Distribution of Different Mutations in Fluoroquinolone-Resistant (FQR) Sequence Types of Uropathogenic Escherichia coli (UPEC)

Abstract

The efficacy of fluoroquinolone for treating a wide variety of infections is considerably constrained due to the rapid emergence and dissemination of fluoroquinolone resistance. The most critical resistance mechanism for reducing susceptibility to fluoroquinolone is the alterations in the target sites, i.e., quinolone resistance determining regions (QRDRs) of gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes. From Pakistan, not much is known about the occurrence of these mutations in emerging uropathogenic Escherichia coli (UPEC) strains. This study aims to investigate the distribution of these target site mutations in different sequence types (STs) in UPEC strains and to establish their correlation with the level of observed fluoroquinolone resistance. We confirm that out of one hundred and fifty-four UPEC isolates, n = 90 showed resistance to ciprofloxacin, levofloxacin, or enrofloxacin. These FQR isolates were sequenced and screened for the mutations in QRDRs of gyrA, gyrB, parC, and parE genes. All seven housekeeping genes were sequenced through MLST to determine the distribution of sequence types (STs) in these isolates. Molecular techniques were used for the phylogenetic grouping, and categorization was done into A, B1, B2, and D groups. ESBL producers were scrutinized by antibiotic susceptibility testing, and molecular screening of blaCTX-M-15 was performed. MIC of enrofloxacin, another fluoroquinolone, was measured for all ninety isolates. Sequencing of QRDRs revealed Ser83, Asp87, and Pro79 position as hotspots for mutations in the gyrA gene, Ser80, Glu84, Ala90, Leu54, and S57 in parC gene, and S458 and D463 for parE mutations. The most abundant mutations were the gyrA gene’s Ser83Leu (92%), Asp87Asn (81.1%), and parC gene’s Ser80Ile (83%), and parE gene’s S458A (43%). The distribution of most frequent double mutations of gyrA and parC gene included Ser83Leu and Asp87Asn (76%) and Ser80Ile, Glu84Val (28%), respectively. The most abundant combination of mutations in 76% of the isolates was double mutations in gyrA (Ser83Leu, Glu84Val), parC (Ser80Ile and/Glu84Val/Leu54Pro/Ala90Val/S57T) with or without parE mutation (S458A/D463N). Triple mutations (Ser83Leu, Asp87Asn, and Pro79Arg) in the gyrA gene were found in 6% of the isolates. The most abundant sequence types were ST-131 (48%), ST-405 (18%), and ST-168 (14%). The MICs for enrofloxacin ranged from 0.125 µg/ml to >256 µg/ml, similar to ciprofloxacin and levofloxacin. Among ST 131 isolates, 95% were ST-131 O25b, 30% were ST-131 H30, and 49% were CTX-M Page | XIII 15 producers. Most STs belonged to B2 (60%) and D (27%) phylogenetic groups. Novel mutations in gyrA (Pro79Arg), parC (Ala90Val, Leu54Pro), and parE (D463N) were found in some FQR isolates. ESBL CTX-M-15 was produced by 29% of the sequence types that were non-ST-131 and showed the same mutation patterns previously associated with the elevated MICs. The same mutations were reported globally and are linked with the elevation in MICs. The patterns of the mutations, at least three or four mutations (double in gyrA and single/double in parC), found in our ninety FQR isolates were the same as the globally accepted resistance pattern for the elevation in MICs to achieve the highest resistance level. Along with FQR, ESBL-producing isolates are on the rise in Pakistan, rendering both fluoroquinolone and β-lactams efficacy against these pathogens. The most prominent ESBL-producing sequence types after ST-131 were ST-405 and ST-168, confirming these as emerging sequence types