Prevalence of ST131 Clone Producing Both ESBL CTX-M-15 and AAC(6′)Ib-cr Among Ciprofloxacin-Resistant Escherichia coli Isolates from Yemen

Introduction

Escherichia coli is a common commensal organism of the gastrointestinal tract of humans and many animals. However, several variants of E. coli have been described that cause infection of the gastrointestinal system (called intestinal pathogenic E. coli). ¹ Besides, E. coli isolates that have the ability to cause infections outside the gastrointestinal system are referred to as extraintestinal pathogenic E. coli isolates (ExPEC). ExPEC incorporates the following variants: avian pathogenic E. coli, uropathogenic E. coli (UPEC) and isolates responsible for sepsis and neonatal meningitis. ² Phylogenetic analyzes have shown that ExPEC belongs mainly to phylogenetic group B2 and, to a lesser extent, to group D. ³ Resistance to fluoroquinolones (FQ-R) and extended-spectrum cephalosporins, introduced in the 1980s, due respectively to the production of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamases (ESBL) by E. coli has been growing steadily over the past 20 years. There is also evidence to suggest that this increase in resistance is related to the global spread of a specific E. coli clone, sequence of E. coli type 131 (ST131). ⁴ E. coli ST131 was originally identified in 2008, but retrospective analyses suggest that the pandemic emergence of this clone took place over a period of fewer than 10 years.^5,6 Most of isolates belonging to ST131 clonal complex exhibited O25b serogroup and H4 antigen. This clonal group has emerged as an important human opportunistic pathogen worldwide because of its combination of successful spread, antibiotic resistance, and virulence. ⁷

The virulence potential of this clone is due to the presence of multiple virulence factor genes, including the genes encoding the mannose-specific type 1 fimbriae adhesin, FimH, which is a major virulence factor in UPEC.^8,9

Today, the largest subclonal lineage of E. coli ST131 is FQ-R and consists of clade C with fimH30 (H30) allele, ¹⁰ which comprises two subclades within clade C named C1/H30R (associated with FQ-R) and C2/H30-Rx (associated with CTX-M-15 ESBL). ¹¹ This last one is often associated with PMQR determinants especially Aac(6′)-lb-cr and/or Qnr ¹² showing an extensive global distribution and causing millions of antimicrobial-resistant infections annually (e.g., up to 30% of all ExPEC, 60–90% of FQ-R ExPEC, and 40–80% of ESBL ExPEC belongs to ST131). ⁴ Thereby, the resistance of E. coli ST131 to antibiotics often used to treat infections (cephalosporins and fluoroquinolones) leads to limited treatment options and frequent recurrences. ²

Although many reports have been published regarding the prevalence of the ST131 clone in the Middle East (including Kuwait, Lebanon, Saudi Arabia, and Iran) and another part of the world, a PubMed search did not identify any published report describing the occurrence of this clone in the region of Yemen. Therefore, the aim of this study was to assess the occurrence of O25b/ST131 clone and to identify its molecular characteristics in ciprofloxacin-resistant E. coli isolates among inpatients and outpatients from Sana'a (Northern Yemen).

Materials and Methods

Results and Discussion

All of the 41 ciprofloxacin-resistant E. coli isolates were found to harbor the aac(6′)Ib-cr gene. In addition, qnrS was detected in five isolates (12.2%). None of the isolates was found to carry qnrA, qnrB, qnrC, qnrD, qepA, or oqxAB genes.

Antimicrobial susceptibility testing results showed that out of the 41 ciprofloxacin-resistant E. coli strains, 97.5% were resistant to cephalosporins, including 63.4% (26/41) ESBL producers, in which we have identified bla_CTX-M-15 in all cases. The resistance rate for tetracycline, amoxicillin/clavulanic acid, gentamicin, cefoxitin, and amikacin were 65.8%, 51.2% 39%, 24.4%, and 12.2%, respectively. All of these isolates, nevertheless, remained susceptible to carbapenems and colistin.

Most of E. coli isolates in the study (90.2%; 37/41) showed a QRDR region with mutations in codons 83 and 87 (Ser83Leu/Asp87Asn) of the gyrA gene. A single alteration Ser83Leu was found in two isolates. The E. coli strains with an additional mutation in codon 80 (Ser80Ile) and associated with an alteration in codon 84 (Glu84Val) of the parC gene were found in 56% and 36.6%, respectively.

Cluster analysis of the PFGE image of restriction fragments revealed diverse genetic profiles suggesting that they were not epidemic cases, except seven groups of isolates from the community which displayed related PFGE profiles with a similarity >85% suggesting a clonal relationship (Fig. 1). The results of phylogroup assessment, fimH typing, and MLST showed that one of these groups contained isolates which belonged to the ST131 clonal group. The prevalence of E. coli strain, ST131, in this set of strains was 36.5% (15/41). The characteristics of ST131 E. coli isolates are shown in Table 1. These isolates contained the fimH30Rx gene and expressed the enzymes Aac(6′)Ib-cr and CTX-M-15. Most isolates (11/15, 73.3%) were recovered from urine samples and were genetically unrelated by PFGE.

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FIG. 1.

Dendrogram of patterns generated by pulsed-field gel electrophoresis of Escherichia coli isolates.

The clonal group ST131 harboring a broad range of virulence and resistance genes on a transferable plasmid has been reported worldwide both in hospitals and in the community, causing multidrug-resistant infections. Following its initial identification in 2008 in a limited number of countries in three continents, North America, Europe, and Asia, this clone was successfully detected in many other countries on these three continents and on the two remaining continents, Africa and Oceania. ⁴ However, to our knowledge, this is the first report of prevalence of E. coli O25b/ST131 from Yemen.

In this study, the ST131 was identified in 36.6% (15/41) among clinical and community FQ-R E. coli isolates from Sana'a in 2013. Several studies carried out in the Middle East had described a high prevalence of this clone. Indeed, a rate of 60.9% (56/92) ST131, including 44 (78.6%) ST131-O25b was reported among FQ-R E. coli isolates causing community-acquired urinary tract infections at Fayoum University Hospital, in Egypt. ²⁰ Moreover, Alghoribi et al. reported that 64.5% (20/31) of ESBL-producing E. coli isolates collected at a tertiary hospital in Riyadh (Saudi Arabia) from 2010 to 2011 belonged to theST131 clone. ²¹ Furthermore, the prevalence of ST131 was described among unselected E. coli clinical isolates with a rate of 10% (83/832) in three hospitals of Kuwait collected between 2011 and 2012. ²² In this study, the principal source of ST131 isolates (11/15) was urine recovered from outpatients. Indeed, E. coli (ST131) are responsible for a high proportion of community- and hospital-acquired urinary tract infections (UTIs) (cystitis and pyelonephritis) in terms of its multiple virulence factors that give it the ability to adhere to intestinal, bladder, and kidney epithelial cells. ⁴

The O25b/ST131 E. coli isolates presented a multidrug-resistance, including ciprofloxacin, gentamicin, cefotaxime, aztreonam, and tetracycline. A recent study carried out in Iran showed that the resistance of these antibiotics was significantly higher in ST131 isolates than non-ST131 isolates. ²³ All E. coli isolates, including ST131 and non-ST131 were aac(6′)Ib-cr-positive associated with bla_CTX-M-15 and qnrS in 63.4% and 12.2%, respectively. The aminoglycoside acetyltransferase gene Aac(6′)Ib-cr was described in a variety of Enterobacteriaceae species, but its usually most common PMQR in E. coli often coproduced CTX-M-15 ESBL. ²⁴

The ST131 isolates were recovered from 2 inpatients and 13 outpatients from the same region (Sana'a) in 2013 most of them displaying diverse genetic profiles, except three clonal groups. E. coli ST131 strains were commonly identified as transmitted within the hospital and between members of the same household. ²⁵ However, the true origin or transmission route of ST131 in the community remains unknown.

In our study, fimH typing revealed that all of E. coli ST131 isolates belonged to the H30-Rx subclone producing both CTX-M-15 and Aac(6′)Ib-cr associated with QnrS in three (20%) isolates. Moreover, these isolates carried two substitutions in GyrA (Ser83Leu/Asp87Asn) and two substitutions in ParC (Ser80Ile/Glu84Va) contributing to the high-level FQ-R. ²⁶ Recent molecular epidemiology showed that H30Rx subsets within ST131-O25-H30 subclones were associated specifically with FQ-R, and CTX-M-15 was widely detected. Johnson and colleagues observed that the fimH30 ST131 lineage consisted of nearly 70% of recent FQ-R E. coli isolates, while it remained infrequent (i.e.,1%) among fluoroquinolone-susceptible ST131 isolate. ²⁷ A previous study reported that the H30Rx subclone was prevalent (61.1%, 30/49) as ST131-O25-H30 from Korea. ²⁸ A proportion of 26/134 (19.4%) of H30 subclone, including 24 (92.3%) H30-Rx was observed in a collection of E. coli isolates from a French hospital in 2013. ²⁹ A further study reported that ST131-H30 (accounted for 26.4%), with its extended-spectrum cephalosporin resistance-associated H30Rx subset, caused most antimicrobial-resistant E. coli infections across the United States in 2011–2012 and, since 2007, increased in relative prevalence by >50%. ²⁷

Conclusion

This is the first report of prevalence of E. coli O25b/ST131 from Yemen. Therefore, the continuous monitoring, rapid detection, and determination of risk factors for acquisition of the ST131 clone across the different clades causing drug-resistant infection is essential to limit their spread. Moreover, deep genome sequencing is required to understand the origin, evolution, and spread of antimicrobial resistance genes related to this clone.