PREVALENCE OF ANTIBIOTIC RESISTANCE AND EXTENDED-SPECTRUM β-LACTAMASE (ESBLs) GENES IN ESCHERICHIA COLI ISOLATED FROM NON-DIARRHOEA STOOLS IN NSUKKA.

ABSTRACT

Antibiotic resistance has emerged as a major threat to global health. Extended spectrum β- lactamases (ESBLs) are enzymes that confer resistance to β-lactam antibiotics and acquisition of ESBL genes and integrons by Escherichia coli is increasingly recognized, being associated with resistance to multiple antibiotics. This study investigated the prevalence of multidrug resistance (MDR), ESBLs genes and integrons in E. coli isolated from non-diarrhoeal human stools. Stool samples were collected from both hospitals and non-hospital locations within the Campus, Town and Onuiyi areas of the university-town of Nsukka. The stool samples were processed by directly inoculating the faecal matter onto MacConkey agar. Dark pink colonies were presumed to be E. coli and subcultured on a chromocult coliform agar (CCA). Colonies that appeared purple on CCA were further characterized using relevant biochemical tests and gram staining. Purple colonies on chromocult coliform agar were confirmed as E. coli by PCR detection of the target tuf gene.   Antibiotic susceptibility was determined by the Kirby-Bauer disc diffusion method, with E. coli ATCC 25922 as control strain and interpreted using the Clinical and Laboratory Standard Institute standards. The prevalence of integrons was determined using PCR and specific primers. Multiplex PCR was used for the screening of genomic DNA for antibiotic resistance genes  (ARGs)  including  blaCTX-M,  blaTEM,  blaOXA,  blaSHV   and  blaNDM.  Differences  in  the resistance of isolates from children and adults, and hospital and non-hospital locations were determined using students’ t-test. A total of 124 E. coli isolates were obtained from 300 non- diarrhoeal stool samples. Sixty-nine (55.64%) out of the 124 E. coli isolates were from female subjects and 55 (44.35%) were from males and 75 (60.48%) and 49 (39.52%) from adults and children respectively. Seventy-five isolates (60.48%) were resistant to tetracycline and 62 (50%) to ciprofloxacin. Only 14 (11.29%) of the isolates were resistant to the beta-lactam antibiotic, cefuroxime. All the isolates (100%) were sensitive to imipenem. Antibiotic resistance was significantly higher (P<0.05) in isolates from adults than those from children, and higher in isolates from non-hospital than those from hospital locations. Multi-drug resistance was observed in 122 (98.34%) of the 124 isolates. ESBLs genes (blaCTX-M and blaTEM) were detected in 85%, blaOXA  in  10% of 20  selected MDR  E.  coli  isolates  (including  all  the  cefuroxime-resistant isolates). Seven (35%) of the 20 isolates screened for ARGs harboured two or more resistance genes (blaCTX-M,  blaTEM, blaOXA  and integrons). Integrons were detected in 5 (25%) of the 20 isolates. The prevalence of MDR, ESBLs genes and integrons in E. coli isolated from non- diarrhoeal human stools is very high in Nsukka and could contribute to resistance dissemination among bacterial species in the gut and the environment. This constitutes public health threat and necessitates continued surveillance of antibiotic resistance.

CHAPTER ONE

INTRODUCTION

1.1 Introduction

Antibiotic resistance has emerged as a major threat to global health (Machado et al., 2013; da Costa et al., 2013; van Schaik, 2015) and puts humans, animals and the environment at risk. Surveillance of antibiotic resistance is a path to ending it, and it has been suggested, about 30 years ago, that commensal microbes could play a role in disseminating bacterial resistance (Antoine, 2003). The human gastrointestinal tract (GIT) provides an ideal combination of factors for antibiotic resistance genes to arise and spread through bacterial populations. One of these factors, in addition to antibiotic exposure, is high cell density (Chaudhary et al., 2014). The human GIT is home of a dense microbiota, housing numerous bacterial cells. This microbial ecosystem is frequently exposed to different antibiotics, because of the regular use of antibiotics in the clinics and farm animals. Hence, it can provide an essential reservoir of antimicrobial resistant microbes which act as opportunistic pathogens or as distributors of resistance genes to other bacteria (de Vries et al., 2011).

Although Escherichia coli exists as a ubiquitous commensal member of the GIT flora (Chen et al., 2011; Sani et al., 2015; Moshtagian et al., 2016), some strains are pathogenic, causing a variety of infections including urinary tract infections, meningitis and gastroenteritis (Filho et al.,

2015). Colonization of healthy subjects with antibiotic resistant E. coli could contribute to the increase  of  resistant  bacteria  both  at  community  and  nosocomial  settings.  Mobile  genetic elements  (plasmids,  integrons,  etc.)  are  widely  present  within  the  gut  microbiota,  often containing multiple genes that provide their bacterial hosts with antibiotic resistance (Arnold et al.,  2016)  factors.  Commensal  strains  can  acquire  antibiotic  resistance  via  horizontal  gene transfer (HGT) (El-Rami et al., 2012; Porse et al., 2017). Acquisition of integrons and beta- lactamase-encoding genes by E. coli is increasingly recognized, being associated with resistance to multiple antibiotics (Machado et al., 2013; Arnold et al., 2016).

Extended spectrum β-lactamases (ESBLs) are enzymes produced by the members of the family Enterobacteriaceae (Poulou et al., 2014; Abike et al., 2018; Peker et al., 2018). They confer resistance  to  β-lactam  antibiotics,  including  penicillins,  cephalosporins,  carbapenems  and

monobactams (Kim et al., 2016; Candan and Aksöz, 2017; Nahar et al., 2018). The inactivation

of the β-lactams antibiotics is achieved by the hydrolysis of the β-lactam ring (Kong et al.,

2010). Antibiotics of ��-lactam derivatives are used worldwide and about 50% of all prescription belongs to this class. The most common of this class of enzymes are CTX-M and SHV types (Bush and Fisher, 2011). ESBLs were basically limited to health care setting, but recently, a rise in the prevalence of ESBL was reported beyond the health care setting (Woerther et al., 2010). The  blaCTX-M   genes  are  often  located  within  integrons,  which  mediate  the  transmission  of antimicrobial resistance genes among the strains of the  Enterobacteriaceae family (Kim et al.,

2016).

Genotypic detection of ESBLs genes in E. coli is important for epidemiological purposes as well as for limiting the spread of resistance mechanisms. However, studies of E. coli tend to focus on diarrhoeagenic pathogens, overlooking the resident, nonpathogenic E. coli community and there is therefore paucity of data on ESBLs genes in E. coli from non-diarrhoea stools.

1.1.1 Statement of the problem

Antibiotic resistance is a growing global problem (Collignon et al., 2015; Purohit et al., 2017), and multi-drug resistance in E. coli has been reported globally (Chigor et al., 2010; Nyanga et al., 2017). Therapeutic failures associated to antimicrobial resistance increases morbidity and mortality, (Bailey et al., 2010; Santajit and Indrawattana, 2016; Purohit et al., 2017), complicate and limit the choice of therapeutic agents (da Costa et al., 2013; Huddleston, 2014). ESBLs genes confer multidrug resistance to β-lactam antibiotics, like penicillins, cephalosporins, and monobactams (Kim et al., 2016; Nahar et al., 2018) and other antibiotics classes. Since antimicrobial resistance of E. coli varies geographically over time (Nyanga et al., 2017), continuous antimicrobial resistance genes examination is a mean of preventing emergence of antimicrobial resistant strains.

1.1.2 Aim

This study was aimed at evaluating the prevalence of antibiotic resistance and ESBLs genes and integrons in E. coli isolated from non-diarrhoeal stools.

1.1.3    The specific objectives

❖ To isolate E. coli from non-diarrhoea stool samples.

❖ To determine antibiotic susceptibility pattern of E. coli isolates.

❖ To characterize the isolates for ESBL genes. ❖ To screen the isolates for integrons.

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