Phenotypic and molecular characterization of antimicrobial resistant Escherichia coli from urinary tract infections in Port-Harcourt, Nigeria

Introduction Multidrug resistance among Escherichia coli causing Urinary Tract Infections (UTIs) is a major public health problem, threatening the effective treatment of UTIs. This study investigated the phenotypic and molecular characteristics of E. coli associated with UTIs in Port-Harcourt, Nigeria. Methods Twenty-five non-duplicate isolates of E. coli from UTIs patients at the University of Port-Harcourt Teaching Hospital, Nigeria were identified using Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry. The antimicrobial susceptibility patterns were determined using Kirby-Bauer disc diffusion technique. Phenotypic expression of Extended Spectrum Beta Lactamases (ESBLs) and AmpC beta-lactamase were determined using standard laboratory methods and polymerase chain reaction (PCR) was used to detect ESBLs, AmpC, Quinolones and Aminoglycosides resistance genes. Results The isolates exhibited high rates of resistance to co-trimoxazole (76%), nalidixic acid (68%), ciprofloxacin (60%), gentamicin (44%) and low resistance to cefotaxime (20%) but were fully susceptible to cefoperazone/sulbactam, amikacin, nitrofurantoin, colistin and carbapenems. Phenotypic expression of ESBLs was recorded in 6(24%) isolates while genotypic detection revealed the highest prevalence of blaTEM 22(88%), followed by blaCTX-M-15 16(64%), blaSHV 7(28%) and blaOXA-1 6(24%) while AmpC (blaCMY-2) gene was detected in 8(32%) isolates. Amongst the quinolone resistant isolates, qnr variants (qnrB, qnrD and qnrS) and aac(6')-Ib genes were detected in 7(28%) and 3(12%) isolates respectively while all gentamicin resistant isolates possessed the aacC2 gene. The co-expression of blaCTX-M-15 with quinolones and aminoglycoside genes were 20% and 40% respectively. The prevalence of multiple drug resistance was 52%. Conclusion A high proportion of the studied E. coli isolates co-expressed ESBLs, quinolones and aminoglycosides resistance genes which call for prompt antibiotic stewardship and preventive strategies to limit the spread of these genes.


Introduction
Escherichia coli strains are common bacteria that inhabit human gastrointestinal tract, whilst they are often harmless commensals; they can cause multitude of infections such as urinary tract infections (UTIs), meningitis, diarrhoea and septicemia [1]. Their harmless strains can remain commensals as long as they do not acquire genetic elements encoding virulence factors which may eventually result in these diseases [2]. The alarming increase in the rate at which these strains acquire antibiotic resistance genes has limited therapeutic options especially for UTIs for which extensive use of antibiotics has been witnessed in both community and hospital settings [1,3].
Extended Spectrum Beta Lactamases (ESBLs) expression among E. coli strains encodes resistance to oxyiminocephalosporins and many other important groups of antibiotics, thereby causing impediment to treatment of its infections [4,5]. Also, the carbapenems which are the last resort in the effective treatment of severe ESBL-producing E. coli infections, have recently witnessed rise in resistance by E. coli strains that produced carbapenem-hydrolyzing enzymes [4,6,7]. Aminoglycosides have been an essential component of the antibiotic armory in the treatment of serious life threatening infections and UTIs caused by E. coli, but the increasing wind of antibiotic resistance across the globe has reduced their effectiveness, rendering some members of this class of antimicrobials virtually useless in certain E. coli infections [8]. The ineffectiveness of aminoglycosides has been attributed to the expression of aminoglycoside-modifying enzymes {nucleotidyltranferases (ANTs), phosphotransferases (APHs), or acetyltransferases (AACs)} which catalyze the modification of the 2-deoxystreptamine nucleus or the sugar moieties [9]. An increase in resistance to gentamicin has been reported amongst isolates of E. coli associated with UTIs in many parts of Nigeria and other African countries [10][11][12].
The advent of fluoroquinolones, the new generation of quinolones antimicrobial agents brought a ray of hope to the treatment of various infections caused by multi-drug resistant bacteria and became the drug of choice for the empiric therapy of most serious life threatening infections [1,3]. However, the extensive use of these agents in clinical settings has made bacteria to develop resistance to them all over the world [3,13]. Fluoroquinolones are one of the most widely used drugs in the treatment of UTIs but their frequent use in both community and hospital settings has led to a dramatic rise in resistance amongst E. coli causing UTIs [7,12,13]. Quinolones inhibit the DNA replication in E. coli strains by targeting the bacterial DNA gyrase (topoisomerase II) and topoisomerase IV (parC) enzymes but mutations in the specific domains of gyrA, gyrB, parC and parE can cause changes in single amino acid of either gyrase or topoisomerase IV leading to the bacterial resistance to quinolones [14]. High-level of fluoroquinolone resistance in E. coli strains has been attributed to multiple mutations in the quinolone-determining resistant regions (QRDR) of topoisomerase enzymes [1,9]. Various community and hospital based studies from Nigeria and other African countries have reported a varying prevalence of phenotypic and genotypic ESBL producing enterobacteriaceae [15][16][17][18][19]

Results
In total, only 25 non-duplicate E. coli isolates from patients with UTIs were identified. The antimicrobial resistance profile of the isolates revealed a high resistance (60 -76%) to co-trimoxazole (folate inhibitor) and quinolones-fluoroquinolones group, moderate resistance to gentamicin and low resistance to taxobactam/piperacillin and the cephalosporins. All isolates were susceptible to cefoperazone/sulbactam, amikacin, nitrofurantoin, colistin and carbapenems as described in Figure 1.   (Table 3).
Amongst the MDR isolates, 10 (76.9%) had at least two ESBLs encoding genes while 3 (30%) isolates also had pAmpC and ESBLs encoding genes. The isolates that possessed blaOXA gene were significantly associated with MDR (p = 0.039) than those that possessed other screened ESBL genes. However, no significant association was observed between the isolates that possessed pAmpC gene (blaCMY-2) and exhibition of MDR (p = 0.411).
On PCR screening of gyrA and parC genes in eight randomly selected isolates, all were found to be positive. Three (37.5%) of these isolates also carried at least two of the screened β-lactamase and aminoglycosides encoding genes ( Table 3). The screening for PMQRs genes revealed the presence of qnr variants (qnrB, qnrD and qnrS) and aac(6')-Ib genes in 7 (28%) and 3 (12%) of the isolates respectively. These were qnrB (n=1), qnrD (n=5) qnrS (n=4). Three isolates carried both qnrD and qnrS while another carried only qnrS.
Two other isolates carrying qnrD also had aac(6')-Ib gene while the isolate carrying qnrB also had aac(6')-Ib. All these isolates had ciprofloxacin MICs of 256 -512 µg/ml, and also carried at least two of the screened ESBLs encoding genes ( Table 3). The prevalence of PMQRs genes among the MDR isolates was 7 (53.8%) and the MDR isolates were observed to significantly possess at least one of the ESBL genes and one of the PMQRs genes (p = 0.015). All the gentamicin resistant isolates possessed aacC2 gene with one or more of the other screened aminoglycosides genes except aphA2 gene ( Figure 3). The isolates that possessed aacC2gene were significantly associated with MDR (p = 0.017) than those that possessed other screened aminoglycosides genes. Four (36.4%) of the isolates with MICs 128 -512 µg/ml had aadA1, aadA2 and aacC2 genes with at least two of the screened β-lactamase encoding genes ( Table 3). The MDR isolates were observed to significantly possess at least one of the ESBL genes and one of the aminoglycosides genes (p = 0.017).  Increasing aminoglycoside resistance among E. coli associated diseases has been widely reported globally. This study revealed the presence of aacC2, aadA1 and aadA2 as the prominent genes among the isolates with high level of MICs 128-512µg/ml. These isolates were equally observed to possess two or more of the screened β-lactamase genes and 8 (88.9%) of them significantly possessed multidrug resistance capability (p = 0.017). This therefore suggests that rapid dissemination of multidrug resistance genes which is favoured by the co-existence of resistance genes on same mobile genetic elements tend to limit therapeutic options against bacterial infections [48].

What this study adds
• There is a strong association between phenotypic expression of ESBL and the detection of blaSHV and/or blaCTX-M-15 gene in the bacteria in the study environment; • The presence of ESBLs encoding genes in a bacterium is a possible risk factor for multidrug resistance since they are located on same plasmids that carry other resistance determinant genes; • The detection of blaCMY-2 gene, a prominent plasmid mediated AmpC gene among UTIs associated E. coli strains carrying other ESBLs genes is the first report in Nigeria.

Competing interests
The authors declare no competing interests.

Authors' contributions
Adebola Onanuga participated in designing the study, performed the experiments, analyzed the data and wrote the manuscript. Neelam Taneja participated in designing the study, contributed to data analysis, interpretation and review of the manuscript for publication.
Jaspreet Mahindroo and Shreya Singh participated in data analysis, interpretation and review of the manuscript for publication. All authors have read and approved the final manuscript.

Acknowledgments
This study was supported by the Ministry of Science and Technology,