BACTERIAL ANALYSIS OF URINE POLLUTED ENVIRONMENT IN FEDERAL POLYTECHNIC NEKEDE, OWERRI

BACTERIAL ANALYSIS OF URINE POLLUTED ENVIRONMENT IN FEDERAL POLYTECHNIC NEKEDE, OWERRI





                                       CERTIFICATION


This project was dully certified and approved for the award of --------------------- in the Department of Science Technology, -----------------


Mr. ------------------    Date

(Project Supervisor)






    ---------------    Date

(Head of Department)

                                        DEDICATION



This research work is dedicated to the most High God for his knowledge and inspiration towards me and my beloved parents, Chief and Chief Mrs. --------------- for their assistance towards making my dreams come true.

                                    ACKNOWLEDGEMENT


My hearty appreciation first of all goes to the Almighty God for his guidance and sustenance throughout the period of this research work till now.


My gratitude as well goes to my beloved parents, Chief and Chief Mrs. ------------- for their financial support all these years.


A special note of thanks go to my supervisor, Mr. ------------- for his close supervision to make this work a successful one.


My appreciation also goes to my elder sister Mrs. -----------, my beloved ---------------, my friends -------,--------- for their installation during the time of this work.

 ABSTRACT


The bacteriological status of soil environment polluted with urine was analyzed, using standard microbiological methods. 

The identification test of bacteria revealed the isolation of proteus spp. Pseudmonas spp, Escherichia coli, Enterobacteria and Staphylococcus aureus from the contaminated soil. Bacillus spp, Pseudomonas spp and Staphylococcus aureus were obtained from the polluted bathroom and klebsiella spp, Pseudomonas spp, and Escherichia coli were isolated from the cleaned bathroom. Pathogenicity test carried out revealed that while all isolates from the polluted bathroom except E. coli to be pathogenic, only proteus from contaminated soil was found to be pathogenic amongst the isolates from contaminated and uncontaminated soil. Susceptibility tests from contaminated and uncontaminated soil susceptibility test revealed that among the disinfectants used for the study namely, Detol, Izal, Lysol and jik, Lysol was found to be effective against all the test pathogens. The study showed the diversity of pathogens in polluted environment and the efficacy of Lysol in decontaminating the polluted environment that harbour pathogenic organisms, but can be controlled through disinfection.


TABLE OF CONTENT

Title page i

Certification ii

Dedication iii

Acknowledgement iv

Abstract v

Table of content vi


CHAPTER ONE

1.1 Introduction 1

1.2 Aim of project 3


CHAPTER TWO

Literature review 4

MATERIAL AND METHODS

2.1   Sample collection 17

2.2.1  Nutrient agar 18

2.2.2  Macconkey agar 18

2.3.1   Isolation of total hetetrophic bacteria count 19

2.4   Identification of bacteria isolates 20

2.5  Gram reaction 20

2.6  Biochemical characterization of the isolates 21

2.6.2  Oxidase test 21

2.6.3  Indole test 22

2.6.4  Motility test 23

2.6.5   Citrate test 23

2.6.6  Spore stain test 24

2.6.7  Mannitol fermentation (Sugar) 25

2.6.8  Coagulase 25

2.6.9  Lactose fermentation 26

2.6.10  Urease test 26

2.7.1  Pathogenicity test 27

2.7.1.1  Medium preparation 27

2.7.1.2  Method of culture 28

2.7.2  Antimirobial susceptibility testing 28

2.7.2.1  Prparation of antibaceterial disc 29 




CHAPTER THREE

RESULT

3.1 Count of distinct colonies of the isolates 30

3.1.1  Microbial load of contaminated soil sample 30

3.1.2  Bacterial count for sample B 31

3.1.3  Microbial load for sample C (contaminated 

   Bathroom) 31

3.1.4  Microbial load for sample D (cleaned bathroom 32

3.3   Microbiological analysis 33

3.3.1  Result of Pathogenicity Tests 33

3.3.2  Result of Antimicrobial susceptibility 35

CHAPTER FOUR

DISCUSSION, CONCLUSION AND RECOMMENDATION 38

4.1 Discussion 38

4.2 Conclusion 43

4.3 Recommendation 44

References 45

 CHAPTER ONE


1.1   INTRODUCTION

Urine is a liquid waste product from the kidney of both animals and humans. It is collected in the bladder and excreted through the urethra. As a waste liquid product, it contains some dissolved substances such as ammonia, urea, uric acid, and creatinine. These constitute the organic solids in the urine. Urine also contains inorganic dissolved substances such as sodium chloride, calcium, potassium, phosphate and sulfates (Cobire and Wewedo, 2002).


The dissolved substances in the urine can be utilized by microorganisms of various groups as nutrients whenever urine finds its way into the environment. This is evidenced by the fact that urine polluted environments usually have very strong odour, signifying that the biological oxygen demand (BOD) is high. This phenomenon is observe in toilets, bathrooms, street corners and fallow grounds. (Deni and Pennick, 1999).

The different groups of microorganisms can represent different microbial functions and activities. Some can be harmful relating to public health risk, or beneficial relating to positive economic value. Urine leach into ground and surface waters often with much of the nitrogen intact. When microorganisms in lakes and other surface waters consume the nitrogen. It results into a great bloom of growth. When this dies and decomposes, it pulls oxygen from the water or euthrophies, which can suffocate fish and other aquatic life. Underground nitrogen can seep into drinking water, posing a potential health hazard.


Urine contains micro pollutants such as synthetic hormones, pharmaceuticals and their metabolites, that  is mainly excreted via urine (Alder, 2002) and may be harmful to the ecosystems and human health (Daughton and Ternes, 1999). Today, many micro pollutants reach the aquatic environments because their degradation in waste water treatment plant is poor (Barker and Jones, 2005).

More than just dirt hanging around the environment especially urine polluted is unhealthy.

This work plans to asses the level of bacterial building in urine contaminated campus environments and thus suggest  control measures to prevent the invasion of our environment by bacterial pathogens especially the campus female hostel bathrooms which are usually polluted with urine. 

1.2   AIM OF PROJECT

The aim of this work is to isolate bacteria from urine contaminated environments in additions, this work would attempt to find out whether the contaminated environments harbour pathogenic bacteria and thus, pose hazard to health.


 CHAPTER TWO

LITERATURE REVIEW

A number of studies relating to the bacteriological analysis of many environments and the sanitation of such environments like bathrooms have already been executed. Sanitary conditions in public places have always been a major problem, especially bathrooms. Health departments are continually checking the cleanliness and safety of these bacteria breeding grounds to prevent the spread of sickness and disease (Nester et, al, 1995)


In a research experiment conducted in 2000, Barter and Bloomfield studied the presence of Salmonella in domestic bathrooms, it was found that in four out of six bathrooms tested, Salmonella bacteria existed in close proximity to toilet. Despite the fact that Salmonella is usually contacted by consumption of contaminated food, this study shows that the bacteria was commonly spread by air and physical contact in four out of six households. It is difficult to remove the bacteria with common household cleaners. This is because it can get embedded into the biofilm on the underside of the toilet bowl and just below the water level (Barter and Bloomfield, 2000). This experiment was pursued after a report of an attack of Salmonellosis in the home of one of the test bathrooms. In recent studies done in England, it was found that infectious intestinal diseases, such as the Salmonella found in Barker and Bloomfields research, occured in one out of five people each year. Also in reports done by the communicable disease surveillance center, (1999) it was calculated that 136 unreported cases in community caused considerable morbidity.


According to the study by (Daharan, et al, 1999), the use ammonia-based detergent promoted the growth of bacteria colonies.

This is because of the nitrogen contained in these products. In addition, because the detergent being used becomes contaminated, it spreads the bacteria to new surfaces as the individual continues to clean other areas.


The levels of bacteria in cold and hot water were compared in another study by Bloomfield and Scott, (2001). They concluded that cold water contained a higher amount of mesophilic bacteria than other parts of the bathroom. However, the decrease in the hot water temperatures coming from a shower-head and the cooling of the standing water promotes increase in microbial growth.

The potential spread of infection caused by aerosol contamination surfaces after flushing a domestic toilet was studied. It is pointed out that although a single flush deduced the level of microorganisms in the toilet bowl, water when contaminated at concentrations reflecting pathogens shedding, larger numbers of microorganisms on the toilet the bowl surface and in the boil water which were disseminated into the air by further flushes.

Large numbers of bacteria which seeded into household toilets were shown to remain in the bowl after flushing, and even continual flushing could not remove a persistent fraction. This is found to be due to the adsorption of the organisms to the porcelain surfaces of the bowl, with gradual elution occurring after each flush. Droplets produced by flushing toilets were found to harbour bacteria which had been seeded, the detection of bacteria falling out onto surfaces in bathrooms after flushing indicated that they remain airborne long enough to settle on surface throughout the bathroom. Thus, there is a possibility that a person may acquire an infection from an aerosol produced by toilet (Gerba, et al, 1975).


A bacteriological investigation of the effectiveness of cleaning and disinfection procedures for toilet hygiene by Bloomfield and Scott, (1985) showed that the effect of daily disinfection with hypochlorine or a quaternary ammonium product, or with a continuous release of hypochlorite disinfectant system, based on the chlorine-releasing agent tricholorosocyanuric acid, produced some reduction in contamination contaminations compared with daily cleaning, the reductions were less than that associated with the continuous release system and indicated the inadequacy of daily disinfection and / or cleaning of toilets where more effective procedures are required.


Although detergent based cleaning using a typical bowl wash routine without rinsing produced some risk reduction (from 100 to 61.4% of contaminated surfaces). It was insufficient to consistently restore surfaces to a hygienic state. By combing detergent-based cleaning with a rinsing step or with hypochlorine at 500 ppm (or available chlorine) some further reduction in microbial risk was achieved, but was not considered satisfactory for food hygiene purposes. By contrast the risk reduction produced by hypochlorite at 500 ppm was highly significant and was sufficient to reduce the number of contaminated surfaces to 2.9%, (Boln, 1995).


Everyone expects that there will be bacteria growth on bathroom surfaces. However, the magnitude pf bacteria found in bathrooms, used so frequently and by many people that live in a community will be alarming. Even though our immune systems can resist these common bacteria, such high exposure could pose a throat to the health of the users. One common bacterium is streptococcus pyognes and is found in the throats of healthy people. This bacterium can be transmitted by air or saliva. However, it can cause diseases such as pharyngnitis, impetigo, meningitis, and toxic shock syndrome (Geldreich, et al 1976). Staphylococcus, E. Coil and many other normal microfolra can be spread among the students potentially causing major problems.

In a research work to compare the bacteria growth in male and female bathrooms, it was reported that female bathrooms contained more bacteria than the male bathroom. Between the two bathrooms they found seven different types of bacteria including: E.Coil, Streptococcus, Streptococcus, Azobacter, proteus, Candida. In order to keep these bacteria quantities at a safe level the bathrooms must be cleaned daily or even twice a day. This cleaning process will eliminate large quantities or a continual build-up of bacteria. This will make for a healthier, safer, and happier bathroom experience (Davis, et al, 2002). 

The most common soil bacterial are rod-shaped, a microorganism (1/25,000 of an inch) or less in diameter and up to a few microns long (Grey, 1986).


Soil bacteria may be divided into two large groups based on their energy requirements. The two group are:

The heterotrophic bacteria, which obtain their energy and carbon source from complex organic substances.

The autotrophic bacteria, which can obtain their energy from the oxidation of inorganic elements or compounds, obtaining their carbon from carbon dioxide, and their nitrogen and other minerals from inorganic compounds (Alexander, 1977). In the autotrophic group are found such organisms as the nitrate formers, the sulfur oxidizing bacteria, the iron oxidizers, and those that act on hydrogen and its compounds. Most of the soil bacteria require oxygen from the soil air and are classified as aerobes. Some aerobic bacteria can adapt to living where the soil is devoid of oxygen, they are facultative aerobes. Other cannot live in the presence of oxygen and are anaerobes. The soil bacteria also differ considerably in their nutrition and in their response to environmental conditions. Consequently, the kinds and abundance of bacteria depend both on the available nutrients present and on the soil environmental condition.

It has been established that the genetic diversity of soil bacteria is high and that soils contain many bacterial specie or lineage for which no known cultivated isolates are available. Many soil bacteria are referred to as uncultured or even nonculterable. Many of these bacteria are in fact culturable using relatively simple techniques (Jansen, et al 2002).

All natural water contains bacteria. The aerobic gram negative rods of the genera Pseudomonas, Alcalignes and Flavobacterium as well as others are common in water. Water has special unique properties that help make it a necessary part of the environment for many bacteria and contributes to its essential function within the living cell (Nester et al, 1995).


Bacteria in particular form quite an essential part of stream water. Some bacteria such as animal pathogens and other soil species are carried into rives and lakes by run off water. Other species of bacteria grow in nature only in such environments, and are called indigenous population Species of representative bacteria in stream water varies considerably in their relative numbers and kinds from one stream to another depending on the source, kinds, activities and population source and types (Nester et al, 1995).


The presence of some bacteria in stream water invariably are essential for the growth of other organisms, for instance, bacteria produce vitamin (B12) that are essential for the growth of many algae. Most bacteria are Gram negative, motile aerobes or facultative anaerobes while strict anaerobes are found only in aquatic animals and sediments where anaerobic conditions exist (Nester et. Al 1995).

Bacteriology of urine from patients with long term urinary catheters by (Hayson and Sharp, 2005) was reviewed. The study pointed out that bacteria associated with long term urinary catheters (those in place for greater than or equal to 30 days) appears to be the most common source of  nosocomial infection in US. Medical care facilities. The bacteriuria is polymicrobial and dynamic and accompanied by fevers, catheter obstructions, bacterimias, and deaths.

           

PATHOGENIC BACTERIA IN URINE

In a healthy individual the urine is sterile in the bladder. When the transported out of the body, different types of dermal bacteria are picked up and freshly excreted urine normally contains up to 10,000 bacteria per ml. In urinary tract infections, which in more than 50% of cases are caused by E. Coli (Ivanov et. al, 2006), significantly higher amounts of bacteria are excreted. However, these have not been reported to be transmitted to other individuals through environments. Pathogens causing veneral diseases may occasionally be excreted in urine but there is no evidence that their potential survival outside the body would be of health significance (Ivanov et. Al, 2006). The pathogens traditionally known to be excreted in urine are Leptospira interogans, Salmonella typhl, Salmonella paratyphi and Schistosoma harmatobium (Ivanov et. al, 2006).

Table 1:  Pathogens that may be excreted in urine and the diseases they cause.

Bacteria

Diseases 


Salmonella Typhi

Typhoid fever


Salmonella paraty[hl

Paratyphoid fever


Leptospora

Leprospirosis


Yersinia

Yersinisiosis


Escherichia coli

Diarrhea


Schistosoma harmatobium

Schistosomiosis


ORGANIC SUBSTANCES IN URINE

The concentration of organic substances in urine is high, about 10,000COD/M3. On a COD basis, organic acids, creatinine, amino acids and carbohydrates are the main organic urine compounds. Nitrification and autotrophic gentrification of source separated urine by pointed out that on a molar basis, urea, which has no COD, is the most important of the organic substances in urine. Biological degradation of the organic compounds will occur, if urine gets in contact with anaerobic microorganisms which can use the organic compounds as electron acceptors. Examples are sulphate reducers. Fermenters may also growin urine.

Hogland et al (1998) on evaluation of facial contamination and microbial die-off in urine separating systems noticed that bacteria of the fermenting gernus Clostridium is persistent in urine. Methane production would be critical, but is unlikely because of the high ammonia concentration. Not only anaerobic, but also aerobic degradation can occurm if substantial amount of oxygen diffuse through the tank walls (Grillies and Dodds, 1976). The organic substances in urine also include micro pollutants.


EFFECTS OF URINE ON THE CONTAMINTED ENVIRONMENTS

 Urine on a clay loam soil increases soil microbial biomass carbon and nitrogen contents by about 20% but there was no specific effect of urine. Urine however, caused an increased in soil respiration of greater than 50% and the average increase greater for cows urine that for artificial urine.

Urine disposition on grassland causes significant N2O losses, which in some cases may result from increased identification stimulated by labile compounds released from scorched plants. In the soil contaminated with urine, the ammonium is nitrite, releasing two protons. The nutrient balance and content of the urine will reflect what the crops have removed from the fields and thus the average need of fertilization. In other words, urine supplies nutrient to the soil, which serves as a fertilizer in the soil.


 MATERIALS AND METHODS 

2.1  SAMPLE COLLECTION

Urine polluted soil sample was collected from a urine polluted area behind biology lab in the campus, of Federal Polytechnic, Nekede. Unpolluted soil sample was collected from a garden located within the polytechnic premises as the soil samples were transferred into sterile comical flasks and wrapped with aluminum foil.


Samples were also collected from the Polytechnics female hostel bathroom using cotton wool swab. This was done by swabbing the floor of the hostel bathroom after cleaning it with diluted disinfectant (Izal) (2000ml of water and 20ml of Izla) and detergent (Omo).

All the samples were immediately sent to the laboratory for analysis.





   PREPARATION OF MEDIA FOR BACTERIAL ISOLATION

2.2.1   NUTRIENT AGAR

Two point eight grams 92.8g) of nutrient agar was dissolved in 100ml of distilled water shaken to dissolve completely, it was then autoclaved at 121oC for 15 minutes at 15psl and allowed to cool. On cooling, 20 ml was aseptically dispensed into sterile Petri dishes, and allowed to solidify. Nutrient agar is a general purpose medium suitable for cultivation of non-fastidious organisms. In this worth the medium was used for isolation of pure cultures and as slants for preparing isolates.


2.2.2  MACCONKEY AGAR 

This medium was prepared by dissolving 4.85g of powdered macconkey in 100ml of distilled water, shaken to dissolve properly and sterilized at 121oC for 15 minutes at 15psi, it was then poured into Petri dishes and differentiates between the enterabacteriaceae based on the ability to ferment lactose pinte colonies indicted lactose fermentation while colourless and indicates non-lactose fermenters.


2.3.1   ISOLATION OF TOTAL HETROTROPHIC BACTERIA      COUNT

One gram each of the soil samples (contaminated and uncontaminated) was weighed out using weighing balance and aseptically added into 9ml of sterile physiological saline contained in the test tube. Using ten fold serial dilution technique, the soil suspension was serially diluted to 10-5 dilution. Aliquots (0.1ml) of appropriate dilutions were inoculated into nutrient agar and into Macconkey agar plates in triplicate (one served as control, and three were triplicate counts) using the spread plate technique. The plates were inculcated at 370C for 24h.

Ten-fold serial dilutions of the bathroom samples were also made, inoculated into nutrient agar and macconkey agar plates in duplicate (one served as control) using the spread plate technique. The plates were incubated at 370C for 24h.

2.4   IDENTIFICATION OF BACTERIA ISOLATES

This was done by the morphological appearance, Gram reaction and biochemical characteristics of the isolates. For biochemical tests, standard inocula were prepared in stocks used when needed and this was done by aseptically subculturing from the stock culture with freshly prepared nutrient agar and incubated at 370C for 18-24th.


2.5  GRAM REACTION

Discrete colonies which developed after 24th picked with a sterile wire loop onto a clean sterile glass slide, with a loopful of physiological saline added to the slide to make a thin film of smear. The smear was allowed to dry and then heat fixed by passing it thrice over the Bunsen blue flame. The fixed smear was then stained for 60 seconds with crystal violet solution. The stain was washed off by gently running tap water and then flooding with Lugols iodine solution (a mordant) for 60 seconds. The iodine was drained and the slide rinsed in running top water. The stained film was then decolourized with 95% (v/v) ethanol until the entire bolet colour disappeared. The smear was counterstained with safrainn solution for 60 seconds. After the slide washed, blotted dry and observed under oil immersion (x100) objective of the light microscope.


2.6  BIOCHEMICAL CHARACTERIZATION OF THE ISOLATES 

This test demonstrates the presence of catalase, an enzymes that catalyzes the release of oxygen from hydrogen peroxide. The glass slide technique was used.  Drop of hydrogen peroxide was placed on a clean grease from glass slide and smeared with a loopful of isolate collected from a 24h fresh culture. The production of gas bubbles was an indication of a positive test.


2.6.2   OXIDASE TEST

This test was adopted from cheesbrough (2000) to differentiate between bacterial groups through the production of oxidase. A few drops of freshly prepared oxidase reagent (1% solution of tetramethylp-phenylene diamine dihydrocholride) were placed on a piece of filter paper and allowed to dry. A small quantity of growth from a fresh culture was smeared across the filter paper. A positive result was indicated by a purple-blue coloration on the filter paper within 10 seconds.


2.6.3   INDOLE TEST

Testing for indole production is important in the identification of enterobacteria. Most strain of E. coli P, Viligaris, P. Rettgeri, M. Morganii and Providencia species breakdown the amino acid trypiophan with the release of indole.


PROCEDURE

The test organism was cultured in a medium which contains tryptophan. Indole production was detected by kovacs reagent which contained 4-(P) dimethy laminobenzldehdyde. Production red colour indicated positive result while no red colour was negative result.

2.6.4   MOTILITY TEST

The medium sued here was a semi-solid agar medium. The medium was prepared by adding 4.0g of bacteriological agar to 15g of nutrient broth and 1 litre and of deionized water. This was then heated to dissolve the agar and 10ml amounts were dispensed into the test tubes and sterilized. The test tubes were allowed to set in a vertical position. Inoculation was done with a sterile straight inoculation wire making a single stab down the centre of the tubes to about half the depth of the medium. The tubes were then incubated and growth examined after 24h. Diffused growth through the medium from the line of inoculation undirected a positive result.


2.5.6   CITRATE TEST

This test was one of several techniques used to assist in the identification of Enterobacteria. The test is based on the ability of an organism to sue citrate as its only source of carbon and NH3 as its only source of nitrogen.


PROCEDURE

The test organism was cultured in a medium which contains sodium citrate, an ammonium salt with the indicator bromothymol blue (simmons citrate agar).

Growth in the medium was shown by turbidity and a change in colour of the indicator from light green to blue, due to the alkaline reaction following citrate utilization.


2.6.6  SPORE STAIN TEST

Smears from cultures were prepared, ail dried and heat fixed. The slide with smear was placed over beaker of boiling water. A small quantity of malachite green (5% v/v) was dropped to cover the smear on the slide. The sildes were washed with water and counter stained with safranin for 30 seconds. The slides were washed with water, allowed to dry and examined under (x100) objective of the microscope. The spore stained green and the vegetative cells red.



2.6.7  MANNITOL FERMETNATION (SUGAR)

This test is based on the fact that some microbes can ferment mannitol to produce either acid or gas or both. One percent weight per volume of sugar solution was prepared and sterilized by autoclaving for 6mins. One milliliter of mannitol sugar solution was then added to the test tube, containing 9ml of tryptone, water and inoculated with a 100pful of the test organism incubation was for 24- 48hrs at 370C. Tubes were examined to indicate change in colour of the medium from blue to yellowish green. 


2.6.8   COAGULASE 

The test was used to differentiate S. auerus which produces the enzyme coagulase, a drop of saline water was placed on a clean slide with a sterile loop, a trace of undiluted plasma was stirred with the bacteria suspension on the slide coarse clumping within 5-10 seconds indicated a positive result.


2.6.9  LACTOSE FERMENTATION

The ability to utilize lactose was determine by observing the growth of organisms on Macconkey agar medium. The specimens were aseptically plated on both media. Lactose fermenters were pink on Macconkey agar and those of non-lactose fermenters were pale on both media.


2.6.10  UREASE TEST

Testing for urease enzyme activity was important in differentiating enterobacteriaecae, proteus strains are urease producers. V. entrodica also shows arease activity (weakly at 35-370C). shigella and Salmonella do not produce urease.


PROCEDURE

The test organism was cultured in medium which contained urea and the indicator, pheonol red. Production of red-pink colour in the medium indicated a positive urease result while no pink colour indicated negative urease result.

MICROBIOLOGICAL ANALYSIS 

The microbiological assays carried out in this study were pathogenicity test and antimicrobial susceptibility tests.


2.7.1  PATHOGENICITY TEST

This experiment was carried out to determine whether the test organisms are pathogenic to humans. This was done routinely using human serum by determining the degree of virulence (haemolysis) by the test organisms. The medium used was blood agar medium. A control was also provided which did not contain the test organism.


2.7.1.1   MEDIUM PREPARATION

The medium was prepared by placing 9.6g of nutrient agar into a sterile 500ml conical flask 200ml of distilled was water was poured into it and shaken thoroughly to dissolve the agar and to mix well. The medium was sterilized by autoclaving at 1210C for 15min at 15psi on cooling at temperature of 450C, 4ml of serum was added and shaken properly to mix well with the nutrient. The prepared medium was then poured into Petri dishes and allowed to solidity.


2.7.1.2   METHOD OF CULTURE

The test organisms were streaked unto the solidified plate and incubated at the temperature of 370C for 24h. The result was then recorded after 24h incubation.


2.7.2   ANTIMICROBIAL SUSCEPTIBILITY TESTING

(USING DISINFECTANT)

This test assesses the ability of the test organism to be susceptible or resistant to antimicrobial agents. The anitimicrobial agents used were four different types of disinfectants, namely; Detol, Izal, Lysol, and Jik. The test was carried out using agar disc diffusion method (the use of filter paper method). The result of this test was determined by measuring the zones of inhibition of the agents on the test organism.





2.7.2.1   PREPARATION OF ANTIBACERIAL DISC 

Discs for sensitivity test were prepared using whatman filter paper No1 Method of preparation was the same was antibiotic disc preparation in the laboratory as stated by Ogbulie et al (1998).


PROCEDURE

The absorbent whatman filter paper No 1 was perforated with the standard office paper perforator, producing discs of 6mm I diameter. The discs were put in one Petri dish and covered very well. Then, it was sterilized using hot air oven at 1060C for 1 hour.

Serial dilution of each of the disinfectants in this work was made by dissolving 1ml of each of the disinfectants in 9ml of sterile water respectively. Then 1ml of this 100 concentration was transferred into 9ml of sterile water again to obtain 10-1 concentration each the sterile perforated disc measuring 6mm was held with sterile forceps and dippened into the dilutions (10-1 of each of the disinfectants used). The dipped disc was placed on cultured plates containing the test organism. The se sensitivity plates were then incubated at 370C for 24h. The zones of inhibition were measured using meter rule.      



 CHAPTER THREE

RESULTS

3.1   COUNT OF DISTINCT COLONIES OF THE ISOLATES 

The result of the total bacterial load per ml of the samples are shown in the tables below.


3.1.1  MICROBIAL LOAD OF CONTAMINATED SOIL SAMPLE

Table 2 Total bacterial count for sample A (contaminated soil)

Plate

No of colonies on nutrient agar

No of colonies on macconeky agar


10-5

6.0x104

5.2x104


10-4

5.6X105

5.4X105


10-5

5.4x106

5.0x105


Average No of colonies (CFLMI)

5.6x105

5.2x105



Total bacterial count for sample B (uncontaminated soil)


3.1.2   table 3:  Total bacterial count for sample B 

(uncontaminated soil)

Plate

No of colonies on nutrient agar

No of colonies on macconkey agar


10-3

5.4x104

4.8x104


10-4

10-5

4.9x105

4.5x104

4.0x105

4.8x105


Average No of colonies (CFLMI)

4.9x105

4.5x105



3.1.3 Microbial load for sample C (contaminated Bathroom) 

Table 4 Bacterial count for uncleaned bathroom 

Plate

No of colonies on nutrient agar

No of colonies on macconkey agar


10-3

4.7x104

4.8x104


10-4

4.4x105

8.0x105


Average No of colonies (CFLMI)

4.5x105

6.4x105





3.1.4   Microbial load for sample D (Cleaned bathroom)

Table 5 table Bacterial count for cleaned bathroom

Plate

No of colonies on nutrient agar

Not of colonies on macconeky agar


10-3

4.2x104

5.9x104


10-4


4.0x105


3.9x105



Average No of colonies (CFLMI)

4.1x105

4.9x105




  3.3   MICROBIOLOGICAL ANALYSIS 

3.3.1   Result of Pathogenicity Tests 

In the contaminated soil, zones of haemolysis were exhibited by only the proteus spp among the five different bacteria isolated. All the three isolates (Proteus, websialla, and staphylecocus aureas) from polluted bathroom showed under zones of haemolysis than the isolates the contaminated soil in the cleaned bathroom only the websiella spp exhibited zones of haemolysis while in the uncontaminated soil no zone of harmolysis was obtained.

  Table 8 result of pathogenicity tests of the all the isolates 

Sample

Isolates 

Location

Diameter

Result


A

Proteus spp 


Pseudomonas

Contaminated soil Contaminated soil

8mm

+



E. coli

Contaminated soil 

-

-



Enterrobacter

Contaminated soil

-

-



S. aureus

Contaminated soil

-

-


B

Bacillus

Uncontaminated soil

-

-



Pseudomonas

Uncontaminated soil

-

-


C

Proteus


S. Aureus

Polluted bathroom

Polluted bathroom 

15mm


12mm

++


++



D

Websiella

Cleaned bathroom

9mm

+



Pseudomonas

Cleaned bathroom 

9mm

+



E. coli

Cleaned bathroom

-

-


      

Key + = Positive (haemolysis); Negative (no haemolysis) ++ = Positive haemolysis (Highly virulence).


3.3.2   Result of Antimicrobial susceptibility Test

The results of antimicrobial susceptibility test using four different disinfectants (Detol, Izal, Lysol and Jik) on the isolates are as follows:


The isolates from contaminated soil were tested using these disinfectants and it showed that proteus species was susceptible to Detol, Izal, Lysol and Jik resistant to Jik, Lysol having the largest diameter of inhibition zone pseudomonas was resistant to Detol, Izal and Jik but susceptible to Lysol. E. Coli was resistant to all the four disinfectants. Enrerobacter was susceptible to Izal, Lysol, and jik but resistant to Detol while staphylococcus aureas was susceptible to Detol, Izal and Lysol but resistant to Jik.

Proteus species from polluted bathroom was susceptible to Detolm Izal and Lysol but resistant to Jik, S. aureaus was susceptible to Detol, Izal and Lysol but resistant to Jik, Lysol and resistant to Izal and Jik.

Websiella from the cleansed bathroom floor was susceptible to Detol and Lysol and resistant to Izal and Jik. Pseudomonas was resistant to Detol, Izal and Jik but susceptible to Lysol, while E. Coli was resistant to all the four disinfectants used table 9 below shows the results with the diameter measurements (in millimeter) of each of the reagents on the isolates from (contaminated soil, contaminated bathroom and cleansed bathroom). The original diameter of the disc used was 6mm.


 Table 9 Antimicrobial Susceptibility Results (0riginal)

Organism

Site

Diameter

(mm)

On the 

Agents


Proteus spp


Contaminated soil

Detol 

8.0

Izal

7.0

Lysol 

9.0

Jik

-


Pseudomonas spp

Contaminated

-

-

8.0

-


E. coil

Contaminated

-

-

-

-


Enterobacter spp

Contaminated

-

8.0

10.0

7.0


Staph aureus

Contaminated soil

7.0

7.0

10.0

-


Proteus spp


Staph aureus

Polluted bathroom

Polluted bathroom

8.0


7.0

9.0


6.5

9.0


10.0

-


-


Websiella spp

Polluted bathroom

7.0

-

9.0

-


Websiella spp

Cleaned bathroom 

7.0

-

10.0

-


Pseudomonas spp

Cleaned bathroom

-

-

7.0

-


E. coil

Cleaned bathroom

-

-

-

-


   Key: = No zone of inhibition

CHAPTER FOUR

4.1  DISCUSSION

The bacteria isolated from soil contaminated with urine were Proteus spp, Pseudomonas spp, Escherichia coil, Enterobacter, and Staphylococcus aureus. Bacillus spp and pseudomonas spp were isolated from uncontaminated soil. 

In the urine polluted bathroom, proteus spp, Klebsiella spp and Staphyloccus aureus were isolated while in the cleaned bathroom Klebsilla spp, Pseudomonas spp and E. Coil were isolated.

The incidence of a close similarity in the genera of bacteria isolated from the soil contaminated with bathroom contaminated with urine suggests the fact that those organism are indeed associated with urine contaminated environment. Though the biomass of the contaminated soil is higher than the contaminated bathroom analogy indicates that the contaminated soil. This is because soil ordinarily is the home of microorganisms. However, urine availability is an additional nutrient, hence the cluster of these species. The bathroom, being a constructed area specific for human use, is amazing to harbour such a load of pathogenic biomass. Therefore, the urine contaminated bathroom is a threat to health. The bacteria isolated from it were Proteus spp, Klebsiella spp and Staphyloccus aureus. These are well established causative agents of human diseases. Proteus spp could be, Proteus mirabills and Proteus vulgaris. They cause urinary and bloodstream infections. Staphyloccous aureaus cases wound infections, food poisoning, abscesses, toxic shock syndrome and even infect the intestine as well as urinary tract, which is most likely where the Staphylococcus isolated came from Klebsiella spp causes chest and urinary tract infections.


The pathogenicity test showed a positive haemolysis by the Proteus spp isolated from contaminated soil after 24hr of incubation, Proteus spp, Klebsiella spp, isolated from contaminated bathroom also exhibited positive haemolysis on blood agar within the same period of incubation. This proves these organisms are pathogenic on humans. The diameter of the haemolytic zones produced by the isolates from contaminated bathroom was wider than that produced by the Proteus spp from the contaminated soil on blood agar. This suggests that the bacteria isolated from contaminated bathroom have higher degree of virulence than that found in the contaminated soil. The Klebsiella spp and Pseudomonas spp isolated from cleansed bathroom exhibited positive haemolysis on blood agar. This bathroom was cleaned with Izal diluted in water and detergent (omo) and yet pathogenic organisms were isolated from it. This shows the ineffectiveness of that disinfectant (Izal) and suggests that Klebsiella and Pseudomonas are resistant to that disinfection. The antimicrobial susceptibility tests of all the isolates to four different types of disinfectants (Detol, Izal, Lysolm and Jik) were al surveyed.

In the contaminated soil Proteus was susceptible to Detol, Izal Lysol and resistant to Jik, pseudomonas was resistant three disinfectant and susceptible to Lysol, E. coli was resistant to susceptible to three disinfections and staphylococcus aureus was susceptible to three disinfectants and resistant to Jik. From the result, Lysol was showed to have the highest susceptible reaction on the isolates therefore proved to be the best disinfectant for exhibition of bacteria in the contaminated soil.

Result showed that Proteus from polluted bathroom was susceptible to three disinfectants (Detol, Izal and Lysol) and resistant to Jik showing Lysol and Izal to have the highest exhibition zones on the proteus, thereby proving to be better disinfectant on Proteus from urine polluted bathrooms. Staphylococcus aureus showed susceptibility reactions on Detol, Izal and Lysol. Lysol had the highest inhibition zone. 

Klebsiella spp was susceptible on Detol and Detol and Lysol and resistant on Izal and Jik. Lysol had the highest inhibition reaction. This suggests that using Lysol on the urine polluted bathroom can eliminate Klebsiella spp, which was resistant to Izal, Pseudomonas pp was resistant to Detol, Izal and Jik but susceptible to Lysol. This suggests none of the disinfectants used in this study can inhibit the E.Coli strain. These results on the susceptible tests suggest that the organisms that were resistant to some of the disinfectants on them but can be eliminated or inhibited through the use of the disinfectants in which they were susceptible to.

This work therefore proves the use of disinfection in the prevention of infectious diseases. This agrees with the work of Cozad and Jones (2003) which pointed out that one of the means of prevention of diseases is through proper disinfection, even though the presence of pathogenic bacteria cannot be eradiated disinfectants like Lysol has been proved to be good disinfectant by this work. This is in agreement with the works of Davis et al, (2002) which pointed out that disinfectants containing a variety of active ingredients demonstrated efficacy against a broad spectrum of pathogens and interrupted microbial transmission (Lysol being the good disinfectant) and the use of disinfectant results in public health benefit.


4.2   CONCLUSION

This study reveals that the urine contaminated environments harbour pathogenic organisms. It is extremely difficult to create an environment completely hostile to all bacteria in order to prevent infection. Nevertheless, it is important to eliminate as much of the pathogenic bacteria as possible by using careful cleaning procedures. When a certain bacteria evolves into an extremely dangerous form or there is an infection that spreads among students grouped in such close quarters-sharing the same fountains, the same toilets, and the same showers the entire community can be infected.


 

RECOMMENDATION

This work will make people aware of the real possible threat of urinating in bathroom and using public bathrooms and therefore recommended the use of disinfectants in prevention of infections diseases in polluted environment such as bathrooms. Even though the presence of pathogenic bacteria cannot be eradicated but the use of disinfectant like Lysol can inhibit the pathogenic organisms to lower numbers. This work also recommends adequate and thorough cleaning of bathrooms as one of the preventive measures. Even if the presence of harmful bacteria cannot be eradicated, students can take precautions to protect themselves from infections on their own.

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