CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION

Water is very important to life. It is the most important and abundant compound in an

ecosystem (Patil et al,2012). It is vital for all life form. Water covers 70% of the earth surface,

mostly ocean and sea. Water is continuous movement above and below the surface of the earth

(USGS, 2019). Water plays an important role in the world economy. Approximately 70% of the

freshwater used by humans goes to agriculture (Baroni L. et al, 2007). Water is important in

many geological processes.

Water quality varies from place to place. Water can be categorized according to the

source such as rain, ground and surface.

Water pollution involves contamination of water by external substances. The existence of

pollutants system can cause serious environment issues and pore threat to the general public

hygiene and health (Ranjit et al,2014).

Water pollution can be caused by chemical compounds which is a key environmental

problem (Bernd et al, 2009). It can also be caused by over population of and environment. Water

pollution can also be caused by microorganism known as microbial pollution. This include

coliform bacteria which are indicator organisms mostly used in bacteria characterization (Jimoh

et al, 2013). Microbial pollutant in many waters supply is the cause of major diseases such as,

typhoid, urinary tract infection, endocarditis.

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 Wastewater can be defined as the liquid end-product of anthropogenic usage of water

(Darvishi and Farahani, 2011). The discharge of wastewater directly to receiving aquatic body

has deleterious effect on aquatic life and the ecosystem as a whole. The component of

wastewater include about 99% of liquid part and 1% of dissolved matter which includes nutrients

(Nitrogenous compounds, phosphorus compounds and heavy metals), dissolved solids and

numerous microorganisms (Mateo-Sagasta et al., 2015). Microorganisms present in waste water

are diverse and covers viruses, fungi, protozoans and different bacterial species.

Hospitals have an important role in the well-being of mankind and other medical research

advancements. Different units/services of hospitals require large volume of water according to

the activities taking place within the hospitals and generates large amount of wastewater (Boillot

et al., 2008). Quantity as well characteristics of hospital wastewater (HWW) is affected by size

(number and type of wards/units), and services provided (kitchen, laundry, and air conditioning),

management policies and awareness of the institution (Kumar et al., 2007). A hospital in

developed country generates 400–1200 L wastewater per bed per day whereas for developing

countries the value is 200–400 L/capita/day as compared to 100–400 L/capita/day of domestic

wastewater generation (Mishra et al., 2016).

In general, characteristics of wastewater generated from hospitals are similar to the

domestic wastewater, but a proportion of the HWW contains toxic/nonbiodegradable/infectious

pollutants (Iweriebor et al., 2014). The hospital effluents contain a large variety of substances

used for medical, laboratories, research purposes, and also include excreta from patients.

Hospital wastewaters are highly complex effluents acting as a hotspot for antibiotic resistant

bacteria. Especially, Gram-negative bacteria bearing multiple antibiotic resistant genes are

increasingly found in hospital wastewaters.

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 Enterococcus spp. are Gram-positive, non-spore-forming organisms that belong to a

group of organisms known as lactic acid bacteria with some species producing a protein called

bacteriocin. They are commensal members of the normal intestinal flora of humans and animals

and female genitourinary tract without causing any infection; enterococci are commonly found in

humans and animal faces, and faecal enterococci e.g., E. faecalis, E. faecium, E. durans and E.

hirae are used as indicators of sewage contamination (Verlicch et al., 2014).

The presence of antibiotic resistant bacteria in waster has been a major threat to public

health, as the antibiotics resistant determinants can be transferred to other bacteria of high human

clinical significance if ingested. Antibiotic resistance is known to impact treatment efficiency of

enterococcus infections negatively.

Hence, this study tends to evaluate the antibiotic susceptibility of enterococcus spp in the

hospital waste water.

1.1.1 Aims and Objectives

The aim of this study is to isolate, identify, characterize and determine the antimicrobial

susceptibility of the enterococcus sp in hospital effluent.

The specific objective of the research are as follows:

i. To determine the physiochemical property of the effluent

ii. To Isolate and identify enterococcus sp from the hospital

iii. To determine the antimicrobial susceptibility of the isolate from the hospital effluent.

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1.2 Literature Review

1.2.1 Water

Water is very essential to life. The access to a good water is very necessary. Most

diseases are caused by bad water quality. Two and a half billion people have no access to

improved sanitation and more than 1.5million children die from water related disease. Most of

which are intestinal disease

Waterborne disease remains important source of morbidity and mortality in the world and

this most common in developing country. Wastewater causes harm to humans. This water

contaminations can be caused by pouring dirt into local water, faeces into water bodies releasing

causing endocarditis caused by enterococcus faecalis.

Around 780 million people do not have access to clean and safe water and around 2.5

billion people do not have proper sanitation worldwide (WHO, 2015). Also, as a result of this,

humans are prone to death due to water related diseases and disasters. Therefore, water quality

control is a top-priority policy agenda in many parts of the world (WHO, 2011)

Water pollution seems to be an inevitable consequence of urbanization and

industrialization, and water resources are overburdened because of contamination from sewage

and pathogenic agents, industrial and trade wastes, agricultural pollutants (e.g fertilizers,

pesticides etc), and thermal pollution. Waterborne diseases have major public health and socio-

economic effects (Pandove et al., 2011).

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 1.2.2 Hospital Wastewater

Hospital is a place where people are treated. It is necessary to monitor the wastewater

released from the areas. Hospital waste water can contain so many hazardous `substances such as

pharmaceutical residues, chemicals, pathogens and radioisotopes. Due to these substances,

hospital wastewater can represent a chemical, biological, and physical risk for public and

environmental health (Fekadu et al, 2015).

Nevertheless, very frequently there are no legal requirements for hospital effluent prior to

its discharge into the municipal collector or directly onto surface water after pretreatment.

Hospital wastewater includes macro and micro pollutant of wide concentration range

from laboratories, research unit, operation unit, where medicine and nutrition solution are

prepared and polyclinic (Verlichhi et al,2010).

Hospital originated wastewater discharged to city sewage system in many countries,

treated together with domestic waste water, and discharged to receiving environments (Gautam

et al,2007). Studies have suggested that even the pretreatment of hospital waste water prior to

discharge to domestic wastewater sewage system for treatment might not be a sufficient solution

due to the micro pollutant content of hospital wastewater (Vieno et al, 2007).

Hospital effluents contain more of multidrug resistant bacteria, posing significant public

health threat through dissemination to the downstream water bodies (Hemen et al,2019).

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1.2.3 Nosocomial Infection

They are also referred to as healthcare associated infections (HAI), are infections

acquired during the process of receiving health care that was not present during the time of

admission. They may occur in different areas of health care delivery such as in hospitals, long

term care facilities and may also appear after discharge. HAIs also include occupational

infections that may affect staff.

Infections occurs when pathogens spread to a susceptible patient host. In modern health

care, invasive procedures and surgery, indwelling medical devices, and prosthetic devices are

associated with this infection.

Pathogens responsible for nosocomial infections include bacteria, viruses and fungi. The

prevalence of infections caused by particular microorganisms varies depending on the healthcare

facility locations. Opportunistic bacterial infections occur when there is a breakdown of the host

immune system functions such as staphylococcus sp, streptococcus sp, enterococcus sp.

Hospital waste waster is potentially infectious and sewer of some healthcare facilities are

not watertight thereby allowing wastewater to leak into groundwater. Improper hospital waste

water management, collections and treatment can lead to contamination of local drinking water

sources and contamination of natural resources (Odiyo et al,2016).

HAI is the most common adverse event in health care that affects patient safety and even

health care workers. They contribute to significant morbidity, mortality and financial burden on

patient and health care system.

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 The emergence of multi drug resistance organisms is another complication seen in the

HAI.

1.2.4 Enterococcus sp

Enterococci are gram-positive, opportunistic bacteria. They widely present in the fecal

contaminants of food and as starters for production of cheese and fermented sausages

Enterococci are very important commensals members of the intestine of microbiota of

human and animals. Enterococci may be opportunistic pathogens and are frequently associated

with nosocomial infections (klein,2003). As a result of the high prevalence of acquired antibiotic

resistance, enterococci are recognized as important active spreading agents of this type of

resistance both intra and at interspecific levels (Klare et al., 2003). Despite the ubiquitous

character of enterococci, their distribution in wastewaters and their fate during water treatment

have been poorly characterized. This strongly limits our understanding of their role as indicators

of fecal pollution, of the ecology of pathogenic stain or of their involvement in antibiotic

resistance transmission.

One of the major attributes of enterococcus species is its propensity to Acquire and

disseminate antimicrobial resistance determinant, enterococci have become resistant to a wide

range of antibiotics and resistance to penicillin, clindamycin, cephalosporins, amino glycosides

and others are due to the various intrinsic traits they express while the resistance to the likes of

chloramphenicol, erythromycin, tetracycline, fluoroquinolones, vancomycin and others are

examples of acquired resistance

1.2.4.1 Epidemiology

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 A large number of studies on enterococcal ecology and epidemiology have been

conducted over the past two decades, especially in clinical settings (Arias and Murray, 2012).

Non-healthcare-associated investigation shows that enterococci are common place colonizers

over wide swaths of the planet (Byappanahalli, Nevers, Korajkic and Harwood, 2012).

The origins of enterococcus spp vary from environmental to animal and human sources.

As enterococci are an essential part of microflora of other animals and humans, their distribution

is very similar in these sources (Klein, 2003).

Studies of the ecology and epidemiology of Enterococcus have reported E. faecalis and

E. faecium are being regularly isolated from food such as pork, fish (Foulquie Moreno et al.,

2006). In one study in the UK, samples taken from urban sewage were found to be 100%

positive for enterococcus spp (Kuhn et al., 2003).

1.2.4.2 Pathogenicity

For enterococci to cause disease several barriers must first be overcome. An initial barrier is the

ability to overcome colonization resistance provided by competing microbes, and host defenses

such as gastric acid and bile, and colonize the intestinal tract. From this reservoir the bacteria can

amplify in number and spread to sites vulnerable to infection. A basic prediction from such a

model is that the probability of infection should be a function of the intestinal burden of bacteria

in the gut reservoir- the more bacteria, the greater the probability of contamination of a potential

infection site in numbers large enough to overcome host defenses. Indeed, colonization of the GI

tract has shown to be directly associated with risk of infection (Taur et al., 2012). Infection

occurs when enterococci overwhelm host defenses, replicate at rates that exceed clearance, and

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when pathologic changes result through direct toxin activity, or indirectly by bystander damage

from the inflammatory response (Garsin et al., 2014)

1.2.5 Antimicrobial Resistance

The antibiotic resistance of Enterococcus is well documented. Bacteria may slow

resistance to glycopepetides such as vancomycin and teicoplanin, which are licensed in the UK,

and to aminoglycosides (Kacmaz & Aksoy, 2005). Antibiotic resistance has been of growing

concern for a number of years, Vancomycin was first used in the clinical arena in 1972 and the

first vancomycin-resistant enterococci were recognized only 15years later. NNIS reported an

increase of 7.6% in VRE between 1989 and 1993 (Metan et al., 2005). It has been reported that if

glycopeptide-resistant enterococci (GRE) are present in an infected patient rather than an

antibiotic-susceptible strain, clinical treatment failure is increased by 20% and mortality is

increased from 27% to 52% (Brown et al., 2006). When assessing the studies that is emerging is

the possible occurrence of multidrug resistant strains (Peter et al., 2003).

In both the Surveillance and Control of Pathogens of Epidemiological Importance

(SCOPE) and SENTRY (Antimicrobial Resistance Surveillance Program) data-bases, figures

show that, of enterococcal isolates from the bloodstream, 2% of E. faecalis and 60% of E.

faecium isolates are resistant to vancomycin (Bearman & Wenzel, 2005). Resistance rates of

Enterococcus species have reached endemic or epidemic proportions in North America, with

Europe having lower, but increasing levels (Mutnick et al., 2003). Enterococcal antibiotic

resistance is not exclusive to the clinical arena but is also prevalent in the food industry. The

presence of VRE in individuals who have been hospitalized, when they have not previously been

in hospital or taken antibiotics, suggests that VRE may have been contracted through food chain.

9
GRA may emerge in the good chain through use of avoparcin in animal feed (Mannu et al.,

2003).

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Glass wares

There are a lot of materials needed to carry out various experiment in the course of this

study. This includes conical flask, glass thermometer, measuring cylinder, beaker, test tubes,

2.1.2 Reagent

Reagents used are ethanol, manganate sulphate solution, alkali-iodide-azide, starch

solution, sulfuric solution, sodium hydroxide, sodium thiosulphate, hypo chloride solution,

oxidase reagent, crystal violet, safranin, grams iodine, hydrogen peroxide.

2.1.3 Equipment and Materials

This includes electric cooker, incubator, refrigerator, measuring cylinder, autoclave,

slides, microscope, stock bottle, cotton wool, weighing scale, spatula, antibiotic disk, test tubes,

thermometer, TDS meter, pH meter, transport media, Universal bottle, colorimeter.

2.1.4 Culture Media

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 Culture media area also known as growth media, are specific mixture of nutrients and

other substances that support the growth of microorganism such as bacteria and fungi. There are

different type of culture media such as selective media, enrichment media,

2.2 METHODS

2.2.1 Media Preparation and Sterilization of Materials

All the prepared media are prepared carefully according to the manufacturer’s instruction

and are autoclaved at 121oc for 15min to make it sterile and left to cool. They are also poured

aseptically into the petri dish to avoid contamination. Sampling bottles are soaked overnight with

10% hypo chloric acid and are rinsed thoroughly with sterile distill water.

2.2.2 Study Area

Samples use in the study were collected from the Obafemi Awolowo University Teaching

Hospital Complex (OAUTHC) wastewater effluent.

2.3 Sampling

2.3.1 Sampling Collection

Samples are carefully and safely collected from the hospital effluent of the selected

hospital (Obafemi Awolowo Teaching Hospital Complex), Ile-Ife, Osun State. The samples are

collected inside a 1litre container which is labelled appropriately. The wastewater from the

11
effluent is used to rinse the container twice. The cap of the container is removed in such a way

that the contamination of both surfaces of the bottle with hands are avoided. The sample is

collected in the opposite flow of water.

The samples were collected by dipping the bottle into the wastewater with a gloved hand

and the sampling bottles were filled and ample air was left in the bottle to mixing before

examination. Samples are also collected into sterile universal bottles. This is transported in a iced

packed container for microbiological analysis (Tanu et al, 2017). All the three samples were

collected at the outlet point of the treatment plant between March and June, 2021.

2.4 Physiochemical Analysis

2.4.1 On-site Analysis

2.4.1.1 pH

pH the wastewater is measure by the pH meter by first tarring the meter to zero and then

dipping it into the wastewater and taking the readings. This is done repeatedly for three times to

get an accurate reading. The meter is then cleaned up after usage. This is to check the acidity or

basicity of the wastewater. This is used to indicate the concentration of hydrogen ions in the

solutions

2.4.1.2 Temperature

It is the manifestation of thermal energy. Temperature is a measure of a quality of a state of

materials. The temperature of the wastewater is measured using a thermometer which is dipped

into the water and the reading is taken. The reading is taking repeatedly for accurate reading.

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2.4.1.3 Turbidity

Total dissolve solvent is a measure of the dissolved combined content of all organic and

in organic substance present in a liquid in molecular and other forms. It is used to measure the

water turbidity. TDS concentration are also reported in part per million water TDS concentration

can be determined using the digital meters. TDS of waste water is measured by inserting the

TDS meter into the waste water and the reading is taken, it is measured repeatedly for accurate

reason

2.4.2 Off-site Analysis

2.4.2.1 Dissolved Oxygen

Winkler method was used to measure DO content in waste water. A 250ml of glass

reagent bottle was filled to the brim with the sample of waste water.2ml of magnate sulphate was

added to the sample using a syringe which is inserted below the surface in order to prevent

oxygen from being reproduced. 2ml of alkaline potassium iodide was added in the same manner

and the bottle was corked in other to prevent air from coming in. the sample is mixed by

inverting the bottle several times and checking for air bubbles.

If oxygen is present a brownish orange color or floc will appear. When the floc settles

down the sample is inverted again several times and the floc is allowed to settle again. Pasteur

pipette is used to drop 2ml of hydrogen concentrated sulfuric acid above the sample water. the

bottle is then corked and inverted several times to dissolved the floc. the sample is then stored for

8 hours after which it is titrated with sodium thiosulphate to a pale straw color. Few drops of

starch solution are added to the sample and a dark blue color is formed and the titration is done

13
by slowly dropping the titrant into the sample until the sample color turns clear. The

concentration of the DO in the sample is equivalent to the number of milliliters of the titrant used

2.4.2.2 Biochemical Oxygen Demand

The procedure for analyzing BOD is the same as that of DO above except with a

difference. At the site, the sample is collected in a brown amber bottle and it is stored in a dark

room for five days then after which it follows the same steps used for measuring dissolved

oxygen. The BOD is expressed in milligram by liter.

2.5 Microbiological Analysis

2.5.1 Enrichment

1 millimeter of the waste water from the hospital effluent is added into 9-millimeter of

the tryptone soy ager broth and it is incubated for 24 hours.

2.5.2 Serial Dilution

After the incubation of the broth, a whitish cloudy precipitate is observed. A series of test

tube containing 9 mils of distilled sterile water is labelled accordingly and a sterile 1 mil pipette

is used to aseptically transfer 1 mil of the enriched sample to test tube 1 and then mixed together

thoroughly to give a dilution of one in ten (10-1). Then a new pipette is used to transfer 1 mil of

the sample from test tube 1 to test tube 2 giving a dilution of one in hundred. This process was

repeated until the desired dilution was reached and the 1 mil is pipetted out of the last test tube

and then discarded.

2.5.3 Isolation of enterococcus sp from hospital effluents

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 The waste water which was enriched and has been serial diluted and highly cut of 10 -3

was picked with a 0.1 mil micro pipette. It is then inoculated and spread on the enterococcus

agar and incubated for 24 hours.

2.6 Characterization And Identification of Bacteria Isolate

2.6.1 Macroscopic morphology

The bacteria isolate is examined for shape, pigmentation, opacity, elevation, surface,

edge, consistency and the surface appearance of the colonies.

2.6.2 Microscopic morphology

Gram staining techniques is used for the identification of the bacteria. This divide the

bacteria into 2 groups namely; gram positive and gram-negative bacteria. A smear of 18-24

hours isolate is prepared on a grease free labelled slide which is air dried after which it is heat

fixed then few drops of crystal violet is added to the smear for 40-60 seconds and then rinsed off

with a low running water then the iodine (mordant) is added left for 40 seconds and then rinsed.

Then a few drops of ethanol decolorizer) is added and rinsed off almost immediately then a few

drops of safranin (secondary stain) is added and left for 60 seconds before it is rinsed off and

then air dried. A drop of oil immersion is added to the smear and examined under objective

lens(x100). gram positive bacteria appear purple while gram negative appears red of pink (Thairu

et al,2014).

2.7 Biochemical test

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2.7.1. Oxidase Test

An 18-24 hours old culture of deisolated oxygen was inoculated unto the oxidase paper.

Then a positive result will give purple color on the oxidase paper and a negative result will show

no color. (Swati et al, 2014).

2.7.2 Catalase Test

A smear is made with an 18-24hrs old culture on a clean grease free slide. then a 3%

hydrogen peroxide is added. If no bubble is observed, it is catalase negative. If bubbles are

observed it is catalase positive

2.7.3 Triple Sugar Iron test

An 18-24hrs old culture is stab inoculated into a slanted gel TSI agar and then incubated

for 18-24hrs. TSI is used to determine the ability of an organism to ferment glucose, lactose and

sucrose and their ability to produce hydrogen sulfide. If the result is red slant/yellow butt it

indicates dextrose fermentation only. If it is yellow slant/yellow butt, it indicates fermentation of

dextrose, lactose, sucrose. Red slant/red butt, it indicates absence of carbohydrate fermentation.

Blackening in the medium indicate H 2S present. Gas production indicate the production of gas

(Sagar et al, 2019)

2.7.4 Indole Test

An 18-24hrs old culture is inoculated into a prepared TSB in a test tube. It then incubated

for 18-24hrs after which a drop of Kovac’s reagent is added to the sample. The result is positive

16
if a red-like colored ring is observed. It is negative if the ring doesn’t change color (Sagar et

al,2019).

2.7.5 Citrate Test

An 18-24 hrs. old culture is inoculated on a slant EMB agar in sterile bottle and then

incubated 18-24hrs. The result is positive if the color change from green to blue and the result is

negative if there is no color change (Sagar et al, 2019).

2.7.6 Hemolysis Test

An 18-24hrs old culture is streaked on a blood agar and then incubated for 18-24hrs. if

there is partial break down in the red blood cells and leaves a greenish color it is an alpha

hemolysis, if the red blood cell is broken down completely leaving a clear zone around the

bacteria it is beta hemolysis. If there is no break down and no color change it is a gamma

hemolysis

2.8 Antimicrobial Susceptibility Test

The antimicrobial susceptibility test for enterococcus sp was done in order to determine

the susceptibility or resistance of the bacteria isolates to some selected antibiotics of certain

concentration. Kirby-Bauer National Committee for Clinical and Laboratory Standard (CLSI,

2014) modified disc diffusion techniques was used to determine antibiotics susceptibility of the

isolates. Using a sterile inoculating loop, a colony of the organism of interest was taken and

inoculum into Mueller Hinton broth and incubated for 24 hours. The broth culture was

standardized with sterile broth to obtain turbidity that is optically comparable to 0.5 McFarland

standard using colorimeter. A sterile swab stick was dipped into adjusted suspension. The swab

stick was related severally and pressed firmly on the side of the wall of the tube to remove excess

17
inoculum from the swab. The dry surface of the Mueller Hinton agar was inoculated by

spreading the swab over the entire sterile agar surface. Later, antibiotic disc of different types

was fixed on the media using sterile forceps. The following antibiotics used were; Cefuroxime

(CRX, 20ug), Gentamicin (GEN, 10ug), Augmentin (AUG, 30ug), Cefixime (CXM, 5ug),

Ciprofloxacin (CPR, 5ug). Each disc was pressed down to ensure complete contact with agar

surface and incubated at 37oC for 24 hours. The zones of inhibition were then measured using a

calibrated ruler in millimeters and are classified on CLSI (2014) (resistance, intermediate and

susceptible).

2.9 Statistical Analysis

Statistical tools were used to compare physiological and microbiological values of the

hospital effluent. Correlation and regression were also used to analyze and compare result using

MS Excel and SPSS

CHAPTER THREE

RESULTS

3.1 Physiochemical Analysis of the Wastewater Samples

The physiological parameters considered in the study were temperature, pH, Total

Dissolved Solid, Dissolved Oxygen and Biological Oxygen Demand.

3.1.1. Temperature

The sample temperature during sample ranged from 22.6±0.03ºC to 28.7+ 0.67ºC. The

highest temperature was recorded at the first sampling while the least was recorded at the second

18
sampling as shown in Table 3.1. The values are statistically significant with p- value 6.9×10ˆˉ4

(p˂ 0.05) hence, the null hypothesis was rejected.

3.1.2. pH

The sample had the lowest pH of 8.7±0.07 and the highest pH was recorded as 8.9±0.15

as shown in table. The null hypothesis was rejected since there is a significant difference

between the values of pH as the p- value (6.9×10ˆˉ4) was lesser than 0.05 as shown in the table

3.1.3. Total Dissolved Solid (TDS)

The values for the total dissolved solids were taken and the mean value obtained for each

sampling was 31±0.006ppm as the lowest at first sampling and 54±0.033 ppm as the highest at

second sampling as shown in Table3.1. The p- value for TDS is 6.9×10ˆˉ4 which rejects the null

hypothesis.

3.1.4 Dissolved Oxygen (DO)

The values of dissolved oxygen obtained ranges from 0.13±0.033 as the lowest to

0.3±0.09 as the highest value as shown in Table 3.1

3.1.5 Biological Oxygen Demand

There was no BOD recorded because

19
20
 Table 3.1 Average values of physiochemical parameters of wastewater samples from Obafemi Awolowo University

Teaching Hospital Complex

Sampling Period pH Value TDS Value (mg/L) Temperature (oc) DO (mg/L) BOD (Mg/L)

1ST 8.7 ±0.37 31±0.006 28.7±0.67 0.13±0.03

2ND 8.9±0.15 54±0.033 22.6±0.03 0.23±0.03

3RD 8.8±0.07 43.9±0.033 23.3±0.03 0.33±0.09

p-value =0.0069

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3.2 Microbiological Analysis

3.2.1 Biochemical Identification of Isolates

A total of 30 isolates were obtained, 15 isolates were found to be Enterococcus sp which

appeared exclusively as creamy, spherical, elevated on the enterococcus agar. Most of the

isolates (28) were gram positive rods while the rest were Gram negative. All Enterococcus sp

were oxidase negative, catalase negative and ferments glucose. The results of other biochemical

tests are summarized in Table3.2. 53% of the isolates were presumptively Enterococcus sp as

shown in fig3.1

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 ISOLATE
CATALA

PRODUC

PRODUC
STAININ

PRESUM
SE TEST
OXIDAS

INDOLE

HAEMO
CITRAT
E TEST

E TEST

SLANT
GRAM

PTIVE
LYSIS
CODE

BUTT
TEST

TEST
TION

TION
GAS
H2S
TSI

TSI
G

S
HO1A1 GPC - - - - Y R - - α Streptococcus sp
HO1A2 GPC - - - - Y Y + - α Streptococcus sp
HO1B1 GPC - - - - Y Y - - Ɣ Enterococcus sp
HO1B2 GPC - - - - Y R - - α Streptococcus sp
HO1C1 GPC - - - - Y Y - - Ɣ Enterococcus sp
HO1C2 GPC - - - - Y Y - - Ɣ Enterococcus sp
HO1D1 GPC - - - - Y Y - - Ɣ Enterococcus sp
HO1D2 GPC - - - - Y Y - - Ɣ Enterococcus sp
HO1E1 GPC - + - - Y Y - - Ɣ Enterococcus sp
HO1E2 GPC - - - - Y R - - α Streptococcus sp
HO2A1 GPC - - - - Y Y - - α Streptococcus sp
HO2A2 GPC - - - - Y R - - Ɣ Enterococcus sp
HO2A3 GPC - + - - R Y - - Ɣ Staphylococcus sp
HO2A4 GPC - - - - Y R - - Α Streptococcus sp
HO2A5 GPC - + + + Y R - - Ɣ Staphylococcus sp
HO2B1 GPC - - - - Y Y - - Ɣ Enterococcus sp
HO2B2 GPC - - - - Y R - + Ɣ Enterococcus sp
HO2B3 GPC - - -- - Y Y - - α Streptococcus sp
HO2B4 GPC - - - - Y Y - - α Streptococcus sp
HO2B5 GPC - + - - Y Y - - Ɣ Enterococcus sp
H03A1 GPC - - - - R Y - + α Streptococcus sp
H03A2 GPC - - - - Y Y - - Ɣ Enterococcus sp
H03A3 GPC - - - - Y Y - - α Streptococcus sp
H03A4 GPC - + - - Y R - - Ɣ Staphylococcus sp
H03A5 GPC - - - - Y Y - - Ɣ Enterococcus sp
H03B1 GPC - - - - Y Y - - α Streptococcus sp
H03B2 GPC - - - - R R - + Ɣ Enterococcus sp
H03B3 GPC - - - - Y R - - Ɣ Enterococcus sp
H03B4 GPC - + - - Y R - + Ɣ Staphylococcus sp
H03B5 GPC - + - - R R - - Ɣ Staphylococcus sp

3.2 Biochemical results of presumptive Enterococcus sp isolated from the hospital effluent in

OAUTHC, Ile-Ife

KEY: GPC = Gram positive cocci GNC= Gram negative cocci -= Negative += Positive Y= Yellow R=
Red α= alpha Ɣ= Gamma

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 no of colonies

13%

53%
33%

streptococci staphylococcus enterococcus

Fig 3.1 Percentage occurrence of isolated bacteria species in wastewater samples from

Obafemi Awolowo University Teaching Hospital Complex

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3.3 Antibiotic Susceptibility Testing Result

Antibiotic susceptibility testing was carried out for the identified Enterococcus sp against

a panel of 8 antibiotics using the Kirby-Bauer disc diffusion method. The antibiotic susceptibility

testing results for enterococcus spp were interpreted as susceptible (S), resistant (R) according to

the recommended guidelines and instructions by the Clinical Laboratory Standard institute

(CLSI, 2018). As shown in fig 3.2, Enterococcus spp was observed to have 81% susceptible of

tetracycline and 63% resistance to amikacin, 68% susceptible to cotrimoxazole, 56% resistance

to vancomycin, 56% resistance to Gentamycin, 69% susceptible to chloramphenicol, 75%

susceptible to ciprofloxacin, 56% resistance to ceftriaxone

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 90
Chart Title
80

70

60

50

40

30

20

10

0
TET COT GEN CHL CTR CIP AMK VAN

SUSCEPTIBLE RESISTANCE Column1

Fig 3.2 Antibiotics Susceptibility pattern of enterococcus sp from OAUTHC, Ile-Ife

TET: Tetracycline, COT: Cotrimoxazole, GEN: Gentamycin, CHL: Chloramphenicol,

CTR: Ceftriaxone, CIP: Ciprofloxacin, AMK; Amikacin, VAN: Vancomycin

26
 CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 DISCUSSION

Water is important in maintaining human health and ecosystem (Awoyemi et al., 2014).

Water is indisputably the most importance to all life forms. The very numerous functions it

serves is a testament to its importance to life ranging from industrial uses down to personal

human consumption for domestic and drinking purposes (Naseem and Munir, 2016).

Wastewater quality is measured through assessment of the physicochemical and

biological properties of the water against a set of standards, in order to determine whether it is

suitable for re-use or disposal into the environment (Khatri and Tyagi, 2015). Wastewater is

composed of contaminants which may be of organic or inorganic origin (Azrina et al. 2011).

Water quality refers to the chemical, physical, biological and radiological characteristics of

water. It is simply a term used to determine the suitability of water to sustain various uses and

purposes (Meybeck et al., 2007). Scientific procedures used to access these water contaminants

involves the analysis of the physical, chemical and biological qualities of the wastewater which

include parameters such as pH, temperature, turbidity, total suspended solids (TSS), total

dissolved solids (TDS), dissolved oxygen (DO), biological oxygen demand (BOD) and heavy

metals (Rahmanian et al., 2015). When these parameters have values that are higher in

concentration than the safe limits set by the World Health Organization (WHO) and other

regulatory bodies, they affect the wastewater quality (WHO, 2011).

27
 Temperature is the degree of hotness or coldness of body. Temperature is an important

physiochemical parameter because at higher temperature, the rate of chemical reactions increases

and therefore, exerts a major influence on the kind of organisms present in the water bodies,

availability of dissolved oxygen, biological activity and growth (Kale, 2016). The temperature

value obtained for the three sampling sites were within the acceptable range according to WHO

(2011) guidelines. The P-value obtained was statistically significance (p<0.05), hence, the null

hypothesis was rejected. The observed temperature which ranged from 25.3 OC to 28.7OC

revealed that the hospital waste water is a suitable habitat for Enterococcus sp.

The pH is a measure of the hydrogen ion concentration in water (Reda, 2016). It is a

measure of acidity (pH < 7.0) or alkalinity (pH >7.0) of the water (Rahmanian et al., 2015). The

pH determines the solubility of chemical constituents such as nutrients (Phosphorus, nitrogen

and carbon) and heavy metals and the amount available for use by aquatic life (Kale, 2016). Low

pH in wastewater can increase the availability of metals since hydrogen ions have the affinity for

competing with metals ions and releasing them in the wastewater (Singh and Agrawal, 2012).

The observed pH of the hospital effluent samples was within the range of8.7 ±0.37 and 8.9 ±

0.07 for wastewater.

Total dissolved solids (TDS) refer to the inorganic matters and small amounts of organic

matter present as solution in water (Rahmanian et al., 2015). TDS can be used as an indicator for

the general water quality because it can limit the suitability of water as a drinking source and

irrigation supply and directly affects the aesthetic value of water by increasing turbidity (Reda,

2016). TDS can be toxic to aquatic animals by causing osmotic stress and affecting the

osmoregulatory capability of organisms (Akan et al, 2008). Total dissolved solids for the

wastewater samples (Table 3.1) weren’t within the recommended standards set by EPA (2006)

28
which set the limit of 1000mg/L of no risk to aquatic life for wastewater released into the

receiving environment. TDS values for wastewater samples varied significantly with a P-value of

0.0006 (P<0.05) as shown in Table 3.1.

Dissolved oxygen is the concentration of molecular oxygen in water and it depends on

the temperature and biological oxygen demand of the system because it is used in aerobic

decomposition of Dissolved oxygen concentrations are directly dependent on oxygen generation

through photosynthesis and consumption by living organisms especially bacteria (Julian et al.,

2018). The biodegradable materials in water are easily oxidized through the use of the dissolved

oxygen (DO) and therefore, the oxygen demand for biological activities (BOD) in the water

depletes the DO and threatens the aquatic life present (Maitera et al., 2010). The standard for

sustaining aquatic life is stipulated at 6mg/L, a concentration below this value adversely affects

aquatic biological life in the water, while concentration below 2mg/L may lead to death for most

Aquatic life (Chapman, 1997). The DO of the hospital effluent is below the standard for

sustaining aquatic life.

BOD is the amount of dissolved oxygen needed by aerobic biological organisms in the

water body to break down organic matter present in a given water sample at certain temperature

over a specific period. It is often used as a Robust surrogate of the degree of organic pollution of

water. In this study, the BOD was below the recommended acceptable standard of 6mg/l by

FME. This suggests that the hospital effluent is effective in the removal of dissolved organic

matter.

The Enterococcus spp in this study was isolated using enterococcus agar and identified

using standard biochemical tests (Table 3.3). The obtained isolates were screened for antibiotic

29
susceptibility using the recommended guidelines by Clinical Laboratory Standard Institute

(CLSI. 2018)

The susceptibility of the isolated Enterococcus spp showed that it was highly susceptible

to most of the antibiotics. They were highly susceptible to ciprofloxacin, tetracycline. This

indicates that infection caused by the bacterial isolates could still be treated by any of this

antibiotic. However, high resistance to amikacin and vancomycin as being observed. This could

be result of altered sites().

4.2 CONCLUSION

In this study, Enterococcus spp was isolated with some other pathogenic microorganism.

The constant use of antibiotics in the hospital environment has increased the probability of

resistance of the microorganism to various antibiotics. The risk of residual antibiotics in the

environment has generated global concern and this risk will continue to increase due to the

continuous use of the antibiotics in the hospital effluent. If proper precaution is not put in place,

the rate of resistance of the microorganism to the antibiotics will increase, which poses a threat

to the public health as well as economic growth. This indicates the need to take effective

measures in the proper discarding of hospital wastewater.

4.3 RECOMMENDATION

There should be continued surveillance of the antibiotic susceptibility profile to ensure

public healthy safety and to enlighten the hospital workers on the proper way of disposing

30
hospital wastewater. There is a need to create more environmental and personal hygiene

awareness among health workers.

The standard disk diffusion tests should be combined with other approaches, such as

modified disk test or E-test for phenotypic detection of ESBL. It is also helpful to formulate

guidelines for the optimal use of antibiotics. Also, it is important that studies such as this should

forwarded to the appropriate sector

31
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39
 APPENDIX I

PREPARATION OF BROTHS AND MEDIA

1. NUTRIENT AGAR

Composition g/L

 Peptone water 5.00

 Beef extract 3.00

 NaCl 8.00

 Agar 15.00

Preparation:

28g of nutrient agar was suspended in 1L of distilled water completely, boiled to dissolve

the medium completely and sterilized by autoclaving at 121oC for 15 min. allowed to

cool to 47oC before pouring into sterilized petri dish.

2. MUELLER HINTON AGAR

Composition g/L

 Beef infusion 300g

 Peptone 17.5g

40
 Starch 1.5g

 Agar 17.0g

Preparation

This was prepared by suspending 38g of the medium in 1L of distilled water. It was

heated to completely dissolve the solution, sterilized by autoclaving at 121oC for 15 min,

cooled to room temperature and poured into sterile petri dish and allowed to set.

3. TRIPLE SUGAR IRON (TSI)

Composition g/L

 Peptone 20.0g

 Beef extract 3.0g

 Yeast extract 3.0g

 Glucose 1.0g

 Lactose 10.0g

 Sucrose 10.0g

 Ferrous sulphate 0.2g

 NaCl 5.00g

 Sodium thiosulphate 0.3g

 Phenol red 0.024g

 Distilled water 1L

Preparation

64.32g was suspended in 1L of distilled water completely, boiled to dissolve the mixture

in order to homogenize completely, sterilize by autoclaving at 121oC for 15 min, allowed

to cool to 50oC before pouring into sterilized petri dish.

4. SIMMON’s CITRATE AGAR

Composition g/L

 Sodium chloride 5.0g

 Sodium citrate 2.0g

 Ammonium dihydrogen phosphate 1.0g

 Dipotassium phosphate 1.0g

 Magnesium sulphate 0.2g

 Bromothymol blue 0.08g

 Agar 15.0g

Preparation

42
 24.28g was suspended in 1L of distilled water completely, boiled to dissolve the mixture

in order to homogenize completely, sterilize by autoclaving at 121oC for 15 min. allowed

to cool to 50oC and shake before pouring into sterilized petri dish.

5. PHYSIOLOGICAL NORMAL SALINE

Composition g/L

 Sodium chloride 8.5g

 Distilled water 1L

Preparation

The salt was weighed and dissolved in 1mL conical flask containing distilled water,

homogenized to mix until completely dissolved, sterilized at 121oC for 15mins.

43
 APPENDIX II

COMPOSITION OF SOLUTIONS AND REAGENTS

1. GRAM STAINING REAGENTS

Composition of reagents

i. CRYSTAL VIOLET SOLUTION

Composition g/L

 Crystal violet 0.5g

 Distilled water 300ml

ii. GRAM’S IODINE SOLUTION

Composition g/L

 Iodine 1.0g

 Potassium iodide 2.0g

 Distilled water 300ml

iii. SAFRANIN SOLUTION

Composition g/L

 Safranin O powder 1.0g

 Distilled water 100ml

44
iv. ETHANOL

Composition g/L

 Ethanol 200ml

 Distilled water 200ml

6. KOVAC’S INDOLE REAGENT

Composition g/L

 Paramenthylaminobenzaldehyde 10ml

 Paraamyl alcohol 150ml

 Concentrated pureHydrochloric acid 50ml

45
Table 3.3 Colony Morphology of Bacterial Isolates

ISOLATE COLOUR SHAPE EDGE SURFACE ELVATION OPACITY

CODE

o Creamy Pink Circular Entire Smooth Convex Opaque

AW15 Creamy Pink Circular Entire Smooth Convex Opaque

AW24 Creamy Pink Circular Entire Smooth Convex Opaque

OS11 Blue Centre Circular Entire Glistering Convex Opaque

OS14 Blue Centre Circular Entire Glistering Convex Opaque

OS21 Blue Centre Circular Entire Glistering Convex Opaque

ASW11 Black Centre Circular Entire Smooth Convex Opaque

ASW15 Black Centre Circular Entire Smooth Convex Opaque

46
ASW22 Black Centre Circular Entire Smooth Convex Opaque

ASW23 Black Centre Circular Entire Smooth Convex Opaque

47
Table 3.4: Antibiotic susceptibility pattern of Enterococcus sp

CODES TET COT CRX CHL CTR CT CIP AMK VAN CPZ
X

48