The occurrence of ESBL-producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia

Abstract

Background: The infection and prevalence of extended-spectrum β-lactamases (ESBLs) is a worldwide problem, and the presence of ESBLs varies between countries. In this study, we investigated the occurrence of plasmid-mediated ESBL/AmpC/carbapenemase/aminoglycoside resistance gene expression in Escherichia coli using phenotypic and genotypic techniques.

Methods: A total of 58 E. coli isolates were collected from hospitals in the city of Makkah and screened for the production of ESBL/AmpC/carbapenemase/aminoglycoside resistance genes. All samples were subjected to phenotypic and genotypic analyses. The antibiotic susceptibility of the E. coli isolates was determined using the Vitek-2 system and the minimum inhibitory concentration (MIC) assay. Antimicrobial agents tested using the Vitek 2 system and MIC assay included the expanded-spectrum (or third-generation) cephalosporins (e.g., cefoxitin, cefepime, aztreonam, cefotaxime, ceftriaxone, and ceftazidime) and carbapenems (meropenem and imipenem). Reported positive isolates were investigated using genotyping technology (oligonucleotide microarray-based assay and PCR). The genotyping investigation was focused on ESBL variants and the AmpC, carbapenemase and aminoglycoside resistance genes. E. coli was phylogenetically grouped, and the clonality of the isolates was studied using multilocus sequence typing (MLST).

Results: Our E. coli isolates exhibited different levels of resistance to ESBL drugs, including ampicillin (96.61%), cefoxitin (15.25%), ciprofloxacin (79.66%), cefepime (75.58%), aztreonam (89.83%), cefotaxime (76.27%), ceftazidime (81.36%), meropenem (0%) and imipenem (0%). Furthermore, the distribution of ESBL-producing E. coli was consistent with the data obtained using an oligonucleotide microarray-based assay and PCR genotyping against genes associated with β-lactam resistance. ST131 was the dominant sequence type lineage of the isolates and was the most uropathogenic E. coli lineage. The E. coli isolates also carried aminoglycoside resistance genes.

Conclusions: The evolution and prevalence of ESBL-producing E. coli may be rapidly accelerating in Saudi Arabia due to the high visitation seasons (especially to the city of Makkah). The health authority in Saudi Arabia should monitor the level of drug resistance in all general hospitals to reduce the increasing trend of microbial drug resistance and the impact on patient therapy.

Keywords: E. coli; ESBL; Genotyping; K. pneumonia; Phenotyping; Saudi Arabia.

Fig. 1

The level of E. coli resistance (in percentages) compared with the major ESBL drugs based on the MIC tests. No carbapenase phenotype was detected but Ciprofloxacin-resistance (fluoroquinolone resistance) was identified among the E. coli isolates

Fig. 2

The distribution of the 58 ESBL-producing E. coli isolates investigated via oligonucleotide microarray-based assay and PCR genotyping against genes associated with β-lactam resistance

Fig. 3

a Quadruples PCR profiles of 58 E. coli MDR isolates. Isolate IDs shown under each lane. Data assignment were performed according to the presence or absence of signals of the following gene order arpA, chuA, yjaA, TspE4.C2. Row 1, lanes 1, 12—group A (+−−−); lanes 2–11, 13, 15—groups A or C (+−+−), lanes 16–22—group B1 (+−+), lane 14—group B2 (−+++), lanes 23–24—group F (−+−); Row 2, lanes 1–2—group F (−+−), lanes 3–24—group B2 (−+++) or (−++−) in lane 7; Row 3, lanes 1–4—group B2 (−+++), lanes 5–8—groups E or D (++−+), lanes 9–10—groups A or C (+−+−). b Groups A/C differentiation. In all lanes, both bands for internal control and Group C specific trpA fragment (219 bp) are present. c Groups D/E differentiation. Lanes 1, 3 and 4—both bands for internal control and Group E specific arpA fragment (301 bp) are present. Lane 2—only internal control present

Fig. 4

Prevalence of genes associated with aminoglycoside resistance

Fig. 5

Phylogenetic tree of 58 MDR E. coli strains constructed using the maximum likelihood algorithm in the MEGA6 software [27] and based on the concatenated alleles of 7 housekeeping genes according to Achtman’s scheme (columns 4–10). The numerals on the branches represent bootstrap values. The phylogenetic analysis identified 24 ST (column 11) that corresponded to 11 ST complexes (STc, column 12). Phylogenetic groups in column 2 were identified using phylo-typing method [24] by quadruplex PCR using four target genes (arpA±, chuA±, yjaA±, TspE4.C2±). Strains incorrectly assigned using the extended quadruplex method are indicated in red. The phylo-group memberships of two isolates (1097, 1125) were ambiguous. Notably, E. coli Clone ST131, ST410, and ST1193, which are disseminated globally, were among the identified STs in this study