Prevalence and Characterization of Quinolone Resistance Mechanisms in Commensal Escherichia coli Isolated from Slaughter Animals in Poland,2009–2012

Introduction

The introduction of quinolones into medical practice was enthusiastically announced as a defeat of pathogens against perfectly efficient synthetic bactericidal antimicrobials. The dramatic decline in efficacy, occurring 50 years later, justifies the preservation of fluoroquinolones, considered critically important for medicine. ³ The change of perception of sustainable quinolone usage resulted from the discovery of chromosomal and transferable mechanisms followed by the spread of resistant clones or the genes alone. ¹³ Spontaneous point mutations lead to amino acid substitutions in the quinolone resistance-determining region (QRDR) of topoisomerase II (gyrase) and IV genes and thus alter quinolone binding sites ending up with the failure of antibacterial activity. ⁴ As a stepwise process in Enterobacteriaceae, single mutation in gyrA, gyrB, parC, or parE reduces susceptibility to fluoroquinolones and multiple substitutions are needed for clinical resistance. ¹³ Additionally, that scheme might be interfered with nonspecific mechanisms, including outer membrane permeability or efflux pumps. ⁴ Plasmid-mediated quinolone resistance (PMQR) confers lower susceptibility to fluoroquinolones and it is considered a background for the selection of chromosome-encoded resistance.^9,13 Three mechanisms are involved in PMQR: Qnr peptides protect topoisomerases from antimicrobial action, variant of aminoglycoside acetyltransferase [AAC(6′)-Ib-cr] modifies antimicrobial molecules, and QepA and OqxAB proteins modulate the quinolone efflux pump.^4,7,10,13 The number of discovered qnr genes is currently approaching 100 alleles divided into several categories (qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC) of which qnrB accounts for more than 70 variants (www.lahey.org/qnrStudies). ¹³ Since all (fluoro)quinolones exert their antibacterial effect by topoisomerase inhibition, basically the same mechanisms confer resistance to all clinically relevant compounds. ⁴

Low dosage and favorable pharmacokinetics, resulted in wide usage of fluoroquinolones in humans and animals.^4,9 Being excreted during treatment mostly as active compounds, their biological action is not limited to the therapeutic site, however, it is moved further as resistance selection pressure into the environment. ⁸ These feature interplays with the chromosomal mutations causing propagation and subsequent spread of quinolone-resistant bacteria. ⁴ Our previous study ²⁰ has shown that antimicrobial resistance is an ever-evolving issue driven by pressure of antimicrobial usage in animals. Nonpathogenic Escherichia coli of animal origin may act as a reservoir of resistance genes for other bacteria, including pathogens. ³

Although no temporal trends were noted, the baseline study ²⁰ indicated on the complex and multivariable nature of (fluoro)quinolone resistance in indicator E. coli and the need for knowledge on its genetic background. Therefore, to fulfill some of the identified knowledge gaps, the current study aimed at the detection and characterization of quinolone resistance mechanisms in a broad range of commensal E. coli, isolated from food animals in Poland.

Materials and Methods

Laboratory testing

MIC_Nal and MIC_Cip were reevaluated against epidemiological cut-offs and clinical breakpoints (Table 1) to reveal the prevalence of microbiological and clinical resistance (nonwild-type and R populations, respectively).

Plasmid-mediated mechanisms (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6′)-Ib-cr) were tested in both groups of E. coli: suspected for PMQR and QRDR substitutions. Mutations of chromosomal genes (gyrA, gyrB, parC, and parE) were tested in 52 isolates representing the latter category. Resistance determinants were identified with polymerase chain reaction (PCR) assays in 25 μl mixtures consisting of Maxima^® Hot Start PCR Master Mix (Thermo Scientific–Fermentas), primers (50 mM), and bacterial boiling lysate (1 μl). All but one primer pair as well as temperature profiles have already been reported and listed elsewhere. ¹⁹ A new primer pair was designed (59F: 5′-ATGGCTCTGGCACTCGTTG-3′ and 58R: 5′-CGATGCCTGGTAGTTGTCCA-3′) to differentiate between few inconclusive qnrB sequences. The relevant amplicons were sequenced (Oligo, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland). Reverse and forward nucleotide sequences were aligned with SeqMan Pro (DNAStar Lasergene), analyzed with MEGA5 software (Center for Evolutionary Medicine and Informatics), and deposited in the GenBank (accession numbers: gyrA: KJ136263–KJ136314, parC: KJ136315–KJ136366, parE: KJ136367–KJ136418, qnrB10/B19: KJ136419–KJ136436, qnrB17: KJ136494, qnrS1/S3: KJ136437–KJ136493).

XbaI Pulsed Field Gel Electrophoresis (PulseNet protocol) was performed as described elsewhere ¹⁹ on a subset of 52 E. coli representing various QRDR alterations (n=27) or PMQR (n=25) mechanisms (Fig. 1).

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FIG. 1.

Phylogenetic similarity of Escherichia coli (n=52) isolated from different animal production groups (Source) from various geographical settings (Area code), with diverse quinolone resistance mechanisms: PMQR, plasmid-mediated quinolone resistance; QRDR, amino-acid substitutions in the quinolone resistance determining region of topoisomerases encoding genes; WT, wild-type gene.

For basic statistical analysis χ² test and 95% confidence intervals were applied.

Results

Clinical ciprofloxacin resistance was recorded in 19.2% of tested E. coli (Table 1), whereas nonwild-type populations accounted for 40.3% (Cip) and 35.7% (Nal). Up to 6.2% isolates with elevated MIC_Cip but MIC_Nal≤32 mg/L represented PMQR-suspected population. The prevalence of that resistance phenotype varied by source of isolation (χ², p≤0.001, Table 2) reaching the lowest values in cattle (1.1%) and the highest in turkey isolates (9.7%). Although 75 out of 219 PMQR-suspected isolates (34.2%) were retrieved in 2012, the increase over the study period was not significant (χ², p>0.05). Of 121 tested PMQR-suspected E. coli, 20 isolates carried qnrB (16.5%), 64 qnrS (52.9%), while the remaining 37 (30.6%) were found negative to all tested targets. Furthermore, seven (11.5%) qnrS-positive isolates were identified within the QRDR group (n=61). Sequencing of qnrS amplicons (n=70, including six from the QRDR group) identified the allele as qnrS1 (or qnrS3, the amplicon too short to differentiate). They were found in E. coli from all tested animal sources (cattle, n=2; pigs and layers, n=13 each; broilers and turkeys, n=21 each). In contrast, qnrB19 (or qnrB10; n=19) occurred only in poultry (4, 5, and 10 in broilers, turkeys, and layers, respectively) and single qnrB17 was found in the laying hen isolate. Few of those isolates have been reported previously. ¹⁶ None of the PMQR-suspected isolates conferred resistance to third-generation cephalosporin, although seven E. coli within the current study group ²⁰ were positive for bla_CTX-M (n=6) or bla_CMY-2 (n=1) (data not shown).

Amplification and sequencing of QRDR revealed relevant substitutions of two amino acids in both GyrA and ParC subunits (Table 3). The most frequent alteration was observed in GyrA (Ser83→Leu, n=44). Asp87 of GyrA (n=30) and Ser80 in parC (n=28) were substituted with various amino acids, occasionally resulting from different codons. Finally, Ala56→Thr substitution in ParC was recorded in three isolates. Altogether 14 patterns comprising up to four substitutions resulted in MIC_Cip shift. Two or more substitutions were observed in 26 out of 30 isolates showing clinically resistant MIC_Cip values.

Each of the 52 tested E. coli carrying various plasmid- or/and chromosome-mediated quinolone resistance mechanisms showed unique XbaI-PFGE profile with phylogenetic similarity ranging from 60.0% to 90.9% (Fig. 1).

Discussion

The reports from various parts of the world indicate the significant scope of quinolone resistance in E. coli and point out differences depending on the source of isolation. Over the last decade, it was found in human and animal sources in countries as distant as Turkey ² and South Korea, ¹² or with a different approach to antimicrobial usage in animals such as the US ¹⁴ and Denmark. ¹ In our previous report, its prevalence in Poland ranging from 3.5% in cattle up to 61.5% in turkey and 80.8% in broilers was explained with antimicrobial consumption and usage preferences. ²⁰ Built on that, the current study identified multiple (fluoro)quinolone resistance backgrounds. A stepwise phenomenon, affecting intially gyrA and then parC genes as a secondary target to reach clinical ciprofloxacin resistance,^12,14 was observed in most of the isolates. All the observed QRDR substitutions have already been reported with variable frequencies.^1,2,6,12,14 The observed broad MIC_Cip range in single mutants (i.e., Ser83→Leu), four E. coli with no mutations in QRDR, or quite a number of negative results in PMQR-suspected isolates indicate on undetected (i.e., unspecific oqxAB or qnrVC currently reported only in Vibrio) or still unrecognized mechanisms contributing to quinolone resistance.^10,12–14

An international study showed the occurrence and dissemination of PMQR genes in several European countries. ¹⁶ Being considered of limited clinical impact, their high prevalence in human cases is striking.^9,11,12 Retrospective surveys tend to identify the beginnings of the phenomenon, ⁷ but the temporal trends based on systematic survey similar to the one described in this study, have been thus far rarely assessed. The Korean studies revealed considerable occurrence of PMQR-positive E. coli in food animals ¹⁵ and higher and increasing frequency of that resistance phenotype in human clinical isolates.^9,12 Current data confirmed Poland as a high PMQR prevalence country compared with the reports from other regions.^1,2,6,16 Another important finding of the study is qnrS occurring in E. coli with chromosomal mutations at the same frequency as in the whole studied population (χ², p>0.05). It proves that plasmid-mediated mechanisms spread independently from chromosomal mutations ¹⁵ under selection pressure of quinolones usage.^11,20

Both predominant qnr alleles described in this study were the most prevalent also in other countries ¹⁶ with switch in time between both variants as described in clinical E. coli from the Republic of Korea. ⁹ Noteworthy, we have not found any other qnr or aac(6′)-Ib-cr occurring in as distinct geographical settings as Europe, Americas, and Far East, ⁷ and ranging from few food-producing isolates in Korea ¹⁵ up to 50% of PMQR-positive E. coli from companion animals in the US. ¹⁴ The gene was occasionally found in European animal-related isolates, ¹⁶ but its occurrence in human patients seems to be higher, reaching 14% in Denmark, ¹ ranging in Italy from 11% at community level to 21% in hospitalized patients, ¹¹ and up to 93% in Pakistan. ⁵ Homogeneity of qnr genes found in the current study might be explained with epidemics of certain plasmids ⁸ often carrying various resistance determinants such as the ones reported from China ¹⁰ and the Netherlands. ¹⁷ The concept is supported by finding of both predominant determinants at comparable frequencies in Salmonella collected from similar sources and time frame^16,19 and other studies reporting basically the same PMQR mechanisms in various bacteria isolated in similar geographical settings.^1,2,9 Plasmid spread might be enhanced with selection pressure due to fluoroquinolones usage in animals. ⁶ It selects also for topoisomerase gene mutants resulting in the observed variability of QRDR substitutions.^9,14 The spread of single quinolone-resistant E. coli clones might be neglected, since similar to other studies, ⁵ we have found both low phylogenetic similarity of tested isolates and numerous codon variability and synonymous mutations observed in each of the sequenced chromosomal regions (GenBank accession Nos.: KJ136263–KJ136418) indicating an independent genetic event leading to quinolone resistance development.

Plasmid-mediated cephalosporin and quinolone co-resistance is often reported. Coexistence of numerous PMQR and bla_CTX-M, bla_SHV, bla_CMY-2 genes on the same plasmids was elucidated in E. coli originating from companion and food animals as well as from humans.^5,10,14,17 Although acquired extended-spectrum cephalosporin resistance is quite often conferred by commensal E. coli in Poland, ¹⁸ none of PMQR-positive isolates tested in the current study, produced that type of enzymes.

Herewith, the variable background of quinolone resistance in commensal E. coli was revealed. The results clearly demonstrated the scale and complexity of the problem and pointed out on multifactorial ecological interactions occurring in animal production. The major knowledge gaps to be addressed in further research include identification of undetected mechanisms influencing quinolone MIC shifts, characterization of plasmids carrying predominant qnr genes, and measures for mitigation of their spread. Answering those questions is essential for further sustainable use of quinolones in animal husbandry and efficient protection of human health.