Chromosomal and plasmid-mediated fluoroquinolone resistance mechanisms among broad-spectrum-cephalosporin-resistant Escherichia coli isolates recovered from companion animals in the USA

Abstract

Objectives

To determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and investigate mutations in gyrase and topoisomerase genes that may contribute to increased fluoroquinolone resistance in canine and feline Escherichia coli isolates in the USA that displayed reduced susceptibility to extended-spectrum cephalosporins. This study was undertaken because previous epidemiological studies identified a potential correlation between extended-spectrum cephalosporins and fluoroquinolone resistance.

Methods

Isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime were screened by PCR for the presence of PMQR determinants and gyrase and topoisomerase genes were sequenced. Isolates were further characterized by conjugation and phylogenetic analyses.

Results

PMQR determinants aac(6′)-Ib-cr, qnrS and qepA were identified in 30, 23 and 5 isolates, respectively. Multiple mutations were identified in the quinolone resistance-determining region, including the novel substitutions of Glu-84 → Ala and Leu-88 → Gln in ParC and Arg-432 → Ser and Glu-460 → Val in ParE. The isolate that exhibited the highest level of enrofloxacin resistance (MIC > 256 mg/L) had a double mutation in gyrA (Ser-83 → Leu and Asp-87 → Asn) and a triple mutation in parC (Ser-80 → Ile, Glu-84 → Gly and a novel mutation, Leu-88 → Gln). The presence of PMQR genes increased the ciprofloxacin MIC values 4-fold to 8-fold in transconjugants relative to the recipient strain. Approximately 39% of the isolates belonged to phylogenetic group D and 30% to group B2, which typically contain an increased number of virulence determinants compared with other groups.

Conclusions

Novel mutations in topoisomerase genes and PMQR determinants aac(6′)-Ib-cr, qnrS and qepA genes were detected among extended-spectrum β-lactamase-producing E. coli in the USA.

Introduction

Escherichia coli are commonly isolated from clinical specimens from dogs and cats, as a cause of urinary tract infection and pyometra.¹ Several fluoroquinolones (FQs) have been approved for treatment of canine and feline clinical infections.² Consequently, increasing resistance to FQs in E. coli has been observed worldwide,³ thereby limiting the therapeutic options or resulting in treatment failure. Treatment can be further complicated when FQ-resistant E. coli isolates exhibit multidrug resistance phenotypes.^2,4 Resistance to quinolones is primarily attributed to chromosomal mutations in DNA gyrase and/or topoisomerase IV genes or mutations in the regulatory genes of the acrAB efflux pump.^5,6 Changes in cell membrane permeability and overexpression of the multidrug efflux pump are complementary mechanisms that contribute to high-level FQ resistance in clinical isolates.^4,5,7 In addition, plasmid-mediated quinolone resistance (PMQR) genes, such as the target protection PMQR gene qnr, the enzymatic modification gene aac(6′)-Ib-cr and the efflux pump gene qepA, have also been shown to reduce the susceptibility to quinolones.⁶

Several epidemiological surveys have been conducted to identify the presence of PMQR in E. coli strains from humans and food animals.^6,8,9 However, limited studies describe the prevalence and the role of PMQR determinants and novel mutations, such as those found in gyrA, parC and parE, in mediating resistance to FQs in E. coli isolates from companion animals in the USA.^5,6,10 Studies have also reported an epidemiological link between PMQR genes and those encoding extended-spectrum β-lactamases (ESBLs) or other β-lactamases in E. coli isolates of human origin.^6,8–10 Therefore, we have determined the prevalence of the PMQR determinants qnr, aac(6′)-Ib-cr and qepA, and investigated mutations in gyrase and topoisomerase genes that may contribute to increased FQ resistance in canine and feline E. coli isolates that displayed reduced susceptibility to extended-spectrum cephalosporins (ESCs).¹¹

Materials and methods

Bacterial isolates and susceptibility testing

Susceptibility testing was performed on 944 canine and feline clinical E. coli isolates collected from clinical veterinary laboratories between May 2008 and May 2009.¹¹ The antimicrobial MICs for all isolates were determined at the Clinical Pharmacology Laboratory at Auburn University using custom microdilution susceptibility plates. The MIC results were interpreted according to the CLSI interpretive standards.^12,13E. coli isolates (n = 54) that exhibited reduced susceptibility to ceftazidime or cefotaxime (MIC ≥ 16 mg/L) were selected for the study.¹¹ These bacterial strains were isolated from urine (n = 38), skin wound (n = 5) and soft tissue (n = 11) specimens.

Detection of resistance genes and DNA sequence analysis

All 54 E. coli isolates were screened for PMQR genes [qnrA, qnrB, qnrC, qnrD, qnrS, aac(6′)-Ib-cr or qepA] and β-lactamase genes (bla_TEM, bla_CMY-2 and bla_CTX-M-1 group) by PCR using specific primers and conditions (Table S1, available as Supplementary data at JAC Online). The qnr-positive, aac(6′)-Ib-cr-positive and qepA-positive control strains, E. coli J53 pMG252 (qnrA1), J53 pMG298 [qnrB1 and aac(6′)-Ib-cr], J53 pMG306 (qnrS1), J53 p2007057 (qnrD) and J53 pAT851 (qepA) and Proteus mirabilis 06-498 (qnrC), were provided by Dr George Jacoby (Lahey Clinic Medical Center, Burlington, MA, USA). All E. coli isolates that tested positive for aac(6′)-Ib-cr were confirmed by enzymatic digestion with BtsCI restriction endonuclease (New England Biolabs, Ipswich, MA, USA) or by DNA sequencing. The PCR products for qepA and qnrS genes were sequenced. PCR amplification and DNA sequencing of the quinolone resistance-determining regions of gyrA, gyrB, parC and parE were performed using primers listed in Supplementary Data.

Conjugation and plasmid analyses

Conjugation experiments were performed on seven E. coli (donor) isolates to determine the transferability and the contribution of PMQR-carrying plasmids in mediating resistance to FQs. Conjugation assays were performed using plate mating experiments with selected donors, including representatives of aac(6′)-Ib-cr-, qnrS- and qepA-positive isolates and an azide-resistant recipient strain of E. coli (E. coli J53 AZ^r) as previously described.¹⁴ All transconjugants were tested for susceptibility to the following antimicrobials using standard veterinary microdilution plates (CMV1AGNF, Trek Diagnostics, Cleveland, OH, USA): amikacin, amoxicillin/clavulanic acid, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline and trimethoprim/sulfamethoxazole. Donors and transconjugants were analysed by S1 nuclease-PFGE and Southern blot analysis as previously described.¹¹

Phylogenetic grouping

E. coli isolates were examined for their phylogenetic groups A, B1, B2 and D based on triplex PCR and the presence or absence of chuA, yjaA and tspE4.C2 genes.¹⁵

Results

Mutations in topoisomerase genes

Point mutations in the quinolone resistance-determining regions of gyrA, parC and parE, involving an amino acid substitution, were detected in 49 (91%) of the 54 E. coli isolates exhibiting reduced susceptibility to ESCs (Table 1). No mutations were identified in gyrB genes. In four isolates, novel substitutions Glu-84 → Ala (NCTR-914) and Leu-88 → Gln (NCTR-915) in parC and Glu-460 → Val (NCTR-930) and Arg-432 → Ser (NCTR-888) in parE topoisomerase IV genes were identified.

PMQR determinants

Thirty-eight (70%) of 54 phenotypic ESBL-producing E. coli isolates were positive for PMQR determinants, with aac(6′)-Ib-cr-type, qnrS-type or qepA-type alleles detected in 30 (56%), 23 (43%) and 5 (9%) E. coli isolates, respectively (Table 1). The qnrA, qnrB, qnrC and qnrD genes were not detected in our study.

PMQR determinants and association with ESBL genotype

Among E. coli that produce ESBLs, bla_CTX-M-15 was prominent and detected in 78% (42/54) of isolates (Table 1). A bla_CTX-M-14 group gene was detected in 11 of 23 qnrS-positive isolates; 8 were bla_CTX-M-24 and 3 were bla_CTX-M-14. Moreover, TEM-1, TEM-33 and CMY-2-type β-lactamases were detected in 15, 2 and 20 of qnrS-positive isolates, respectively. The bla_CTX-M-24, bla_CTX-M-14, bla_CMY-2, bla_TEM-1 and bla_TEM-33 genes were detected in 11, 4, 26 and 21, respectively, and in 3 of 30 aac(6′)-Ib-cr-positive isolates. Of the five qepA-positive isolates, bla_CTX-M-24, bla_CTX-M-14, bla_CMY-2 and bla_TEM-1 genes were detected in four, one, five and four isolates, respectively.

Phylogenetic analysis

Nearly 69% (37/54) of E. coli isolates belonged to phylogenetic group D (21/54) or B2 (16/54), while the remaining isolates belonged to group A (9/54) or group B1 (8/54) (Table 1).

Conjugation experiments

Plasmids were transferred by conjugation from all seven isolates used as donors. The ciprofloxacin MICs for seven transconjugants harbouring PMQR aac(6′)-Ib-cr or qepA ranged from 0.06 to 0.12 mg/L, representing an increase of 4-fold to 8-fold compared with the recipient, E. coli J53 AZ^r (Table 2). Southern blotting of S1-PFGE products revealed plasmids ranging from 33 to 160 kb and probes for the aac(6′)-Ib-cr gene were hybridized with plasmids from donor strains and their respective transconjugants (n = 7; Figure S1, available as Supplementary data at JAC Online).

Discussion

Nearly 6% (54/944) of isolates selected for this study exhibited reduced susceptibility to ESCs. Of these, 85% (46/54) were also resistant to FQs, thus ∼ 5% (46/944) of E. coli strains isolated from the companion animals exhibited co-resistance to FQ and ESCs. Few studies have investigated the prevalence of PMQR determinants in E. coli from companion animals.^10,16 In our study, PMQR determinants aac(6′)-Ib-cr (56%) and qnrS (43%) were most prevalent, followed by qepA (9%), in the E. coli isolates exhibiting reduced susceptibility to ESCs. The qepA gene, which codes for an efflux pump, was first identified from E. coli strains of human origin in Japan.¹⁷ To the best of our knowledge, the current study is the first report of the prevalence of qepA gene among E. coli strains from companion animals in the USA. The detection of aac(6′)-Ib-cr and qepA has been reported elsewhere in E. coli from dogs¹⁰ and pigs.¹⁸ These PMQR determinants contributed only minimally to FQ resistance in isolates devoid of mutations in the quinolone resistance-determining region of gyrA (Table 1). However, previous studies have indicated that PMQR determinants in strains can facilitate the selection of higher-level FQ resistance compared with plasmid-free strains.⁶ Additionally, several studies have indicated a high prevalence of PMQR determinants among ESBL-producing Enterobacteriaceae.^6,8–10 Our study showed that PMQR determinants can co-exist with bla_CTX-M, bla_TEM and bla_CMY alleles, which are the most prevalent types in the USA.¹¹

Previous studies have shown that the primary target of FQ in E. coli is DNA gyrase and mutations in the gyrase genes can lead to development of decreased susceptibility to FQs, thus complicating the treatment of infections.^4,5 Typically, a mutation in gyrA leading to the Ser-83 → Leu substitution is the first gyrA mutation observed, which leads to a reduction in susceptibility; however, high-level FQ resistance (ciprofloxacin MIC > 4 mg/L) requires an additional mutation in gyrA at position 87 and at least one parC mutation at either position 80 or 84.^19,20 In the present study, no isolates with a single mutation in parC at Ser-80 → Ile without the concurrent gyrA double mutation at Ser-83 → Leu and Asp-87 → Asn were detected (Table 1). Hence, the present study could not directly characterize the impact of just the concurrent gyrA mutations at Ser-83 → Leu and Asp-87 → Asn without the parC mutation. However, the presence of all three mutations in the isolates was associated with high-level enrofloxacin resistance (≥ 16 mg/L; Table 1), which correlates with the results of other studies.^5,19,20 In addition, the high percentages of groups D and B2, which generally contain more virulence factors,¹⁵ found among E. coli isolates from companion animals potentially reflect a higher level of virulence in these isolates.

In summary, the PMQR genes, which are located on conjugative plasmids, conferred reduced susceptibility to ciprofloxacin relative to the recipient E. coli strain. Mutations in the gyrase and topoisomerase IV genes play a greater role in mediating increased FQ resistance in E. coli than PMQR determinants. The emergence and dissemination of conjugative PMQR determinants in canine and feline E. coli isolates can serve as a potential reservoir of multidrug resistance genes. Because FQs and ESCs remain some of the more effective treatment options in companion animals,^2,4,11 it is of concern that their clinical use may drive the dissemination of PMQR determinants in E. coli isolates from companion animals. Judicious use of FQs and ESCs by veterinarians is warranted to preserve the efficacy of these drug classes in treating diseases in dogs and cats.

Funding

This work was supported by US FDA funds. B. W. S. is supported by the Oak Ridge Institute for Science and Education.

Transparency declarations

None to declare.

Disclaimer

The views presented in this manuscript do not necessarily reflect those of the US FDA.

Acknowledgements

We thank Dr George A. Jacoby at Lahey Clinic for kindly providing qnr-positive control strains and the E. coli J53 AZ^r strain. We thank Idexx Laboratories and the Morris-Animal Foundation for their collaborations with D. M. B. We also thank Allen Gies of the University of Arkansas for Medical Sciences Core Sequencing Facility for DNA sequencing. We are grateful to Drs John B. Sutherland, Mark Hart, Fatemeh Rafii and Carl E. Cerniglia for their critical review of the manuscript before submission. We thank Dr Fatemeh Rafii and Miseon Park for their technical assistance with Southern blotting and Dr Carl E. Cerniglia for his encouragement and support of this work.

References