patients) or with existing respiratory conditions, such as

allergies and asthma are at increased risk of exposure to airborne bacteria and their

derivatives(Bunger et al., 2000; Nasir et al., 2012).

The South Asian region has a high burden of infectious disease. These diseases form a leading

cause of morbidity and mortality and are responsible for 42% of all disability-adjusted daily’s

life in the region. Some infectious diseases seem to be influenced more by the social

environment than others. Leprosy and tuberculosis are examples of diseases in which the role of

social determinants is well recognized. Factious diseases, like tuberculosis and leprosy

transmitted from person to person, both diseases are caused by Mycobacteria that use an airborne

route by small droplets as main mode of transmission (Feenstra et al., 2013).

Exposure to airborne pathogens is a common incidence of all human life. With the improvement

of research methods for studying airborne pathogens has come evidence indicating that

microorganisms (e.g., viruses, bacteria, and fungal spores) from an infectious source may

separate over very great distances by air currents and ultimately be inhaled, ingested, or come

into contact with individuals who had no contact with the infectious source. Airborne pathogens

present a unique challenge in infectious disease and infection control, for a small percentage of

infectious individuals appear to be responsible for distribution of the majority of infectious

particles(Fernstrom et al., 2012).

In this paper begins by reviewing the microbial elements of atmosphere and physical

contamination that allow infectious particles to be transmitted via airborne and droplet means.

Building on the basics of air environment, we then explore the common origins of droplet and

airborne infections, as these are factors critical to understanding the epidemiology of diverse

airborne pathogens. We then discuss several environmental considerations that in degree of the

airborne transmission of disease, for these greatly impact particular environments in which

airborne pathogens are commonly believed to be problematic. Finally, we discuss airborne

pathogens in the environment of several specific examples: at Mogbazar Restaurant, Dhanmondi

Star Kebab, Mouchak Restaurant, Mogbazar Wireless Fish & Vegetable Market, Mailbag Rail

Gate Fish & Vegetable Market, Agora Supermarket in Mogbazar & Stamford University

Bangladesh, Siddeshwari Campus; during November and December 2015. Surface organisms

table (3) was collected from the location of Mogbazar Wireless Fish Market, Mogbazar Wireless

Vegetable Market, Mogbazar Restaurant, Agora Supermarket in Mogbazar, & Stamford

University Bangladesh area. Susceptible individual’s respiratory tract, eyes, mouth, nasal

passages, and so forth. In contrast, airborne transmission is defined as the transmission of

infection by expelled particles that are comparatively smaller in size and thus can remain

suspended in air for long periods of time. Airborne particles are particularly troublesome simply

because they can remain hanging in the air for unlimited periods of time. Seminal studies from

the 1930s and 1940s demonstrated that airborne particles can remain airborne for as long as one

week after initial aerosolization, and suggested further that these particles likely remained

airborne for much longer. They thus potentially expose a much higher number of susceptible

individuals at a much greater distance from the source of infection. Depending on environmental

factors (e.g., meteorological conditions outdoors and fluid dynamic effects and pressure

differentials indoors), airborne particles are easily measured 20 m from their source. These

factors would be of no concern but for the fact that airborne bacterial, viral, and fungal particles

are often infectious(Fernstrom et al., 2012).

Thee aerosolized transmission of disease occurs through both “droplet” and “airborne” means.

Droplet transmission is defined as the transmission of diseases by expelled particles that are likely

to settle to a surface quickly, typically within three feet of the source. Thus, for example, in order

for an infection to be caused by droplet transmission, a susceptible individual must be close

enough to the source of the infection (e.g., an infected individual) in order for the droplet

(containing the infectious microorganism) to make contact with the In recent years, opportunistic

fungal diseases have been characterized by their increased incidence, as well as the diversity

of Bacteria & fungi isolated and infections greater severity(Fernstrom et al., 2012)..

City-level airborne epidemics are constant threats to healthy living. With the fast growth of the

world’s population and the constant increase in human mobility, the danger of outbreaks of

epidemics is rising. For example, the pandemic influenza A (H1N1), also known as Human Swine

Influenza/Swine Flu, caused an international outbreak in Mexico in 2009, and it caused a serious

epidemic in China. Indeed, China is highly susceptible to pandemic influenza A (H1N1) due to the

large population and high residential density. According to the report by the Ministry of Health of

China, the provinces in mainland China had reported 19,589 confirmed cases, 14,348 cured cases,

10 severe cases and several deaths up to 30th September 2009 (Ministry of Health of China, 2009).

In urban areas with high density such as public transportation and transfer points, where people

frequently experience close proximity to one another, we observe a striking increase in the

transmission of airborne viruses and related pathogens. To correctly model and simulate airborne

epidemics, it is critical that the city infrastructure, which causes these hot spots of transmission, be

analyzed and captured in detail. We utilized Geographic Information Systems (GIS) to model the

infrastructure of a city that is likely to be threatened by epidemic attacks. GIS facilitates storing,

querying and visualizing city infrastructure including roads, regions with diverse functionality,

public transportation, and other attributes. To model airborne disease spread, it is important to

understand how city infrastructure is used by the inhabitants and acts as the container of infection.

We addressed path routing based on city transportation to capture mobility of people and

transmissions that occur in localities, especially public transit. This approach was used because in

many developing countries such as China, the overly crowded public transportation system can

greatly exacerbate airborne epidemics. Based on geo-spatial information, it is essential to model a

local population that dwells in a city with their spatio-temporal behavior. ‘‘There is growing

recognition that the solutions to the most vexing public health problems are likely to be those that

embrace the behavioral and social sciences as key players’’ (Mabry et al., 2008). ‘‘Human

behavior plays an important role in the spread of infectious diseases, and understanding the

influence of behavior on the spread of diseases can be key to improving control efforts’’ (Funk et

al., 2010). Obtaining a strong understanding of the ‘transmission highways’ in urban areas

regarding the transmission locations and relevant behavior of people is vital to predict and prevent

the spread of infectious diseases. Therefore, investigating the patterns that are relevant to social

contacts, and resulting airborne virus transmissions, is a great importance.

In this study, we exclusively considered 5 types of regions – Fish Market, Vegetable Market,

University, Restaurant, and Supermarket according to the general function assignment of city

regions.

A region contains a set of sub locations of different classes. For example, a university region (UR)

contains office sub locations (offices), residential sub locations (student dormitories and faculty

members’ homes), classroom sub locations (classrooms, labs and library space), recreation sub

locations (cafeteria, clubs, shops, refectories and restaurants) and possibly Supermarket sub

locations.

Specifically, storage section, shelf section, Managing office room, CCTV control section, meat,

vegetable, cloth & cosmetics section etc. In this study, we classified sub locations as Wall area,

Floor, Table, ceiling, storage section, Fish or Meat containing Bowl or Pate etc. Sub locations are

virtually created with 2 dimensions inside each region. Because sub locations are usually beyond

the resolution of GIS files. Accordingly work places, people interact with only those who stay

inside the same sub location, although visually sub locations can overlap. Additionally, each sub

location is characterized as being either indoor or outdoor, conveying different transmission

probabilities of bacteria, fungi &viruses inside the space. For many airborne bacteria, fungi &

viruses, outdoor conditions such as sunshine, heat, wind blowing and air circulation can lower the

infection probability between the infected and the susceptible. Although room ventilation and other

environmental factors are known to be important in the airborne transmission of Mycobacterium

tuberculosis, little attention has been given to these factors in the transmission of B. anthracis.

The spread of infectious disease is of global concern for social and economic reasons. For

example, seasonal influenza kills 200–500 thousand people annually. In 2009-2010, influenza A

(H1N1) caused 17,000 deaths worldwide, many among whom were healthy adults. In 2002-2003,

severe acute respiratory syndrome (SARS) killed more than 700 people and spread into 37

countries causing a cost of $18 billion in Asia. These recent outbreaks remind us of the potential

for a pandemic such as the Spanish flu of 1918–1920 which killed 50–100 million people.

Diseases can spread wherever people have direct or indirect contact, but this paper focuses on

infections that occur in health care facilities, because they often contain a large proportion of

infectious or vulnerable people, and because governments and other health care providers have a

clear responsibility to mitigate infections that occur within their walls.

Human-human transmission of disease can result from direct contact with an infected person or an

indirect contact through an intermediate object. A direct contact infection could be caused by

caregivers not washing hands prior to attending patients. Another common direct contact

transmission is due to large infectious aerosols that travel a short distance from the source to the

receptor. An important mode of indirect contact is airborne transmission occurring via the spread

of fine aerosols, skin flakes, and fungal spores in room air over long distances and time scales.

Aerosols can be generated and released by human expiratory actions (speech, coughing, and

sneezing), skin shedding, or resuspension from surfaces.

To Advances in Preventive Medicine Aerosol disease transmission is known to be the main route

for many diseases such as Tuberculosis and Aspergillosis. Also, recent research has shown that the

importance of aerosol infection is underrated for common diseases such as influenza, especially

during cold and dry seasons. For example, modern experimental techniques have detected

infectious aerosols produced by infected patients while breathing, coughing, or sneezing. As far as

building ventilation design is concerned, the greatest impact of any ventilation design would be on

the airborne route of infection, which is the focus of this paper.Infection control involves blocking

any stage of the infection pathway (Aliabadi et al., 2011). For airborne transmission, this can mean

reducing the generation of pathogens from an infectious person, using disinfection techniques to

kill pathogens released to the air, or simply isolating infectious people in rooms (Mei et al., 2013).

Although room ventilation and other environmental factors are known to be important in the

airborne transmission of Mycobacterium tuberculosis(Fennellyet al., 2004).

Small particles that can become airborne are typically generated by coughing, sneezing, shouting,

and to a lesser extent by singing and talking. Even breathing may generate such particles in sick

and highly infectious individuals. Particle size distributions of coughed materials are thought to

encompass a broad range of diameters, from very small to large droplets, depending on differences

in patients and disease. Fennelly et al. (2004) measured cough aerosol emanating directly from

tuberculosis patients. The patients generated infectious aerosol that contained from three to four

colony forming units (CFU, a direct measure, using culturing techniques, of the number of viable,

growing, and infectious organisms) to a maximum of 633 CFU. The size distributions that were

measured in this study suggest that most of the viable particles in the cough-generated aerosols

were immediately respirable, ranging from 0.65 to 3.3 μm. Wainwright et al. (2009) also measured

cough aerosols from cystic fibrosis patients and documented that 70% of viable cough aerosols

containing Pseudomonas aeruginosa and other Gram-negative bacteria were of particles 3.3 μm.

Positive room air samples were associated with high total counts in cough aerosols. There are not,

however, enough data to fully describe or predict cough particle size distributions2 for many

diseases, and research is needed to better characterize them (Xie et al. 2009). In the 1950s, the

relationship among particle size, airborne suspension, and transmission implications began to

become clear. The different routes require different control strategies, which have evolved over

many years of infectious disease practice, Small particles that can become airborne are typically

generated by coughing, sneezing, shouting, and to a lesser extent by singing and talking. Even

breathing may generate such particles in sick and highly infectious individuals (Bischoff 2013).

Particle size distributions of coughed materials are thought to encompass a broad range of

diameters, from very small to large droplets, depending on differences in patients and diseases

(Riley and Nardell 1989). Fennelly et al. (2004) measured cough aerosol emanating directly from

tuberculosis patients. The patients generated infectious aerosol that contained from three to four

colonyforming units (CFU, a direct measure, using culturing techniques, of the number of viable,

growing, and infectious organisms) to a maximum of 633 CFU. The size distributions that

weremeasured in this study suggest that most of the viable particles in the cough-generated

aerosolswere immediately respirable, ranging from 0.65 to 3.3 μm. Wainwright et al. (2009)

alsomeasured cough aerosols from cystic fibrosis patients and documented that 70% of

viablecough aerosols containing Pseudomonas aeruginosa and other Gram negative bacteria

wereof particles3.3 μm. Positive room air samples were associated with high total counts in

coughaerosols.There are not, however, enough data to fully describe or predict cough particle size

distributions2for many diseases, and research is needed to better characterize them (Xie et

al.2009). In the 1950s, the relationship among particle size, airborne suspension, and transmission

implicationsbegan to become clear. The different routes require different control strategies, which

haveevolved over many years of infectious disease practice,

Methicillin-resistantStaphylococcus aureus&Clostridium difficilecan survive on surfaces or skin

scales for up to80 days. can be transmitted in aerosol from the respiratory tract but commonly

attaches to skin scales of various sizes. The distance of travel depends on the size of the scale,

the larger falling to the floor within 1–2 m, the smaller travelling the entire length of the one

contribution of 10 to a Theme Supplement ‘Airborne transmission of disease in hospitals ward.

Establishing colonization depends not only on the number of organisms but also the site of

inoculation, e.g. an open wound or mucous membrane could generate colonization with under 10

organisms compared with several hundred on intact skin. Methicillin-resistant Staphylococcus

aureus disseminates widely throughout the ward and is commonlyfound in dusty, inaccessible

high surfaces. Clostridiumdifficile spores are thought to spread in the air andcan be found near a

patient carrying the organism. The key steps discussed here understand the sources of pathogens

in the air, the effect of environmental factors on their survivability and the potential for

expressing infection. Pathogens in the air are spread on particles or droplets. The solid matter

may come from skin, while the droplets may be generated from the upper or lower respiratory

tract, mouth, nose. The physical mechanism of the generation of droplets and particles carrying

pathogens is largely unknown (Eames et al., 2009).

3. Methods

3.1 Location Of Sampling:

Air sampling was carried out at Mogbazar Restaurant, Dhanmondi Star Kebab, Mouchak Restaurant,

Mogbazar Wireless Fish & Vegetable Market, Mailbag Rail Gate Fish & Vegetable Market, Agora

Supermarket in Mogbazar & Stamford University Bangladesh, Siddeshwari Campus; during November

and December 2015. Surface organisms table (3) was collected from the location of Mogbazar Wireless

Fish Market, Mogbazar Wireless Vegetable Market, Mogbazar Restaurant, Agora Supermarket in

Mogbazar, & Stamford University Bangladesh area. The RCS air sample collecting table (1) locations

were Mouchak Restaurant, Mailbag Rail Gate Fish & Vegetable Market, Agora Supermarket in Mogbazar

& Stamford University Bangladesh, Siddeshwari Campus area. The table (2) falling microorganisms were

collected from Dhanmondi Star Kebab, Mogbazar Wireless Fish Market, Mogbazar Wireless Vegetable

Market, Agora Supermarket in Mogbazar, & Stamford University Bangladesh Siddeshwari Campus area.

3.2 Measuring airborne bacteria & fungus:

Reuter Centrifugal Sampler (RCS) standard microbial air sampler was used to measure airborne bacteria.

The RCS machine has air flow rate 40 litter per minute. Airflow measurement range from 20 litters to

320 litters. RCS have motor speed 4096 RPM. Air samples were collected using two type of agar strip,

one of which is Nutrient Agar (NA) and another Sabouraud Dextrose Agar (SDA). The RCS machine was

held for two minutes to collect air sample from the sampling locations. Sample collected on the agar

strip was incubated at 37º C for 24 hours for bacteria (NA) & for 48 hours at 25º C for Fungus (SDA). The

resulting colonies were counted using the following formula;

CFU/L= Colonies on Agar Strip/ 40 × Sampling Time (minutes).

3.3 Measuring falling microorganisms with Petri dish:

Falling organisms in the environment of Bangladesh was measured with Petri dish. Mainly two types of

agar medias were used in this experiment (1) Nutrient Agar (NA) media for bacterial growth count & (2)

Sabouraud Dextrose Agar (SDA) media for fungal growth. The Petri dish was kept open for 30 minutes in

the sampling locations, to collect microorganisms into Agar Media. They were places at three different

locations at each sampling area, to gather an average count of organisms present. After collecting the

air samples, the media plates were incubated for 24 hours at 37º C for bacterial growth (NA) & 48 hours

at 25º C for fungal growth (SDA). The resulting colonies were counted.

3.4 Measuring surface Microorganisms of Open area in Bangladesh:

In this experiment some different type of agar media was used for measuring the normal microbial

growth count & pathogenic microbial growth on the surface area. Such as Nutrient Agar (NA) media

used for bacterial growth count, Sabouraud Dextrose Agar (SDA) media used for fungal growth count,

Mannitol Salt Agar (MSA) media used for Staphylococcus spp. growth count, McConkey Agar (MAC)

media used for E.Coli & Klebsiella spp., & S-S agar media used for Salmonella spp. & Shigella spp growth

count. Some Glass Test tube with normal saline was used to collect surface sample with cotton swab.

This experiment was performed to detect how many microorganisms are present in the surface of

Restaurants, Supermarkets, Fish & Vegetable Markets and University Area. The Eosin Methylene Blue

(EMB) media was performed to confirming the presence of E.coli & Klebsiella spp.

4. Results

4.1 Airborne Bacteria

Table 1 shows all outdoor area including University Area, Vegetable Market & Fish Market area had

higher number of bacterial & fungal colonies presence. In other hand the indoor area such as Restaurant

& Supermarket area had lower number of bacterial & fungal colonies presence. The University Area had

31 bacterial & 84 fungal colonies present; Vegetable Market area had 63 bacterial & 51 fungal colonies

present; Fish Market area had 44 bacterial & 28 fungal colonies present; Restaurant area had 20

bacterial & 37 fungal colonies present & the Supermarket area had 19 bacterial & 25 fungal colonies

present. Table 1 also shows the cfu/l count of airborne bacteria. The cfu/l results for all areas were

0.3875 cfu/l in Nutrient Agar media (NA) & 1.05 cfu/l in Sabouraud Dextrose Agar (SDA) for University

Area; 0.7875 cfu/l in Nutrient Agar media (NA) & 0.6375cfu/l in Sabouraud Dextrose Agar (SDA) for

Vegetable Market area; 0.55 cfu/l in Nutrient Agar media (NA) & 0.35 cfu/l in Sabouraud Dextrose Agar

(SDA) for Fish Market area; 0.25 cfu/l in Nutrient Agar media (NA) & 0.463 cfu/l in Sabouraud Dextrose

Agar (SDA) for Restaurant area; & 0.2375 cfu/l in Nutrient Agar media (NA) & 0.3125cfu/l in Sabouraud

Dextrose Agar (SDA) for Supermarket area

Table 1: Airborne Bacteria

4.2 Falling Organisms

This table is formed to count falling organisms in different places with NA media & SDA media, which

was collected in 30 munities time. As seen in Table 2 Vegetable Market & Fish Market area had higher

number of falling bacterial & fungal colonies present. In other areas like University area, Restaurant &

Supermarket area had lower number of falling bacterial & fungal colonies presence. Such as in

University Area had 43 bacterial & 11 fungal colonies presence; in Vegetable Market area had 172

bacterial & 29 fungal colonies presence; in Fish Market area had 288 bacterial & 47 fungal colonies

Location Count in colonies cfu/l

NA SDA NA SDA

University Area 31 Colonies 84 Colonies 0.3875 1.05

Restaurant 20 Colonies 37 Colonies 0.25 0.463

Vegetable Market 63 Colonies 51 Colonies 0.7875 0.6375

Fish Market 44 Colonies 28 Colonies 0.55 0.35

Supermarket 19 Colonies 25 Colonies 0.2375 0.3125

presence; in Restaurant area had 13 bacterial & 27 fungal colonies presence & the Supermarket area

had 15 bacterial & 6 fungal colonies presence .

Table 2: Falling Organisms

Air sample Location Microbes count in Nutrient agar Microbes count in Saboured Dextrose Agar

University Area 43 Colonies

11 Colonies

Restaurant 13 Colonies

27 Colonies

Vegetable Market 172 Colonies

29 Colonies

Fish Market 288 Colonies

47 Colonies

Supermarket 15 Colonies

6 Colonies

4.3 Surface Organisms

The surface organism’s growth result of different area was recorded on Table 3. five different media

plate was used to count microbial growth for different surface sample collecting area. Such as, for the

University Area result was 2×103 cfu/ml bacterial colonies in NA media, 1.5×103 cfu/ml fungal colonies in

SDA media, 7×102 cfu/ml golden yellow color Staphylococcus aureus in MSA media, 9×102 cfu/ml gummy

pink color Klebsiella spp in MAC media. The Vegetable Market area result was 2.7×103 cfu/ml bacterial

colonies in NA media, 1.33×104 cfu/ml fungal colonies in SDA media, 1.28×104 cfu/ml golden yellow

color Staphylococcus aureus in MSA media, 1.8×103 cfu/ml gummy pink color Klebsiella spp in MAC

media. The Fish Market area result was 2.90×104 cfu/ml bacterial colonies in NA media, 2.4×103 cfu/ml

fungal colonies in SDA media, 8.9×103 cfu/ml golden yellow color Staphylococcus aureus in MSA media,

4×102 cfu/ml dry pink color Escherichia coli spp in MAC media; The Restaurant area result was 2×102

cfu/ml bacterial colonies in NA media, 2.7×103 cfu/ml fungal colonies in SDA media, 9.4×103 cfu/ml

golden yellow color Staphylococcus aureus in MSA media, & no growth in MAC media. And the

Supermarket area result was 1.6×103 cfu/ml bacterial colonies in NA media, 4×102 cfu/ml fungal colonies

in SDA media, 8×102 cfu/ml golden yellow color Staphylococcus aureus in MSA media, & no growth in

MAC media.

Table 3: Presence of pathogens in surface samples

Surface sample Location

Nutrient agar Saboured Dextrose Agar

Samonella-Shigela Agar

Manitol Salt Agar

MacConkey Agar

University Area 2×103 1.5×103 No growth 7×102 9×102

Restaurant 2×102 2.7×103 No growth 9.4×103 No growth

Vegetable Market 2.7×103 1.33×104 No growth 1.28×104 1.8×103

Fish Market 2.90×104 2.4×103 No growth 8.9×103 4×102

Supermarket 1.6×103 4×102 No growth 8×102 No growth

Table 4 we can see that the NA agar media of University area, Vegetable Market area, Fish Market area

& Supermarket area shows higher bacterial growth, except Restaurant area showing medium growth.

The SDA agar media of University area, Vegetable Market area, Fish Market area & Restaurant area

exhibited higher fungal growth, also supermarket exhibited medium fungal growth. The S-S agar media

shows no growth for all five sampling areas. The MSA agar media of University area, Vegetable Market

area, Fish Market area & Restaurant area having higher Staphylococcus aureus spp bacterial growth, But

Supermarket area having medium Staphylococcus aureus spp bacterial growth. The MAC agar media of

University area & Vegetable Market area shows higher coliform bacterial growth, and the Fish Market

area shows medium coliform growth. But The Restaurant & Supermarket area shows no coliform

bacteria growth in MAC agar media.

Table 4: Degree of contamination of surface samples

Surface sample Location

Nutrient agar Saboured Dextrose Agar

Samonella-Shigela Agar

Manitol Salt Agar MacConkey Agar

University Area +++ +++ - +++ +++

Restaurant ++ +++ - +++ -

Vegetable Market +++ +++ - +++ +++

Fish Market +++ +++ - +++ ++

Supermarket +++ ++ - ++ -

(+++) high growth

(++) medium growth

(+) low growth

(-) no growth

The table (5) result is also taken from taken from table (3). The percentage of isolated pathogens was

described in this result table (5).

The percentage of specific pathogens present in surface was calculating basis on five areas is

demonstrated in Table 5. As our calculation we have found 100% Staphylococcus aureus spp, Escherichia

coli spp 20%, Klebsiella spp 40%.

Table 5: Percentage of specific pathogens present in surface.

Isolated organisms Agar media percentage

Staphylococcus aureus MSA 100%

klebsiella sp MAC 40%

E.Coli sp MAC 20%

5. Discussion:

Air quality of the University area, Fish Market, & Vegetable Market area contains higher bacterial load

than the Supermarket area, & Restaurant area. And also the Supermarket area, fish market &

Restaurant area having less fungal growth than the University area, & Vegetable Market area. As a result

we can see that the University area & Vegetable Market poses more contamination points than the

Supermarket area, fish market & Restaurant area. Falling organisms count in air environment of

University area, Supermarket area, Vegetable Market area, & Fish Market area were containing higher

bacterial growth than fungal growth. But only the Restaurant area was containing higher fungal colonies

than bacteria. As a result, we can see that the Fish market area & Vegetable Market area poses more

contamination points than the Supermarket area, University area & Restaurant area.

Observations of contamination of surface samples revealed, the microbial presence in the University,

Vegetable Market, Fish Market & Supermarket to be numerous, on the contrary Restaurant surface area

exhibited medium growth. The fungal presence at the University, Vegetable Market, Fish Market &

Restaurant have high growth, but Supermarket have medium fungal growth. Staphylococcus spp. was

predominant in all areas. After observing the two result tables of surface microbial growth count we can

state that, these five surface sampling areas of Dhaka city are not a safe zone for public.

From surface sample Percentage Table (5) we can see that, In MAC media of Vegetable Market &

University area we have found Klebsiella spp 40%. In MAC media of Fish Market area we have found

Escherichia coli spp 20%, In MSA media of all five areas we have found commonly Staphylococcus

aureus 100% . Here Escherichia coli spp & Staphylococcus aureus microbes are gastrointestinal

pathogens. these bacteria pathogens is the causative agent of bacillary dysentery in most developing

countries today, which could be fatal in children if not diagnose and treated on time.

The Fish & Vegetable Market, Supermarket & Restaurant area should maintain the clean & hygiene rules

very strictly. Cause many fecal contamination & food contamination agents could cause disease through

this area. As we know that fish, vegetable, & meats are swiftly perishable food; As a result many

pathogenic microbes could arise from these perishable food items. And also we know that, the

restaurant area is the food serving area & public gathering place; As a result many foods contaminating

agent could contaminating the food from the unhealthy environment of Restaurant.

When compare to the results of Badri et al., (2014) we can see that, the NA & SDA Agar media of

bacterial & fungal count table (1) & (2) shows similar results with Badri et al., (2014) results. In this

(Badri et al., (2014)) research journal was written about indoor microbial count like microbial

count on inside of some homes in Bagdad city. If we compare with Badri et al., (2014) research result

with our research result, then we have found that the Badri et al., (2014) research table shows; NA agar

media for bacterial count having high growth in maximum indoor area, the MAC agar media coliform

bacteria count shows low growth in maximum indoor area, but S-S agar media for the salmonella sp &

shigella sp growth count was shows negative result. In other hand if we follow our research result table

(1) & (2), then we can see that NA agar for all five palaces shows high growth in maximum indoor &

outdoor area, but the MAC agar media for Coliform bacteria count shows high growth result in

maximum area than Badri et al., (2014) research result table. But both Badri et al., (2014) & our research

result shows negative growth of salmonella spp & shigella spp on S-S agar media.

We could had more specific microbial count if we completed our further research with more areas of

Dhaka city like the Hospital area, Bank & office area, food factory area, cloth industries area etc. We

didn t found much bacterial & fungal growth count from RCS sampling areas.

Because we had collected microbial sample by RCS air sampler which was set in 2 munities time. If we

could gather sample with more times, then we could had more microbial count from these air sampling

area. And also we didn t found the count of pathogenic microbes in air sampling areas. Because we

didn t performed our experiment with MAC, MSA, & S-S agar media. We had only bacterial & fungal

growth count from air sampling areas. We have to do further more experiment to identify the specific

pathogenic microbes from air environment of different places in Dhaka city.

In addition, the bacteria isolated were usually of human and soil origins. The bacteria that come from

human are mainly from the skin and respiratory tract, and were released by occupants into the sites

through shedding off from skin, sneezing or talking; whereas the soil bacteria were usually dispersed in

the air by dust. Therefore, the bacteria isolated from the sampling sites were contributed by human and

the outdoor environment. Many pathogenic known & Unknown, common & uncommon bacteria which

is causing regular disease in our regular life. Determining the presence of organisms in air and surface of

food purchase and consumption are gathering knowledge which can be used to take some

precautionary steps to prevent this harmful microorganism from our open environment & surface. If we

can t prevent these microbes at least we could save ourselves from harmful disease causing microbes.

By gathering knowledge about presence of microorganisms in our environment we could take some

important steps to acquire a healthy & peaceful life.

Conclusion:

From the result of this research conducted, it is observed that E.coli, klebsiella spp, & Staphylococcus aureus

had the highest occurrences in each of the sampling zones. This is true, looking at the fact that these

bacterial pathogens are commonly associated with gastrointestinal infections as a result of eating food

contaminated by these pathogens. These bacteria pathogens is the causative agent of bacillary dysentery in

most developing countries today, which could be fatal in children if not diagnose and treated on time. Also

looking at the fact that some airborne bacterial pathogens are associated with respiratory tract infection

that if left untreated could be life threatening both in children and adult as reported. Klebsiella sp one of the

respiratory tract pathogens. from Open area of Dhaka constitute a great concern to the health of the

inhabitants residing in Dhaka because these bacterial pathogens could be life threatening both in children

and adult if not diagnose on time and appropriate antibiotic administered to treat these infections

associated with these pathogens. Also considering the fact that proper ventilation system should be

provided when constructing Market, Supermarket, Restaurants to permit the in-flow and out-flow of air

inside the Building to minimized not only the concentration of pathogenic bacteria also chemical substances

such as carbondioxide which could also have an adverse effect on the inhabitants of Dhaka city. Finally, this

research focus on the isolation of bacterial pathogens alone, but there is the need to carried out further

research with the view to find out the possibilities of isolating fungal pathogens which could be a good

source of air pathogens and also to evaluate these microbes (bacteria and fungi) against some of the

commonly used antimicrobial agents used in the treatment of infections associated with these pathogens.

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Acknowledgement

Appendices