i

ANUAGASI, FRANCISCA EBELE PG/M.Sc./09/51140

MULTIDRUG RESISTANCE PROFILES OF CLINICAL AND ENVIRONMENTAL ISOLATES OF PSEUDOMONAS AERUGINOSA AND ESCHERICHIA

COLI

FACULTY OF BIOLOGICAL SCIENCE

DEPARTMENT OF MICROBIOLOGY

Ebere Omeje

Digitally Signed by: Content manager’s Name

DN : CN = Webmaster’s name

ii

MULTIDRUG RESISTANCE PROFILES OF CLINICAL AND ENVIR ONMENTAL

ISOLATES OF PSEUDOMONAS AERUGINOSA AND ESCHERICHIA COLI

BY

ANUAGASI, FRANCISCA EBELE PG/M.Sc./09/51140

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

AUGUST, 2015

i

TITLE PAGE

MULTIDRUG RESISTANCE PROFILES OF CLINICAL AND ENVIR ONMENTAL ISOLATES OF PSEUDOMONAS AERUGINOSA AND ESCHERICHIA COLI

BY

ANUAGASI, FRANCISCA EBELE PG/M.Sc./09/51140

A DISSERTATION SUBMITTED TO THE SCHOOL OF POST GRAD UATE STUDIES, UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILMENT OF THE

REQUIREMENT FOR THE AWARD OF MASTER OF SCIENCE (M.S c.) DEGREE IN ENVIRONMENTAL MICROBIOLOGY

SUPERVISOR: PROF. C. U. ANYANWU

AUGUST, 2015

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CERTIFICATION

Miss Anuagasi, Francisca Ebele, a postgraduate student in the Department of

Microbiology majoring in Environmental Microbiology, has satisfactorily completed the

requirements for course work and research work for the degree of Master of Science (M.Sc.) in

Microbiology. The work embodied in this dissertation is original and has not been submitted in

apart or full for any other diploma or degree of this University or any other University.

…………………………………. …………………………………. Prof. A. N. MONEKE Prof. C. U. ANYANWU Head, Supervisor, Department of Microbiology Department of Microbiology University of Nigeria, Nsukka University of Nigeria, Nsukka

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DEDICATION

This work is dedicated to God Almighty, for His goodness and mercies bestowed on me.

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ACKNOWLEDGMENTS

My profound gratitude goes to my supervisor, Prof. C. U. Anyanwu, for his support,

encouragement and criticism during the course of this work. May God reward you.

I am also thankful to my lecturers: Prof. C. U. Iroegbu, Prof. A. N. Moneke, Prof. J. C. Ogbonna,

Prof. J. U. Ugwuanyi, Prof. (Mrs.) I. M. Ezeonu, and Rev. Sr. (Dr.) Dibua for all their assistance

in ensuring the completion of this work. I owe immense thanks to my dad, Engr. P. Udoye and

my sisters for their financial encouragement throughout the period of my studies. Worthy of my

gratitude are also some of my friends Oti, Nchedo, Albert, Uju, Gabriella, Ijeoma and Emma who

motivated me by their advise and encouragement and other well wishers for their help and prayers

towards making this work a success. May the Almighty God reward you all, Amen.

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TABLE OF CONTENTS

Title page ------------------------------------------------------------------------------------------------ i

Certification --------------------------------------------------------------------------------------------- ii

Dedication ----------------------------------------------------------------------------------------------- iii

Acknowledgments ------------------------------------------------------------------------------------- iv

Table of Contents -------------------------------------------------------------------------------------- v

List of Tables ------------------------------------------------------------------------------------------- vii

List of Figures ------------------------------------------------------------------------------------------ viii

Abstract -------------------------------------------------------------------------------------------------- x

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction ----------------------------------------------------------------------------------------- 1

1.1.1 Statement of problem --------------------------------------------------------------------------- 4

1.1.2 Research objective ------------------------------------------------------------------------------ 4

1 .2 Literature Review --------------------------------------------------------------------------------- 4

1.2.1 Antibiotics and resistance mechanisms ------------------------------------------------------ 4

1.2.2 Resistance to β-lactams ------------------------------------------------------------------------- 6

1.2.3 Resistance to sulfonamides and trimethoprim ---------------------------------------------- 7

1.2.4 Resistance to macrolides ----------------------------------------------------------------------- 7

1.2.5 Resistance to tetracyclines --------------------------------------------------------------------- 8

1.2.6 Resistance to nitroimidazoles ------------------------------------------------------------------ 9

1.2.7 Resistance to glycopeptides -------------------------------------------------------------------- 9

1.2.8 Disseminating antibiotic resistance ----------------------------------------------------------- 11

1.2.9 Epidemiology of resistance ------------------------------------------------------------------- 13

1.3 Development of Multidrug Resistance of P. aeruginosa ----------------------------------- 15

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1.4 Plasmids --------------------------------------------------------------------------------------------- 16

1.4.1 Plasmids and bacterial resistance ------------------------------------------------------------- 18

CHAPTER TWO: MATERIALS AND METHODS

2.1 Sample Site ---------------------------------------------------------------------------------------- 20

2.2 Collection of Samples ----------------------------------------------------------------------------- 20

2.3 Isolation Procedure ------------------------------------------------------------------------------ 20

2.4 Identification of the Isolates ---------------------------------------------------------------------- 21

2.4.1 Gram staining ------------------------------------------------------------------------------------ 21

2.4.2 Oxidase test ------------------------------------------------------------------------------------- 22

2.4.3 Sugar fermentation ----------------------------------------------------------------------------- 22

2.4.4 Indole test ----------------------------------------------------------------------------------------- 22

2.4.5 Citrate test --------------------------------------------------------------------------------------- 23

2.4.6 Catalase test -------------------------------------------------------------------------------------- 23

2.4.7 Methyl red (MR) test -------------------------------------------------------------------------- 23

2.4.8 Voges Proskauer test ---------------------------------------------------------------------------- 24

2.5 Antibiotic Susceptibility Test -------------------------------------------------------------------- 24

2.6 Plasmid Curing ------------------------------------------------------------------------------------- 25

2.6.1 Use of sodium dodecyl sulphate (SDS) ------------------------------------------------------ 25

2.7 Statistical Analysis -------------------------------------------------------------------------------- 25

CHAPTER THREE: RESULTS

3.1 Number of Isolates from different Sites ------------------------------------------------------- 26

3.2 Antibiotic Resistance Pattern of the Isolates from different Sites ------------------------- 28

3.3 Effect of Plasmid Curing ------------------------------------------------------------------------ 40

CHAPTER FOUR: DISCUSSION

References ----------------------------------------------------------------------------------------------- 45

vii

LIST OF TABLES

Table Title Page

1: Isolates from both urban and rural hospitals and environment ---------------36

2: Effect of SDS mediated plasmid curing on resistant bacteria-----------------50

viii

LIST OF FIGURES

Figure Title Page

1: Antibiotics resistance mechanism----------------------------------------- 19

2: Horizontal exchange of genetic material like antibiotics resistance through plasmid by bacteria-------------------------------------------- 19

3: Percentage antibiotic resistance of urine isolates from urban hospital----------------------------------------------------------------------- 28

4: Percentage antibiotic resistance of wounds isolates from urban hospital---------------------------------------------------------------------- 29

5: Percentage antibiotic resistance of urine isolates from rural hospital---------------------------------------------------------------------- 31

6: Percentage antibiotic resistance of wounds isolates from rural hospital-------------------------------------------------------------------- 32

7: Percentage antibiotic resistance of water isolates from urban environment------------------------------------------------------------------ 34

8: Percentage antibiotic resistance of soil isolates from urban

environment------------------------------------------------------------------ 35

9: Percentage antibiotic resistance of water isolates from rural environment-------------------------------------------------------------- 37

10: Percentage antibiotic resistance of soil isolates from rural environment---------------------------------------------------------------- 38

ix

Abstract

The emergence of multiple antibiotic resistance in bacteria and the indiscriminate use of

antibiotics contribute to the dissemination of resistant pathogens in the environment which may

cause problems in therapy and is a serious public health issue. This study was conducted to

determine the incidence of Pseudomonas aeruginosa and E.coli isolates in certain clinical and

environmental samples as well as to determine the susceptibility patterns of these isolates to some

commonly used antibiotics. The organisms were isolated using standard microbiological

techniques and the antibiotic susceptibility was determined using disc diffusion method while

plasmid curing was done using sodium dodecyl sulphate (SDS). The result of this studies showed

that most of the clinical and environmental isolates were more resistant to amoxacillin and

augumentin but clinical isolates showed higher resistance. It was also observed that clinical

isolates showed least resistance to gentamycin, ofloxacin, and ciprofloxacin; similar least

resistance were observed in environmental samples with gentamycin and ciprofloxacin. There was

a significant difference (P≥ 0.05) in the percentage resistance between the clinical and

environmental isolates. Thirteen isolates that were resistant to more than seven antibiotics were

subjected to plasmid curing using 1% and 5% SDS. It was observed that at treatment with 1%

SDS some of the isolates became resistant to more than one antibiotic; when SDS was increased

to 5%, some of the isolates that were resistant become completely sensitive to all the antibiotics

used. However, one of the P.aeruginosa that was initially sensitive to chloramphenicol became

completely resistant at 5% SDS and another isolate of P.aeruginosa that was initially sensitive to

septrin, sparfloxacin and ciprofloxacin became completely resistant at 1% and 5% SDS. This

study indicates that P.aeruginosa and E.coli isolated from clinical samples were more resistant to

antibiotics than those isolated from environmental samples. It has as well shown that there may be

a possible transfer of resistance from one strain to another.

1

CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

The discovery of antibacterial agents had a major impact on the rate of survival from

infections. However, the changing patterns of antimicrobial resistance caused a demand for

new antibacterial agents. Therefore, the emergence of bacterial resistance to most of the

commonly used antibiotics is of considerable medical significance (Khan and Malik, 2001;

Oteo et al., 2002).

Antibiotic resistance genes in most bacteria are frequently found in extra chromosomal

elements known as R-plasmids. Pseudomonas aeruginosa is naturally resistant to many of the

widely used antibiotics, so chemotherapy is often difficult (Dubois et al., 2001).

Resistance is due to a resistance transfer plasmid (R-plasmid) which is a plasmid

carrying gene encoding proteins that detoxify various antibiotics (Poole, 2004). Antibiotic

resistant bacteria are widespread. Several antibiotic resistant genes can be carried by a single

R-plasmid or alternatively, a cell may contain several R plasmids. In either case, the result is

multiple resistance (Madigan et al., 2009).

Escherichia coli is a Gram negative bacterium and the main aerobic commensal

bacterial species (Alhaj et al., 2007; Von and Marre, 2005). The native habitat of Escherichia

coli is the enteric tract of humans and other warm-blooded animals. Therefore, Escherichia

coli is widely disseminated in the environment through the faeces of humans and other

animals and its presence in water is generally considered to indicate faecal contamination and

the possible presence of enteric pathogens. Esherichia coli is able to acquire antibiotic

resistance easily. Antibiotic resistant Esherichia coli may pass on the genes responsible for

antibiotic resistance to other species of bacteria, such as Staphylococcus aureus, through a

process called horizontal gene transfer (Dubois et al., 2001). Esherichia coli often carry

2

multidrug resistant plasmids and under stress readily transfer those plasmids to other species.

Thus, Esherichia coli is an important reservoir of transferable antibiotic resistance (Salyers et

al., 2004). It has been observed that antibiotic susceptibility of bacterial isolates is not

constant but dynamic and varies with time and environment (Hassan, 1995).

Escherichia coli is an opportunistic pathogen in neonatal and immuno-compromised

patients (Annette, 1998). Bacteremia, wound infections, urinary tract infection, and

gastrointestinal infections are the diseases associated with Escherichia coli and are often fatal

in newborns (Raina et al., 1999). The organism is of clinical importance due to its

cosmopolitan nature and the ability to initiate, establish and cause various kinds of infections

(Okeke et al., 2000; Olowe et al., 2003; Tobih et al., 2006). Infections with antibiotic

resistant bacteria make the therapeutic options for infection treatment extremely difficult or

virtually impossible in some instances (El-Astal, 2004). Therefore, the determination of

antimicrobial susceptibility of clinical isolates is often crucial for optimum antimicrobial

therapy of infected patients.

A high-density patients’ population in frequent contact with health care staff and the

attendant risk of cross-infection contributes to the spread of antibiotic-resistant

microorganisms in the environment (Bataineh et al., 2007). Occurrence and prevalence of

these resistant strains in the environment is, therefore, a usual kind of thing in developing

countries. The Gram negative bacterium Pesudomonas aeruginosa is a ubiquitous aerobe that

is present in water, soil and on plants (Banerjee and Stableforth, 2000). Naturally, this

organism is endowed with weak pathogenic potentials. However, its profound ability to

survive on inert materials, minimal nutritional requirement, tolerance to a wide variety of

physical conditions and its relative resistance to several unrelated antimicrobial agents and

antiseptics, contributes enormously to its ecological success and its role as an effective

opportunistic pathogen (Gales et al., 2001). The organism is pathogenic when introduced into

3

areas devoid of normal defenses (Jawetz et al., 1991) and infections are both invasive and

toxigenic (Todar, 2002).

Pseudomonas aeruginosa has been incriminated in cases of meningitis, septicaemia,

pneumonia, ocular and burn infections, hot tubs and whirlpool-associated folliculitis,

osteomyelitis, cystic fibrosis-related lung infection, malignant external otitis and urinary tract

infections with colonized patients being an important reservoir (Hernandez et al., 1997).

Cross-transmission from patient to patient may occur via the hands of the health care staff or

through contaminated materials and reagents (DuBois et al., 2001). However, it is believed

that Pseudomonas aeruginosa is generally environmentally acquired and that person-to-

person spread occurs only rarely (Harbour et al., 2002). As such, contaminated respiratory

care equipment, irrigating solutions, catheters, infusions, cosmetics, dilute antiseptics,

cleaning liquids, and even soaps have been reported as vehicles of transmission (Joklik et al.,

1992; Berrouane et al., 2000; DuBois et al., 2001).

Increase in antibiotic resistance level is now a global problem. Pseudomonas

aeruginosa is naturally resistant to many of the widely used antibiotics, so chemotherapy is

often difficult. Resistance is due to a resistance transfer plasmid (R-plasmid) which is a

plasmid carrying genes encoding proteins that detoxify various antibiotics out of the cell.

Low antibiotic susceptibility, which is a worrying characteristic, is attributable to a concerted

action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes

e.g. mexAB-oprM,mexXY, etc (Poole, 2004), and low permeability of the bacterial cellular

envelopes. Besides intrinsic resistance, Pseudomonas aeruginosa easily develops acquired

resistance either by mutation in chromosomally-encoded genes, or by the horizontal gene

transfer of antibiotic resistance determinants. Development of multidrug resistance by

Pseudomonas aeruginosa isolates requires several different mutations and/or horizontal

transfer of antibiotic resistance genes.

4

Hypermutation favours the selection of mutation-driven antibiotic resistance in

Pseudomonas aeruginosa strains producing chronic infections, whereas the clustering of

several different antibiotic resistance genes in integrons favours the concerted acquisition of

antibiotic resistance determinants. Some recent studies have shown that phenotypic

resistance associated with biofilm formation or to the emergence of small-colony-variants

may be important in the response of Pseudomonas aeruginosa populations to antibiotic

treatment (Cornelis, 2008).

1.1.1 Statement of problem

Massive quantities of antibiotics are being prepared and used each day. As a result of

this, an increasing number of diseases are resisting treatment due to the spread of drug

resistance as a result of drug misuse. Patients with Pseudomonas aeruginosa and Escherichia

coli infections may inherently develop resistant to many classes of antibiotics as a result of

misuse and improper disposal of drug in the environment and this may cause difficulty in

treatment and may lead to life-threatening diseases and possibly death.

1.1.2 Research objective

To determine the incidence of Pseudomonas aeruginosa and Esecherichia coli isolates in

certain clinical and environmental samples.

To determine the susceptibility patterns of the isolates to some commonly used antibiotics.

To determine if the resistance is on the chromosome or on the plasmid.

1 .2 Literature Review

1.2.1 Antibiotics and resistance mechanisms

Antibiotics are biochemical substances produced by living organisms, which are able to

kill or inhibit the growth of other microorganisms. They are natural substances that inhibit the

growth or proliferation of bacteria or kill them directly (Guardabassi and Dalsgaard, 2002;

5

Levy et al., 1988). The introduction of antimicrobial agents in the mid 1930’s heralded the

opening of an era in which literally millions of people that would have faced early death or

invalidism were spared. Thus the development and use of antimicrobial agents was one of the

most important measures leading to the control of bacterial diseases in the 20th century.

Antimicrobial therapy provided physicians with the ability to prevent some infections, to cure

others and to curtail the transmission of certain other diseases. The concept of untreatable

bacterial diseases became foreign to most physicians especially in the developed world.

Natural antibiotic producers are inherently resistant to the antibiotics they produce.

Other bacteria survive by developing or acquiring antibiotic resistance mechanisms. Some of

the prominent means of resistance include: altered permeability barriers across bacterial outer

membranes, preventing uptake of the compound by inhibiting its corresponding transport

carrier, modifying the target’s binding sites so that it no longer recognizes the antibiotic(s),

and the ability to chemically and/or enzymatically degrade the antibiotics. Antibiotics must

enter the bacterial cell to access a target site in order to exert their bactericidal (cell death) or

bacteriostatic (slow bacterial growth) action. Gram negative bacteria are resistant to a greater

number of antibiotics compared to Gram positives, largely because they possess an outer

membrane. Migration of antibiotics between the external environment and a cell’s periplasm

occurs via ‘porin channels’. Mutations within genes encoding for such porin channels could

reduce the ability of the antibiotic to reach its target site as well as physical barriers such as

extracellular gums and/or biofilms.

Efflux pumps are found in both Gram positive and Gram negative bacteria and

actively transport toxic substances from within the bacteria to its surrounding environment.

DNA operons encoding for efflux genes are found either on chromosomes and are indicative

of intrinsic resistance, or on plasmids which are suggestive of acquired resistance.

6

There are five major efflux transporter families in prokaryotes, some selective, whilst

others are involved with expelling an array of compounds including antibiotics. The latter

class of efflux exporters is cause for major concern since they can lead to multi-resistant

bacteria. Mutations in the efflux repressor genes prompt over-expression of the structural

genes which may lead to an increasing level of antibiotic tolerance. Although over expression

of efflux pump genes do not necessarily afford high level resistance; they guard against lower

drug exposures and prolong survival until further possible mutations take place (such as

within the antibiotic target site), potentially leading to highly resistant progeny.

1.2.2 Resistance to β-lactams

β-lactams such as penicillins and cephalosporins are narrow spectrum antibiotics,

effective against the Gram positive genera Streptococcus, Neiseria and Staphylococcus

(Todar, 2002). Prior to its introduction in the 1940s, almost all hospital acquired

Staphylococcus aureus strains were sensitive to penicillin G, whilst today virtually all strains

show resistance. Methicillin-resistant S. aureus (MRSA) produce a low affinity penicillin

binding protein PBP2a, encoded by the mecA gene, which provides resistance to virtually all

β-lactams. Neisseria and Streptococci spp. also have reduced affinity for β-lactams due to

altered penicillin binding proteins (PBPs). Another important resistance mechanism is the

production of β-lactamase enzymes which inactivate the antibiotic molecule by hydrolysing

the β-lactam ring (Deshpande et al., 2004). The ability to produce β-lactamase enzymes is

common amongst Gram negative bacteria, encoded on either plasmid or chromosomal DNA.

Clinical introduction of cephalosporins initially halted the spread of plasmid-encoded β-

lactamase resistance. However, bacteria quickly acquired modifications to their β-lactamase

genes (extended-spectrum β-lactamases) conferring resistance to penicillins and

cephalosporins.

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1.2.3 Resistance to sulfonamides and trimethoprim

Sulfonamides and trimethoprim are synthetic competitive inhibitors of bacterial

enzymes required for the synthesis of tetrahydrofolic acid (THF), which is necessary for the

production of DNA and proteins (Masters et al., 2003). Sulfonamides act as competitive

inhibitors of dihydropteroate synthase (DHPS), while trimethoprim inhibits dihydrofolate

reductase (DHFR). Resistance to sulfonamides and trimethoprim is almost exclusively

associated with plasmid-encoded genes. To date, three sulfonamide resistance genes coding

for different types of DHPS (insensitive to sulfonamides) have been identified: Sul1, Sul2 and

the more recently described Sul3 gene (Graps et al., 2003). Most multi-resistant Gram

negative bacteria harbor Class 1 integrons which carry the Sul1 gene. The Sul2 gene is

frequently associated with the small, multi-copy, non-conjugative IncQ plasmid group.

Although less common, resistance can also be due to mutations within the chromosomally

located dihydropteroate synthase gene (folP). Trimethoprim resistance is widespread amongst

pathogenic bacteria, with up to 29 dihydrofolate reductase (dfr) resistance genes identified

(Graps et al., 2003). Most of these genes are associated with integrons and use elaborate

transfer mechanisms to laterally spread and proliferate within the bacterial community.

1.2.4 Resistance to macrolides

Macrolide antibiotics, such as erythromycin, inhibit protein synthesis in most Gram

positive bacteria by binding to the 50S ribosomal subunit (Todar et al., 2010). Gram

negative bacteria are intrinsically resistant to macrolides, which cannot traverse their outer

membrane. Gram positive bacteria may employ any one of three resistance mechanisms to

negate the effects of these antibiotics; including alteration of the target ribosomal binding

site, expulsion of the macrolides via efflux pumps, and direct inactivation by enzymes

encoded on transmissible plasmids (Mankin, 2008).

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Fluroquinolones on the other hand, are effective against both Gram negative and

Gram positive bacteria. Unlike other antibiotics, fluoroquinolones selectively inhibit nucleic

acid synthesis by inhibiting bacterial DNA topoisomerase II and thus prevent bacterial

growth. They enter Gram negative cells via porin channels and ram positives by lipophilicity

(ability to diffuse through the lipid bilayer of the cell membrane). Resistance may be

conferred by reduced internal drug build up owing to diminished cell wall permeability

and/or increased efflux expulsion (Mankin, 2008). The main mechanism of resistance is

believed to involve the modification of fluroquinolone’s target, chiefly DNA gyrase and

topoisomerase IV. These enzymes consist of two subunits: GyrA and GyrB for DNA gyrase

and ParC and ParE for topoisomerase IV, all of which are encoded by the genes gyrA, gyrB

and parC, parE respectively. Resistance occurs in response to chromosomal mutation of

these genes. In clinical isolates, quinolone resistance genes (qnrA) are either chromosomally

located or plasmid-borne as in qnrB, qnrS and qnrS2. The qnrA gene is located within an

integron and is associated with the sul1 gene and confers resistance to nalidixic acid but not

to fluoroquinolones. The gene product essentially binds to DNA gyrase subunits and

minimises quinolone action. Plasmid-mediated resistance has been shown to enhance pre-

existing quinolone resistant mechanisms such as the efflux system (Mankin et al., 2008).

Consequently, bacteria resistant to fluoroquinolones are often multi-resistant.

1.2.5 Resistance to tetracyclines

Tetracycline antibiotics act by blocking the binding of aminoacyl tRNA to the

ribosome thereby inhibiting protein synthesis. There are 38 known tetracycline (tet) and

oxytetracycline (otr) resistance genes (Roberts, 2005). Of these, 23 encode for efflux pumps,

11 encode for ribosomal proteins, 3 for an inactivation enzyme and 1 for an unknown

resistance mechanism. Efflux genes from Gram positive bacteria are associated with small

plasmids, whilst those from Gram negatives are often linked to large conjugative plasmids.

9

Given that any of the latter harbor resistance determinants for several antibiotic drugs and

heavy metals, selection for resistance to tetracycline will generally render the recipient multi-

resistant (Chopra et al., 2001). Genes encoding for ribosomal protection proteins are usually

located within conjugative transposons, which account for their large host range. Other

resistance mechanisms, conferred by the tet(X) genes, are responsible for enzymatic

alteration of the drug (Roberts, 2005). Point mutations within the 16S rRNA gene and

mutations which alter the permeability of the porin channels also increase tetracycline

resistance (Chopra et al., 2001).

1.2.6 Resistance to nitroimidazoles

Nitroimidazoles such as metronidazole are microbiocidal drugs active against most

anaerobic bacterial species (Theron et al., 2004) and a range of pathogenic anaerobic

protozoa causing infections such as giardiasis, amoebiasis and trichomoniasis. They bind to

macromolecules including DNA and inhibit its synthesis. Metronidazole is administered in an

inactive form and enters the cell by diffusion. Its activation is subject to a reduction of the

molecule’s nitro group by the ferredoxin mediated electron transport system, thereby creating

toxic free radicals which kill sensitive strains (Quon et al., 1992). This reductive mechanism

appears to be unique to the anaerobes. Resistance is associated with the nim genes encoding

5-nitroimidazole reductase enzymes located on the chromosome (nimB) or on low copy self-

transmissible plasmids. Nitroimidazole reductase acts by removing an electron from the

intermediary ferredoxin compound, thus eliminating the drug’s trigger mechanism. Some

organisms may impart resistance by their reduced intracellular concentrations of ferredoxin,

leading to reduced activation of the drug.

1.2.7 Resistance to glycopeptides

Vancomycin is a bacteriocidal glycopeptide antibiotic which until recently was used

as the final safeguard against multi-resistant Gram positive bacterial infections such as

10

MRSA and multi-resistant enterococci (Iversen et al., 2002). Vancomycin inhibits cell wall

formation of Gram positive bacteria by binding to its target site within the peptidoglycan

assembly preventing cross-linking. Vancomycin resistance has been associated with seven

van genes, which code for the promotion of an abnormal target site with lower affinity for the

drug (Poole, 2004). The vanA genotype also confers a high level of resistance to the

glycopeptide teicoplanin. Vancomycin resistant enterococci (VRE) are resistant to

vancomycin and teicoplanin due to a gene cluster which encodes for the synthesis of a novel

cell envelope with a 1000-fold reduced affinity for glycopeptide binding. Thickening of the

cell wall or slowing cell growth is also implicated with vancomycin resistance, especially in

Lactobacillus casei, Pediococcus pentosaceus and Leuconostoc mesenteroides which are

intrinsically resistant to the glycopeptide.

The glycopeptide avoparcin has been used as a growth promoter in animals, thereby

giving rise to a large van resistance gene pool (Philip, 2007). Banning avoparcin use in

Denmark did not lead to reduced levels of glycopeptide-resistant enterrococci (GRE) in pigs

and it wasn’t until all macrolide-based growth promoters were also barred that GRE levels

substantially dropped. This apparent co-selection of resistance genes has been explained by a

genetic linkage between the glycopeptide resistance gene vanA and the macrolide resistance

gene ermB originating from pigs (Boerlin et al., 2001). Aminoglycosides such as

kanamycin, gentamycin and streptomycin bind to bacterial ribosomes and prevent the

initiation of protein synthesis. Bacterial resistance is generally due to chemical alteration of

the drug thus preventing it from binding to its ribosomal target site (Wright, 1999).

Resistance genes are commonly found on self-transmissible plasmids and transposons but

may also reside in the chromosome (Poole et al., 2004). Mutations associated with ribosomal

genes and efflux systems may also be linked to aminoglycoside resistance.

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1.2.8 Disseminating antibiotic resistance

There are two routes for acquired resistance, vertical evolution via mutation and

selection or horizontal evolution via exchange of genes between similar and different species.

Vertical evolution is determined by natural selection whereby a spontaneous mutation in the

bacterial chromosome bestows resistance to a bacterium and its progeny within the

population. Horizontal evolution (or lateral gene transfer) generally occurs via three routes;

transformation (DNA uptake), conjugation (direct contact transfer of mobile plasmids) or

transduction (uptake of naked DNA). Lateral gene transfer is believed to be the major route

for widespread global dissemination of antibiotic resistance and is responsible for transfers of

plasmids carrying antibiotic resistance genes (R plasmids) in 60-90% of Gram negative

bacteria (Levy et al., 1988). Lawrence and Ochman (2006) deduced that 17.6% of E. coli

genes have been acquired by lateral gene transfer. Regardless of their physical location, i.e.

chromosome, plasmid or integrons within transposons, antibiotic resistance genes can

undergo lateral gene transfer. Transposons, are the most conducive means of transferring

antibiotic resistance genes amongst bacterial populations. They typically carry a selectable

phenotype (antibiotic resistance) bordered by two insertion sequences, and are unique in

their ability to ‘jump’ from one genetic locus into another, irrespective of taxanomic class.

Transposons often contain integrons, genetic elements which harbor a range of antibiotic

resistance genes, a promoter site, a recombination site downstream of the resistant genes and

an integrase coding gene. They are transferred between bacteria, integrating into bacterial

genomes and/or plasmids. Multi-resistance is achieved when several antibiotic resistance

cassettes are inserted into the integron. There are five major classes of integrons (Mazel et al.,

2006). Class 1 integrons are derived from transposon Tn402 that can insert into the large

Tn21 transposon; Class 2 is exclusively derived from the Tn7 transposon which is highly

adept at integrating into the chromosome of E. coli and other Proteobacteria thus

12

disseminating its resistance genes throughout a large community of bacteria; Class 3 is

probably transposon-associated; and Classes 4 and 5 are linked to trimethoprim resistance in

Vibrio species. Integrons are thought to play a major role in the spread of bacterial antibiotic

resistance. Some resistance genes reside within highly efficient transfer elements. For

example dfr1, the most common trimethoprim resistance gene, is located on both Class1 and

Class 2 integrons. Class 1 integrons have been identified in 40-70% of Gram negative

bacteria isolated from humans and animals.

A well studied group of highly promiscuous plasmids are the IncP-1 plasmids. In

addition to self-transfer, they are capable of coordinating the movement of non-mobilisable

plasmids, which in some cases enables genetic material to be transferred across taxonomic

barriers. This was verified by Schluter et al. (2007) who compared the entire DNA sequence

of nineteen IncP-1 plasmids isolated from STPs, environmental and clinical isolates. These

plasmids were found to contain mobile genetic elements (MGEs) carrying resistance to most

antibiotics, heavy metals such as mercury and chromate, and quaternary ammonium

compounds. They found genes responsible for replication, conjugation, mating pair

formation, plasmid stability and control present on all of the plasmids. They also found

similarities within the IncP- 1α and IncP-1β subsets which were not common to both.

Interestingly, their comparative study showed that the backbone sequences of the IncP-1β

plasmids were highly similar (in one instance 100%) to an IncP-1 degradative plasmid. As a

general rule, similar incompatibility plasmid groups are unable to co-exist within a bacterium

at any one time. It follows that IncP-1β could transfer antibiotic resistance or degradative

genes to other IncP-1 plasmids provided appropriate selective pressures are maintained on

the host. The study also found significant identities within genomes of bacteria from human,

animal and plant origins, suggesting that bacteria from a wide range of environments had

access to a common gene pool at some point in their evolution.

13

Multi-drug resistance is often achieved by the acquisition of a single mobile genetic

cassette harboring several different resistance mechanisms. In addition to the selective

pressure exerted by antibiotic drugs themselves, other antibiotics and/or agents such as

disinfectants and heavy metals may also contribute to the maintenance of antibiotic resistance

(Schluter et al., 2007). Consequently, bacteria can retain resistance to drugs such as

streptomycin and sulphonamides which are rarely used today, simply because their resistance

genes are closely associated with contemporary antibiotics or heavy metal resistance

mechanisms. Resistance gene transfer rates are affected by factors both internal and external

to the bacterium. External influences include those which facilitate DNA transferability such

as temperature, pH, detergents and organic solvents. Internal influences include the ‘SOS’

response to DNA damage which appears to increase the frequency of transfer of certain

resistance traits. An SOS response regulates transcription in reply to external stresses such as

UV radiation and certain antibiotics (ciprofloxacin, trimethoprim and β-lactams), thus

causing metabolic changes and mutations facilitating survival and resistance (Cirz et al.,

2002).

1.2.9 Epidemiology of resistance

Bacteria respect no country’s borders. Because of this there is progressive

intercontinental spread of drug resistant bacteria. At one level, the epidemiology of resistance

is described with reference to the confinement of a geographical unit and referred to as local.

Here most outbreaks and clusters involve a few patients in a unit, and the prevalence of

resistance is often highest in those units where the most vulnerable patients are congregated

and where antibacterial use consequently is heaviest. In a study by Livermore (2002), 2–fold

higher rates were reported of methicillin resistance among staphylococci, ceftazidine

resistance among E.coli and P. aeruginosa, imipenem resistance among P. aeruginosa and

vancomycin resistance among entrococci in patients in intensive care units (ICUs) than in

14

patients in general wards or outpatient wards at the same hospitals. In virtually all European

countries, the prevalence of methicillin resistant S. aureus is higher in ICUs than in general

wards.

At another level, the epidemiology of resistance is national. In Europe, the common

pattern is for resistance to increase in prevalence as one moves southward: it is lowest in

Scandinavia and highest in the Mediterranean countries (Banquero, 1995). In Nigeria, data

have shown that the prevalence of resistance to most drugs tested against E. coli isolates from

apparently healthy students is within a high range and has increased from 1986 to 1998. The

observed increase in prevalence of resistance to streptomycin and tetracycline was

statistically significant (Okeke et al., 2000). For tetracycline, the proportion of resistant

strains increased from <40% to 100% in a 13-year period. In North America, resistance rates

are mostly higher in the United States than in Canada. Some of the worst resistance rates are

in the newly prosperous countries of East Asia and South America. In Korea, Japan, Taiwan,

and Vietnam, 70% - 80% of S, pneumoniae are resistant or intermediately resistant to

penicillin, compared with 30% - 40% in France and Spain, 5% - 10% in the United Kingdom

and 1% - 2% in Scandinavia (Baquero, 1995).

The epidemiology of resistance is partly international with some transferable

determinants prevalent worldwide. The epidemiology is also international to the extent that

some resistant strains spread between countries and continents. For example, report has

shown that multidrug-resistant pneumococci of serotype 6B were imported from Spain into

Iceland, apparently by nasopharyngeal carriage in the children of returning holiday makers

(Kristinsson, 1995). These penumococci then became established in child care centers in

Iceland, causing an increase in the penicillin-resistance rate from 1% in 1988 to 17% in 1993.

Other penicillin-resistant pneumococci of serotype 23F have spread from Spain to the far

East, America, and South Africa (Munoz et al., 1991). Many of the few E. coli and Klebsiella

15

spp with plasmid-mediated AmpC β-lactamases in the United Kingdom are

epidemiologically linked to the Indian subcontinent, where there is evidence of local

frequency in Punjab. Also PER-I ESBL was first recorded from a P. aeruginosa isolate

collected in France and shorlty afterwards was found in numerous P. aeruginosa, Salmonella

and Acinetobacter spp isolates from several cities in Turkey. An inquiry revealed that the

original source patient in France was a Turk, visiting for treatment (Danel et al., 1995).

1.3 Development of multidrug resistance of P. aeruginosa

P.aeruginosa is a major cause of opportunistic infections among immuno-

compromised individuals. The spread of this organism in healthcare settings is often difficult

to control due to the presence of multiple intrinsic and acquired mechanisms of antimicrobial

resistance. Multidrug resistance is increasingly observed in clinical isolates of P.aeruginosa

collected in the United States (Karlowsky et al., 2002).

Multidrug resistance often reflects not one but a combination of resistance

mechanisms. Efflux pumps are common components of multidrug-resistance in P.

aeruginosa isolates, and prevent accumulation of antibacterial drugs within the bacterium,

extruding the drugs from the cell before they have the opportunity to achieve an adequate

concentration at the site of action. The efflux pumps often work together with the limited

permeability of the P. aeruginosa outer membrane to produce resistance to β -lactams,

fluoroquinolones, tetracycline, chloramphenicol, macrolides, TMP, and aminoglycosides

(Schweizer, 2003; Pole and Srikumar, 2001). The multidrug efflux systems of P. aeruginosa

are composed of three proteins that are structurally and functionally joined. P. aeruginosa

and other Gram negative bacteria possess both an outer membrane and a cytoplasmic

membrane, which flank the periplasmic space. The tripartite efflux system is required for

effective removal of compounds across both membranes of the cell. The three components of

16

the efflux system include an energy-dependent pump located in the cytoplasmic membrane

(e.g., MexB), an outer membrane porin (eg., OprM) and a protein joining them (eg., MexA).

The four major efflux systems of P. aeruginosa are MexAB-OprM, MexXY-OprM,

MexCD-OprJ, and MexEF-OprN (Schweizer, 2003; Pole and Srikumar, 2001). MexAB-

OprM and MexXY-OprM contribute to intrinsic multidrug resistance, whereas

overexpression of MexXY-OprM or MexCD-OprJ has been associated with acquired

multidrug resistance. MexAB-OprM and MexXY-OprM may also be overexpressed. In each

case, overexpression is caused by a mutation in one of the genes encoding a protein

regulating expression of efflux system components. The fact that the efflux systems can

mediate resistance to a variety of drug classes makes them very effective mechanisms of

resistance.

1.4 Plasmids

Pasmids are genetic elements that replicate independently of the host chromosomes

(Madigan et al., 2009). Like chromosomes, most plasmids are double-stranded DNA

molecules that have an origin of replication and therefore can be replicated by the cell before

it divides (Nester et al., 2009). Both circular and linear plasmids have been documented, but

most known plasmids are circular (Prescott et al., 2008). Linear plasmids possess special

structures or sequences at their ends to prevent their degradation and to permit their

replication.

Plasmids have relatively few genes, generally less than 30. Their genetic information

is not essential to the host, and cells that lack them usually function normally. However many

plasmids carry genes that confer a selective advantage to their hosts in certain environments

(Prescott et al., 2008).

Plasmids play many important roles in the lives of the organisms that have them. They also

have proved invaluable to microbiologists and molecular geneticists in constructing and

17

transferring new genetic combinations and in cloning genes. Plasmids are able to replicate

autonomously. Single-copy plasmids produce only one copy per host cell. Multiple plasmids

may be present at concentrations of 40 or more per cell. Some plasmids are able to integrate

into the chromosome and are thus replicated with the chromosome. Such plasmids are called

episomes.

Plasmids are inherited stably during cell division, but they are not always equally

apportioned into daughter cells and sometimes are lost. The loss of a plasmid is called curing.

It can occur spontaneously or be induced by treatments that inhibit plasmid replication but

not host cell reproduction. Some commonly used curing treatments are acridine mutagenes,

UV, and ionizing radiation, thymine starvation, antibiotics and growth above optimal

temperatures. Plasmids may be classified in terms of their mode of existence, spread and

function. We have R-plasmids, col plasmids, virulence plasmids and metabolic plasmids.

R-plasmid confers antibiotics resistance to the cell that contains them; they have

genes that code for enzymes capable of destroying or modifying antibiotics. R-plasmids are

of major concern to public health because they spread rapidly throughout a population of

cells. This is possible for several reasons. One of the reasons is that many R-factors are also

conjugative plasmids. However, a non-conjugative R-factor can be spread to other cells if it is

present in a cell that also contains a conjugative plasmid. In such a cell, the R- factor can

sometimes be transferred when the conjugative plasmid is transferred. Even more troubling is

that R-factor is readily transferred among species (Prescott et al., 2008).

Col plasmid contains genes for the synthesis of bacteriocins known as colicins which

are directed against E.coli. Virulence plasmids encode factors that make their hosts more

pathogenic. Metabolic plasmids carry genes for enzymes that degrade substances such as

aromatic compounds and sugars (Prescott et al., 2008).

18

1.4.1 Plasmids and bacterial resistance

Genes for drug resistance may be present on bacterial chromosomes, plasmids,

transposons and integrons. Because they are often found on mobile genetic elements, they

can freely exchange between bacteria. Spontaneous mutations in bacterial chromosones,

although they do not occur often, can make bacteria drug resistant.

Frequently, a bacterial pathogen is drug resistant because it has a plasmid bearing one

or more resistance genes; such plasmids are called R-plasmids. Plasmid resistance genes

often code for enzymes that destroy or modify drugs; plasmid-associated genes have been

implicated in resistance to aminoglycosides, chloramphenicol, penicillin, erythromycins,

sulphonamides and others (Prescott et al., 2008). Once a bacterial cell possesses R-plasmid,

the plasmid may be transferred to other cells quite rapidly through normal gene exchange

processes such as transduction, conjugation and transformation. Because a single plasmid

may carry genes for resistance to several drugs, a pathogen population can become resistant

to several antibiotics even though the infected patient is treated with one drug. Bacteria can

resist the action of antibiotics by preventing the access to the target of the antibiotics,

degrading the antibiotics or rapid extrusion of the antibiotics. The mechanism is shown in

figure 1.

19

Fig 1: Antibiotics resistance mechanism (Prescott et al., 2008).

Fig 2: Horizontal exchange of genetic material like antibiotic resistance through

plasmid by bacteria.

20

CHAPTER TWO

2.0 MATERIALS AND METHODS

2.1 Sample Site

A total of 290 samples were examined of which 170 were of clinical origin

constituting of 140 samples of urine (from in- and out-patients with urinary tract infection)

and 30 wound swabs (from patients with wound, burns). Also, 120 environmental samples

were randomly collected from water, soil, and sewage effluent and were examined. These

specimens were collected from both urban and rural areas of Nsukka.

2.2 Collection of Samples

Urine, sewage effluent, and water samples were aseptically collected with sterile

containers. Wound specimens were collected with sterile swab sticks and soil samples were

collected with sterile polythene bags from the top 0-15cm layer.

2.3 Isolation Procedure

Clinical sample: Samples were processed as follows:

Urine: The samples were mixed thoroughly by inverting the containers several times. Using

a sterile wire loop, the samples were inoculated on MacConkey agar plates. The plates were

incubated at 370C for 24 h. Distinct colonies were subcultured on nutrient agar repeatedly to

obtain pure cultures. The isolates were stored on nutrient agar slants for further use.

Wound: The wound swabs were collected from patients using sterile swab sticks. The swab

sticks were inoculated into tubes of nutrient broth and incubated at 37oC for 24 h. Ten-fold

serial dilutions of the culture broth were prepared. Diluents were plated out on MacConkey

agar using the spread plate method. The plates were incubated at 37oC for 24 h. Different

colonies were further purified to obtain pure cultures. The pure isolates were stored on agar

slants for further use.

21

Environmental samples: Samples were processed as follows:

Water sample: Ten-fold serial dilution method was used with sterile distilled water in a test

tube. Diluents were plated out on Pseudomonas base agar and MacConkey agar. The plates

were incubated at 370C for 24 h. Discrete colonies were picked from the agar plates based on

size and colour of colonies and were stored on agar slant for further identification.

Soil sample: One gram of each soil sample was mixed with 9 ml of sterile distilled water and

shaken for some minutes. The resulting suspension was allowed to settle and the supernant

was serially diluted and plated. The plates were incubated at 370C for 24 h and then,

subcultured repeatedly on nutrient agar.

2.4 Identification of the Isolates

All isolates were Gram stained and examined microscopically. Biochemical tests were

carried out based on Gram reactions. Among the tests carried out were oxidase, sugar

fermentation, indole, citrate, catalase, methyl red test and Voges Proskauer test.

2.4.1 Gram staining

Smears from fresh pure cultures of the isolates were made on grease-free slides

labeled appropriately, dried in the air and fixed by passing it over the Bunsen burner flame

thrice. The smear was flooded with crystal violet and allowed to act for 30 seconds. This was

washed off with water. The smear was flooded with Lugol’s iodine, which acts as a mordant

for 60 seconds. The iodine was washed off the slide, decolourized with ethyl alcohol for 20

seconds before washing it off again. A counter stain, safranin was added and allowed to act

for 30 seconds and washed off quickly with water. The smear was allowed to dry and a drop

of immersion oil was added and the slides were viewed under the microscope using oil

immersion objective lens. Purple colour indicates Gram positive organisms while pink or red

colour indicates Gram negative.

22

2.4.2 Oxidase test

This test was done to differentiate species of Pseudomonas (oxidase positive) from

members of the enterobacteriacea (oxidase negative). A few drops of 1% aqueous solution of

tetramethyl-p-phenylene-diamine hydrochloride reagent were added to a piece of filter paper

in a Petri dish. A smear of the culture was impregnated on the filter paper using sterile

platinum loop or glass rod. Purple colouration indicates oxidase positive result.

2.4.3 Sugar fermentation

This test was carried out to determine the ability of the isolates to metabolize sugar with the

production of acid/gas or gas. The following sugars were prepared and used for the test:

glucose, maltose, lactose and mannitol. In the test, 0.2g of each of the sugars was dissolved in

20 ml of peptone water. A pinch of bromocresol purple was added as indicator and 5 ml

aliquots dispensed into Bijou bottles containing Durham tubes and autoclaved at 115oC for 10

mins. It was allowed to cool and then inoculated with the test organism using sterile wire

loop and afterwards incubated at 37oC for 48 h. A change in colour from purple to yellow

indicated positive result while gas production was shown by the downward displacement of

liquids in the Durham tubes.

2.4.4 Indole test

Indole is a nitrogen-containing compound formed when the amino acid tryptophan is

hydrolyzed by bacteria that have tryptophanase. The test was carried out by inoculating one

loopful of each test isolate separately into pre-sterilized Bijou bottles containing 3 ml of

tryptone water. These were incubated at 37oC for 48 h after which 0.5 ml Kovac’s reagent

was added. The set up was examined by shaking after 1min. A red colour at the interphase

was indicative of indole production.

23

2.4.5 Citrate test

This test uses a medium in which sodium citrate is the only source of carbon and

energy. The medium used was Simmon’s citrate medium. The medium was prepared

according to the manufacturer’s instruction and dispensed into Bijou bottles and autoclaved at

1210C for 15 mins. The bottles were allowed to solidify as slanting slopes. They were

inoculated with cultures of the isolates and incubated for 24 h at 370C. It is positive when it is

blue and negative when it appears green which is original colour.

2.4.6 Catalase test

This test is used to differentiate those bacteria that produce the enzyme catalase such

as staphylococci from non-catalase producing bacteria such as streptococci. The principle of

this is that catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and

water. In this test, 2 ml of hydrogen peroxide solution was poured into a test tube and a glass

rod was used to remove some colonies of the test organism and immersed in the hydrogen

peroxide solution. Bubbles of oxygen gas appeared from those bacteria that produce catalase

whereas no bubbles were formed in those that do not produce catalase.

2.4.7 Methyl red (MR) test

This test depends on the ability of the isolates to produce acid by fermentation of

carbohydrate present in the growth medium. The medium for this test is glucose phosphate

medium. It was prepared by mixing 5 g of peptone and 5g of dihydrogen phosphate in one

litre of distilled water. The mixture was steamed until the solid dissolved and was then

filtered and adjusted to pH of 7.5. Five grams of glucose was added and mixed well. It was

then distributed in 5ml portions into test tubes and sterilized at 115 oC for 10 min. During

sterilization, the tubes were placed on a solid bottom container to protect them from contact

with steam as this may make the medium become straw yellow in colour. After sterilization,

the medium was allowed to cool and the test organism was then inoculated into the broth and

24

incubated for five days at 37oC. After incubation, 5 drops of indicator (methyl red) were

added to the 5 ml culture. Formation of red colour indicated positive result while yellow

colour was taken as negative result.

2.4.8 Voges Proskauer test

The medium for this test is glucose phosphate. The medium was prepared, sterilized,

inoculated and incubated as stated in MR test. After incubation, 0.6 ml of 5 % α-naphtol and

0.2 ml of 40 % aqueous KOH were added and shaken. The result was recorded after 15 mins.

Formation of red colour indicated positive result.

2.5 Antibiotic Susceptibility Test

The antimicrobial susceptibility test was performed by using Disc diffusion method

according to Bauer (1966) on Mueller-Hinton agar medium. The following antibiotics were

used augmentin(30µg), gentamycin(10µg), pefloxacin(30µg), ofloxacin(10µg),

streptomycin(30µg), chloramphenicol(30µg) sparfloxacin(10µg), ciprofloxacin(10µg),

amoxacillin(30µg) and septrin(30µg) (Maxicare medicals). Pure cultures of isolates were

inoculated in nutrient broth and incubated at 37 0C for 24 h. The growth was standardized by

diluting the culture with normal saline to match the turbidity of 0.5 McFarland standards.

Then 0.1 ml was collected and spread on the surface of Mueller-Hinton agar using sterile

wool swab of each of the cultures and allowed to dry. The antibiotic disks were placed

carefully to make good contact with the agar surface using sterile forceps and sufficiently

separated from each other in order to prevent overlapping of the zones of inhibition. The

agar plates were left on the bench for 30 min to allow for diffusion of the antibiotics and were

incubated at 37 0C for 24 h. Results were recorded by measuring the zone of inhibition and

comparing with the NCCLS susceptibility testing (NCCLS, 2004).

25

2.6 Plasmid Curing

Resistance curing was conducted on multidrug resistant isolates. This was done to

determine whether the gene coding for resistance is carried in the chromosomes or plasmids.

Plasmid being an extra-chromosomal DNA molecule, is eliminated from host bacteria after

exposure to sub-lethal concentrations of intercalating agents such as acridine orange,

ethidium bromide and detergents such as sodium dodecyl sulphate. The curing agent used in

this work was sodium deodecyl sulphate. The experiment was done according to the method

of Tomoeda et al. (1968).

2.6.1 Use of sodium dodecyl sulphate (SDS)

Two concentrations (1% and 5%) of SDS in nutrient broth were used in this

experiment. Nutrient broth was prepared and supplemented with 1g of SDS in one batch of

99 ml and 5 g of SDS in the second batch of 95 ml to achieve a final concentration of 1% and

5% (w/v) SDS, respectively. It was then sterilized by autoclaving at 121 oC for 15 min.

Selected overnight cultures of isolates were standardized to 0.5 McFarland turbidity

standard using sterile saline solution. From these, 0.1ml of each culture was inoculated

separately into 5 ml of SDS-supplemented nutrient broth in test tubes and incubated at 37oC

for 24 h. After incubation, cultures were diluted to 0.5 McFarland’s standard and spread on

Mueller-Hinton agar and susceptibility testing carried out.

2.7 Statistical Analysis

The data obtained was analyzed using one-way and two-way analyses of variance

(ANOVA) to check the level of significance.

26

CHAPTER THREE

3.0 RESULTS

Isolation of organisms

The organisms were isolated from urine, wound, water and soil using standard

bacteriological procedure. The results of the number of isolates from different samples are

shown in Table 1.

Table 1: Isolates from both urban and rural hospitals and environment.

Site Sample No. of samples E. coli P. aeruginosa

Urban hospital Urine 70 43(61.4%) 30(42.9%)

Wound 15 7(46.7%) 8(53.3%)

Rural hospital Urine 70 37(52.9%) 24(34.28%)

Wound 15 4(26.7%) 6(40%)

Urban-

Environment Water 30 12(40%) 4(13.3%)

Soil 30 5(16.7%) 15(50%)

Rural-

Environment Water 30 9(30%) 3(10%)

Soil 30 7(23.3%) 19(63.3%)

Out of 290 samples analyzed, 233 isolates were obtained of which E .coli was 124 and P.

aeruginosa was 109. Table 1 shows the number of isolates from different samples of both

urban and rural hospitals and environment. The highest prevalence of E. coli was shown in

urine from urban hospital with percentage prevalence of 61.4 % and least from urban soil

with percentage prevalence 16.7 %. The highest prevalence of P. aeruginosa was shown in

27

soil from rural environment with percentage prevalence of 63.3 % and least from water in

rural environment with percentage prevalence of 10 %

Antibiotic resistance of urine and wound isolates from urban hospital

Figure 1 shows the antibiotic resistance patterns of E. coli and Pseudomonas

aeruginosa isolated from urine samples from urban hospital. The results showed that both E.

coli and Pseudomonas aeruginosa exhibited the highest resistance to septrin with percentage

resistance of 83.7 % and 90 %, respectively. E. coli showed the least resistance to

ciprofloxacin with percentage resistance of 41.9 while Pseudomonas aeruginosa showed

least resistance to sparfloxacin with percentage resistance of 23.3 %.

The results of the antibiotic resistance of the wound isolates from urban hospital to

different antibiotics are presented in Figure 2. The isolates showed 90% resistance to

augumentin and 87 % amoxicillin. E. coli also showed 66.7 % resistance when

chloramphenicol and pefloxacin were used. Pseudomonas aeruginosa showed 87.5 %

resistance when pefloxacin was used and also showed high resistance to septrin and

chloramphenicol with percentage resistance of 87.5 and 75 %, respectively.

E. coli showed the least resistance to gentamycin, ofloxacin and streptomycin with

percentage resistance of 16.7 % for each of them. P. aeruginosa exhibited the least resistance

when Streptomycin was used with percentage resistance of 12.5 %.The result showed there

was a significant difference (p≥0.05) in the percentage resistance exhibited by the isolates at

different concentrations of antibiotics used.

28

0

10

20

30

40

50

60

70

80

90

100

AU CN PEF OFX S SXT CH SP CPX AM

% R

esista

nce

Antibiotics

E.coli

P. aeruginosa

Figure 1: Percent resistance to antibiotics of urine isolates from urban hospital.

AU- augumentin, CN-gentamycin, PEF-pefloxacin, OFX-ofloxacin, S-streptomycin,

SXT-septrin, CH-chloramphenicol,SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

29

Figure 2: Percent resistance to antibiotics of wounds isolates from urban hospital.

Au-augumentin, CN-gentamycin, PEF-pefloxacin, OFX-ofloxacin, S-streptomycin, SXT-

septrin, CH-chloramphenicol,SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

0

10

20

30

40

50

60

70

80

90

100

AU CN PEF OFX S SXT CH SP CPX AM

% R

esis

ta

nce

Antibiotics

E.coli

P.aeruginosa

30

Antibiotic resistance of urine and wound isolates from rural hospital

The results of antibiotics resistance of urine and wound isolates from rural hospital is

shown in Figures 3 and 4.

In Figure 3, the results of antibiotic resistance by the urine isolates are presented. The results

showed that E. coli has the highest resistance to amoxacillin with percentage resistance of

67.6 % while Pseudomonas aeruginosa showed the highest resistance when amoxacillin was

used with percentage resistance of 83.3 %, E. coli showed 62.2 % resistance when

augumentin was used while Pseudomonas aeruginosa showed 79.2 % resistance when each

of augumentin and pefloxacin was used. E. coli and P. aeruginosa showed the least resistance

when septrin was used with percentage resistance of 10.8 % and 12.5 %, respectively.

Figure 4 showed the resistance of isolates from wound to different antibiotics.

Both isolates showed 89 % resistance to augumentin and amoxicillin, P. aeruginosa also

showed 66.7 % resistance to pefloxacin while E. coli showed 75% to the same pefloxacin. E.

coli showed least resistance of 25 % to each of gentamycin, ofloxacin, septrin, streptomycin

and chloramphenicol while P. aeruginosa showed the least resistance of 33.3% to

chloramphenicol, gentamycin and streptomycin.

The results showed that there was significant different (P≤ 0.05) in the resistance of the

isolates from urine and wound when their resistance to different antibiotics were compared.

31

0

10

20

30

40

50

60

70

80

90

100

AU CN PEF OFX S SXT CH SP CPX AM

% R

esista

nce

E.coli

P. aeruginosa

Figure 3: Percent resistance to antibiotics of urine isolates from rural hospital.

AU-augumentin, CN-gentamycin, PEF-pefloxacin, OFX-ofloxacin, S-streptomycin, SXT-

septrin, CH-chloramphenicol, SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

32

Figure 4: Percent resistance to antibiotics of wound isolates from rural hospital

AU-augumentin,CN-gentamycin,PEF-pefloxacin,OFX-ofloxacin,S-streptomycin, SXT-septrin,CH-

chloramphenicol,SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

0

10

20

30

40

50

60

70

80

90

100

AU CN PEF OFX S SXT CH SP CPX AM

% R

esis

ta

nce

Antibiotics

E.coli

P.aeruginosa

33

Antibiotic resistance of isolates from water and soil from urban environment

The results of the resistance of the isolates from urban samples, soil and water to the

antibiotics are presented in Figures 5 to 6.

The results of the percentage resistance of the isolates from water to different

antibiotics are presented in Figure 5. The results showed that E. coli had the least resistance

of 8.3 % with each of ciprofloxacin and gentamycin and highest resistance of 41.7% with

septrin. P. aeruginosa had the highest resistance when augumentin, pefloxacin, sparfloxacin

and amoxicillin were used with resistance of 75 % for each antibiotics.

Figure 6 showed the results of the percentage resistance of the isolates from soil to

different antibiotics. E. coli showed least resistance to ofloxacin and ciprofloxacin and

highest resistance to augumentin and amoxacillin with percentage resistance of 50 % while P.

aeruginosa showed different degrees of resistance to all the antibiotics. There was a

significant difference (P≤ 0.05) in the percentage resistance to different antibiotics by the

isolates.

34

Figure 5: Percent resistance to antibiotics of water isolates from urban environment.

AU-augumentin, CN-gentamycin, PEF-pefloxacin, OFX-ofloxacin,S-streptomycin,SXT-septrin, CH-chloramphenicol, SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

0

10

20

30

40

50

60

70

80

90

AU CN PEF OFX S SXT CH SP CPX AM

% R

esis

ta

nce

Antibiotics

E.coli

P.aeruginosa

35

0

10

20

30

40

50

60

70

80

AU CN PEF OFX S SXT CH SP CPX AM

% R

esis

ta

nce

Antibiotics

E.coli

P.aeruginosa

Figure 6: Percent resistance to antibiotics of soil isolates from urban environment

Au-augumentin, CN-gentamycin, PEF-pefloxacin, OFX-ofloxacin, S-streptomycin, SXT-

septrin, CH-chloramphenicol, SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

36

Resistance of isolates of water and soil from rural environment to different antibiotics

The resistance of isolates from water and soil from rural environment to different

antibiotics were also evaluated. The results are presented in figures 7 and 8.

The results of percentage resistance of water isolates to different antibiotics are

presented in Figure 7. E. coli showed no resistance to ofloxacin and ciprofloxacin and

highest resistance to augumentin with percentage resistance of 33.3 %. P. aeruginosa from

water in the rural environment showed highest resistance to augumentin and amoxicillin with

percentage resistance of 66.7 % and no resistance to streptomycin. The result showed a

significant difference (P≤ 0.5) in the percentage resistance by the isolates to different

concentrations of antibiotics.

The percentage resistance of soil isolates from rural environment are presented in

Figure 8. The isolates from soil in rural environment showed low resistance to the antibiotics.

The highest resistance showed by E. coli was 40 % which occurred when amoxacillin and

augumentin were used whereas the highest resistance shown by Pseudomonas aeruginosa

was 46.7 % which occurred when augumentin and amoxicillin were used.

37

Figure 7: Percent resistance to antibiotics of water isolates from rural environment.

AU-augumentin,CN-gentamycin,PEF-pefloxacin,OFX-ofloxacin,S-streptomycin, SXT-septrin,CH-chloramphenicol,SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

0

10

20

30

40

50

60

70

80

AU CN PEF OFX S SXT CH SP CPX AM

% R

esis

ta

nce

Antibiotics

E.coli

P.aeruginosa

38

0

10

20

30

40

50

60

AU CN PEF OFX S SXT CH SP CPX AM

% R

esis

ta

nce

Antibiotics

E.coli

P. aeruginosa

Figure 8: Percent resistance to antibiotics of soil isolates from rural environment

AU-augumentin,CN-gentamycin,PEF-pefloxacin,OFX-ofloxacin,S-streptomycin, SXT-septrin,

CH-chloramphenicol,SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

39

Effect of SDS mediated plasmid curing on antibiotics resistance pattern of E. coli and P.

aeruginosa

The effect of SDS mediated plasmid curing on antibiotic resistance pattern of E. coli

and P. aeruginosa was also evaluated. The results are presented in Table 2. The results

showed that there was a significant difference (P≤ 0.05) in the resistance pattern at 1 % and 5

% SDS when the resistance at the above concentration were compared. At 1 % SDS, the

isolates showed greater resistance to the antibiotics while at 5 % SDS, the isolates were

sensitive to most of the antibiotics. However , one of the P. aeruginosa that was initially

sensitive to chloramphenicol become completely resistant at 5 % SDS and another isolate

of P. aeruginosa that were initially to septrin, sparfloxacin and ciprofloxacin became

completely resistant at 1% and 5 % SDS.

40

Table 2: Effect of SDS mediated plasmid curing on antibiotic resistance Isolate

Resistance 1%SDS concentration

5%SDS concentration

E. coli Resistant to all All resistance except

CN, S

Sensitive to all

E. coli Resistant to all except S Resistant to all

except CN,S,OFX

All sensitive

E. coli Resistant to all Resistant to all

except CN, S

Sensitive CN, S

E. coli Resistant to all expect CH, All resistance except

CN,S,OFX,CH

All resistance except

CN, S,CH,OFX,

E. coli Resistant to all except

CN,OFX,CH

All resistance except

CN,OFX,S,CH

All sensitive

E. coli All resistance except

CN,OFX,S

All resistance except

CN,OFX,S,CPX

All sensitive

P. aeruginosa All resistance except CN, S All resistance except

CN,OFX, S

All resistance except

CN,OFX,S,

P. aeruginosa All resistance except CN All resistance except

CN,OFX,S

All resistance except

CN,OFX,S

P. aeruginosa All resistance except

CPX

All resistance except

CN,S,CPX

All resistance except

CN,OFX,S,CPX

P. aeruginosa All resistance except

CN,OFX,CH

All resistance except

CN,OFX,CH

All resistance except

CN,OFX,S,CH

P. aeruginosa

P.aeruginosa

All resistance except S,CH

All resistance except SXT,SP,CPX

All resistance except

to CN,OFX, S,CH

All resistance except CN,S

All resistance except

CN,OFX,S

All resistance except

CN, S

P. aeruginosa All resistance except S,CH All resistance except

CN,S, CH

All resistance except

CN,S,

AU-augumentin, CN-gentamycin, PEF-perfloxacin, OFX-ofloxacin, S-streptomycin, SXT-septrin, CH-chloramphenicol, SP-sparfloxacin, CPX-ciprofloxacin, AM-amoxacillin

41

CHAPTER FOUR

4.0 DISCUSSION

Multiple antibiotic resistance in bacterial population is currently one of the greatest

challenges in the effective management of infections. Antimicrobial drugs have been proved

remarkably effective for the control of bacterial infections. However, it was soon evidenced

that bacterial pathogens were unlikely to surrender unconditionally and some pathogens

rapidly became resistant to many antibiotics (Cheesbrough, 2006).

In this study, several clinical and environmental samples of urine, wound, water and

soil were examined for the presence of multidrug resistant E. coil and Pseudomonas

aeruginosa and the effect of SDS-mediated plasmid curing on antibiotic resistance patterns of

the isolates were evaluated using 1% and 5% concentrations.

The results of this showed that there was higher prevalence of E. coli in urine than

wound. But when wound was analyzed there was higher prevalence of P. aeruginosa. The

results agreed with the findings of Anuratha et al. (2008) who reported that P. aeruginosa

was the most frequent of isolates obtained from burn wound. Evaluation of environmental

samples showed that E. coli was also more frequent in water when compared to soil, whereas

P. aeruginosa was more frequent in soil; this occurred in both urban and rural environments.

The isolates were tested for resistance to different antibiotics. The result showed that E. coli

isolated from urine from urban hospital showed higher resistance to septrin with percentage

resistance of 83.7 %. This isolate also showed higher resistance to amoxacillin and

augumentin with percentage resistance of 76.7 and 72.1 %, respectively; P. aeruginosa had

highest resistance when amoxacillin was used and least resistance when sparfloxacin was

used. The isolates showed some degree of resistance to all the antibiotics. This agrees with

the result of Manikandan et al. (2011) which reported multidrug resistance by bacteria

isolated from UTI. This also in line with the result of Uchenna (2005) who reported

42

multidrug resistance by Gram negative bacteria. The evaluation of resistance of isolates of

wounds from urban hospital also indicated different degrees of resistance to the antibiotics.

These are in keeping with the results of the studies conducted by Okesola and Oni (2009) in

south western Nigeria. E. coli showed complete resistance to augumentin, amoxicillin,

chloramphenicol and pefloxacin, with percentage resistance of 90 %, 87 %, 66.7 %, and 66.7

%, respectively while P. aeruginosa showed similar action when augumentin and amoxacillin

were used. The result obtained also agreed with the work of Gales et al. (2001) who reported

presence of multidrug resistance of P.aeruginosa against various antibiotics. Harbottle et al.

(2006) reported that the overuse of antibiotics has become the major factor for the emergence

and dissemination of multi-antibiotics resistance strain of several bacteria. In these regards,

P. aeruginosa antibiotic resistance was raised from both intrinsic and acquired resistance.

Gehan et al. (2011) reported complete resistance to amoxacillin by Pseudomonas aeruginosa.

The resistance pattern of the urine isolates from rural hospital was not indifferent from the

ones from urban hospital; they also displayed varying degrees of resistance to different

antibiotics. E. coli and P. aeruginosa showed higher resistance when amoxicillin was used

with percentage resistance of 67.6 % and 83.3 % respectively. They also showed high

resistance when augumentin was used, both of the isolates showed least resistance when

septrin was used with percentage resistance of 10.8 % and 12.5 %, respectively. This result

agrees with the findings of Olowu et al. (2008) who reported multidrug resistance by E. coli.

Fred (2006) also reported multidrug resistance by P. aeruginosa. He also reported that

multidrug resistance often reflects not one but a combination of resistance mechanisms.

Efflux pumps are common components of multi-drug resistant Pseudomonas aeruginosa

isolates and they prevent accumulation of antibacterial drugs within the bacterium, extruding

the drugs from the cell before they have the opportunity to achieve an adequate concentration

at the site of action (Fred, 2006). This study also investigated the resistance of wound isolates

43

from rural hospital to different antibiotics. All the isolates showed different levels of

resistance to the antibiotics. E. coli and Pseudomonas aeruginosa showed complete

resistance to augumentin and amoxacillin to be 89 %. This deviated from the work of Iheanyi

et al. (2009) who reported 100% resistance to several antibiotics by bacterial isolates. E. coli

also showed low resistance to septrin, ofloxacin, gentamycin and chloramphenicol with each

of the antibiotics having percentage resistance of 25%. This agrees with the work of Onifade

et al. (2005) and Aiyegoro et al. (2007) who reported sensitivity to ofloxacin to be 25 %.

This is a deviation from the report of Chikere et al. (2008), who reported the sensitivity of

Gram negative isolates to pefloxacin, gentamycin and ciprofloxacin to be 100 %.

The isolates of water from rural environment showed little or no resistance to the

antibiotics, E. coli showed no resistance to pefloxacin, ofloxacin, septrin and ciprofloxacin

and low resistance to other antibiotics whereas P. aeruginosa showed no resistance to the

entire antibiotics.

This result agreed with the work of Tambekar et al. (2008) which reported that P.

aeruginosa was highly sensitive to ofloxacin and gentamycin. This result also agreed with the

work of Aiyegoro et al., 2007 and Chikere et al. (2008). The isolates of soil from rural

environment also showed low resistance to the antibiotics. There was a significant difference

between the isolates from soil and that from water. The highest resistance record by E. coli

was 42.8 % which occurred when amoxacilin and augumentin were used whereas the highest

observed for Pseudomonas aeruginosa was 47.4 % which occurred when augumentin and

amoxacillin were used. This is far below what was obtainable when clinical isolates were

tested. This was in line with the findings of Shahid and Malik (2005) who reported that 96 %

of clinical Pseudomonas aeruginosa isolates was multidrug resistant and the majority (71.4

%) were resistant to five or more antibiotics, the clinical isolates were more resistant to the

antibiotics than environmental isolates (Shahid and Malik, 2005).

44

In general, the clinical isolates are more resistant to the antibiotics than environmental

isolates. The high level resistance to these antibiotics might be attributed to antibiotic

bacterial emergence because of improper and extensive use of these antibiotics, antibiotic

discharge in various amounts in the environment, indiscriminate use of antibiotics in medical,

veterinary and agricultural practices leads to multiple antibiotic resistance in bacterial

pathogens (Diabet al., 2002).

Thirteen isolates of E. coli and P. aeruginosa that were resistant to at least seven

antibiotics were subjected to plasmid curing. Result showed that when the isolates were

treated with 1 % SDS, some of the isolates become susceptible to most of the antibiotics.

When it was increased to 5% SDS, the number that was susceptible increased but it was also

noticed that some of the strains become resistant at this high increase. This result showed

that some of the strains had resistant plasmid which can promote the transfer of resistance to

other strains. This is in line with the work of Davidson (1999) who reported possible transfer

of resistance between bacteria in clinical and environmental samples. These studies have

revealed the prevalence of antibiotic resistant E. coli and P. aeruginosa in the clinical and

environmental samples and possible transfer of resistance to other strains.

Conclusion

This study has shown that there are multiple antibiotic resistant E. coli and P.

aeruginosa in clinical and environmental samples; the clinical isolates are more resistant to

antibiotics than the environmental isolates. The isolate from water had little or no resistance

to antibiotic and the study has also shown that there is a possible transfer of resistance from

one strain to another.

45

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