Abstract

Objectives

To determine the prevalence of qnr genes in selected Salmonella enterica and Escherichia coli isolated in the UK.

Methods

One hundred and eighteen S. enterica and 103 E. coli were screened for qnrA, qnrB and qnrS by PCR. Transferability of qnr plasmids was assessed and isolated plasmids compared with previously identified qnr plasmids by restriction fragment length polymorphism analysis and hybridization experiments. PCRs and sequencing identified co-transferred β-lactamase genes and mutations in the quinolone resistance-determining region of gyrA.

Results

Only six S. enterica strains belonging to four serotypes (Stanley, Typhimurium, Virchow and Virginia) were positive for qnrS1. qnrS1 was present on plasmids of 13.5 kb (TPqnrS-1a and -1b) in Typhimurium and Virginia isolates, 44 kb (TPqnrS-2) in two Virchow isolates and >148 kb (TPqnrS-3a and -3b) in two Stanley isolates. blaTEM-1 and a group 9 blaCTX-M were co-transferred on TPqnrS-2 and TPqnrS-3b. Hybridization of a qnrS1 probe to digested qnrS1 plasmids suggested qnrS1 on TPqnrS-2 may be located in a similar genetic environment to Shigella qnrS plasmid pAH0376, but in a different environment in the other plasmids.

Conclusions

This is the first report of plasmid-mediated quinolone resistance in a Salmonella isolate from the UK; five isolates were associated with foreign travel to, or food imported from, the Far East. The presence of qnrS1 on different plasmid backbones in several Salmonella serotypes suggests successful dissemination of plasmids or qnrS1. It is of concern that qnrS1 is being identified in Salmonella serotypes that are commonly implicated in human infection in the UK. Coupled with β-lactam resistance, it may compromise treatment of vulnerable patient groups.

Introduction

Fluoroquinolone resistance usually arises spontaneously due to mutations within the type II topoisomerase genes, often in combination with decreased expression of outer membrane proteins and overexpression of multidrug efflux pumps. Plasmid-mediated quinolone resistance was first identified in a clinical isolate of Klebsiella pneumoniae in 1998.1 The gene responsible, qnrA, encodes a protein that protects DNA gyrase from inhibition by ciprofloxacin.2qnrA confers resistance to nalidixic acid and reduced susceptibility to ciprofloxacin, but the basal level of quinolone resistance provided by qnr genes is low and strains can appear susceptible to quinolones according to CLSI breakpoints. Their clinical importance lies in facilitating selection of quinolone resistance mutations in the presence of levels of quinolone that would otherwise be lethal.1 New qnr genes, qnrB and qnrS, have been identified in K. pneumoniae from the USA and India and Shigella flexneri from Japan.3,4 Although all are members of the pentapeptide repeat family, qnrB shares only 39.5% amino acid identity with qnrA and 37.4% amino acid identity with qnrS.5qnrA has been found in Enterobacteriaceae worldwide, with a particular association with Asian isolates. qnrB and qnrS genes appear to be more widespread; qnrB having been identified in Senegal, the USA and Korea,6–9 while qnrS genes have been found in Enterobacteriaceae in Germany, the USA, Taiwan, Vietnam and France.7,10–14qnr genes are often associated with strains that produce extended-spectrum β-lactamases (ESBLs).3–5 This observation may explain in part why many isolates resistant to β-lactams are also quinolone resistant.

In this study, we investigated the prevalence of qnr genes in clinical isolates of Salmonella enterica and Escherichia coli, assessed the transferability of quinolone resistance, characterized the β-lactamases co-transferred and compared qnr plasmids with those previously identified.

Materials and methods

Bacterial strains

One hundred and eighteen archived clinical isolates of S. enterica and 103 of E. coli received between 1993 and 2005 were screened. Antimicrobial resistance was determined using a breakpoint method in Iso-Sensitest agar (Oxoid, Basingstoke, UK).15 All E. coli and 102/118 S. enterica were resistant to cefotaxime (MIC >1 mg/L), and 94/103 E. coli and 54/118 S. enterica were resistant to ciprofloxacin (MIC >0.125 mg/L). Forty-two S. enterica strains were from patients reporting recent travel abroad.

Screening for qnr genes

qnrA, qnrB and qnrS were screened for by PCR using primers QP1 and QP2,3 FQ1 and FQ25 and qnrS-F (5′-TGGAAACCTACAATCATACATATCG) and -R (5′-TTAGTCAGGATAAACAACAATACCC, designed using GenBank accession number AB178643). pMG252, pMG298 and pAH0376 were used as positive controls for qnrA, qnrB and qnrS respectively.3–5 All positive results were confirmed by sequencing of amplicons.

PFGE

All qnr-positive strains were typed. Genomic DNA was digested with XbaI and the gel run for 48 h at 5.4 V/cm, with switch times of 5–60 s in buffer pre-chilled to 12°C.

Mutations in the quinolone resistance-determining region (QRDR) of gyrA

The QRDR of gyrA was examined in qnrS-positive isolates by amplification and sequencing using primers P1 and P216 and comparison with the wild-type sequence of gyrA (GenBank accession number X78977).

Transferability of resistance

Conjugations were performed using plasmid-free, rifampicin-resistant E. coli K12 20R764 as the final recipient and Salmonella Typhimurium 48R626, which harbours a 72.5 kb incFII conjugative plasmid with mobilizing functions in a tri-parental mating. Transconjugants were selected on MacConkey agar containing rifampicin (100 mg/L) and ampicillin (100 mg/L) or ciprofloxacin (0.125 mg/L). Where conjugation was unsuccessful, transformation experiments were conducted using ElectroMAX™ DH10B cells (Invitrogen, Paisley, UK) and plasmids purified as described previously.17 Transformants were selected on MacConkey agar containing ciprofloxacin (0.125 mg/L). Plasmid sizes were determined with reference to plasmids of 148, 63, 36 and 7 kb carried in E. coli K12 strain 39R861. All conjugants and transformants were confirmed as carrying qnrS by PCR.

PCR-based plasmid replicon typing

PCR-based incompatibility (Inc) group testing of qnr plasmids was carried out as described previously.18

PCR detection of β-lactamase genes

Primers TEM-F (5′-CATTTTCGTGTCGCCCTTAT-3′) and -R (5′-TCCATAGTTGCCTGACTCCC-3′) and CTX-M-F (5′-CGATGTGCAGTACCAGTAA-3′) and -R (5′-TTAGTGACCAGAATCAGCGG-3′), designed to amplify a 585 bp fragment from all CTX-M genes,19 were used for identification of blaTEM and blaCTX-M genes.

Restriction fragment length polymorphism (RFLP) plasmid analysis

Transferred plasmids were digested with HpaI and PstI (New England Biolabs, Hertfordshire, UK). Fragments were separated by electrophoresis in 0.8% agarose gels at 1.2 V/cm for 20 h at 20°C. Dendrograms were constructed in Bionumerics software (version 3.00, Applied Maths, Kortrijk, Belgium) using the Dice similarity coefficient and the unweighted pair group method with arithmetic averages (UPGMA).

RFLP fragments were transferred to Hybond-N+ membrane (Amersham Biosciences, Buckinghamshire, UK) using vacuum blotting apparatus. The qnrS PCR product was labelled with DIG-11-dUTP by PCR using a DIG PCR Probe Synthesis Kit (Roche Diagnostics Ltd, East Sussex, UK). After hybridization with the qnrS probe, hybridized DNA was detected using a DIG Nucleic Acid Detection Kit (Roche Diagnostics Ltd).

Nucleotide sequence accession numbers

The qnrS1 sequences have been submitted to the GenBank database and assigned accession numbers DQ885570 (S197318), DQ885571 (S206705), DQ885572 (P557570), DQ885573 (H043840529), DQ885574 (H050540160) and DQ885575 (H060340262).

Results and discussion

All E. coli were negative for qnr genes; however, six isolates of S. enterica were positive for qnrS (Table 1). However, it should be noted that primers FQ1 and FQ2 will not detect qnrB4. Additional primers would need to be designed and the strain panel tested for this gene. Sequencing showed complete identity to qnrS1 previously identified on pAH0376 from a S. flexneri strain.4 The six isolates belonged to serotypes Stanley, Typhimurium, Virchow and Virginia. Stanley and Typhimurium isolates were associated with recent travel to Thailand or Malaysia, whereas the two Virchow isolates were part of an outbreak associated with imported cooked chicken meat from Thailand.20 The Virginia strain was isolated from a 1-year-old child living on a UK farm, with no history of recent foreign travel. qnrS has previously been identified only in human isolates of serotypes Anatum and Bovismorbificans from the USA7 and an avian Infantis isolate from Germany.10

Table 1.

Properties of qnrS-positive S. enterica identified in this study and their qnrS plasmids

Strain number Serotype and phage typea Date isolated Resistance patternb Plasmid designation Plasmid size (kb) Transferred resistanceb 
S197318 Stanley 2001 CHL, GEN, NEO, KAN, STR, SUL, TET, CIPL TPqnrS-3a >148 CHL, GEN, NEO, KAN, STR, SUL, TET, CIPL 
S206705 Stanley 2003 AMP, LEX, RAD, CXM, CRO, CTX, CHL, GEN, NEO, KAN, STR, SUL, TET, CIPL TPqnrS-3b >148 AMP, LEX, RAD, CXM, CRO, CTX, CHL, GEN, NEO, KAN, STR, SUL, TET, CIPL 
P557570 Typhimurium DT193 2002 AMP, CHL, GEN, NEO, STR, TET, CIPL TPqnrS-1a 13.5 CIPL 
H043840529 Virchow PT8 2004 AMP, NAL, CIPL TPqnrS-2a 44 AMP, CIPL 
H050540160 Virchow PT8 2005 AMP, NAL, CIPL TPqnrS-2b 44 AMP, CIPL 
H060340262 Virginia 2006 SUL, TET, CIPL TPqnrS-1b 13.5 CIPL 
Strain number Serotype and phage typea Date isolated Resistance patternb Plasmid designation Plasmid size (kb) Transferred resistanceb 
S197318 Stanley 2001 CHL, GEN, NEO, KAN, STR, SUL, TET, CIPL TPqnrS-3a >148 CHL, GEN, NEO, KAN, STR, SUL, TET, CIPL 
S206705 Stanley 2003 AMP, LEX, RAD, CXM, CRO, CTX, CHL, GEN, NEO, KAN, STR, SUL, TET, CIPL TPqnrS-3b >148 AMP, LEX, RAD, CXM, CRO, CTX, CHL, GEN, NEO, KAN, STR, SUL, TET, CIPL 
P557570 Typhimurium DT193 2002 AMP, CHL, GEN, NEO, STR, TET, CIPL TPqnrS-1a 13.5 CIPL 
H043840529 Virchow PT8 2004 AMP, NAL, CIPL TPqnrS-2a 44 AMP, CIPL 
H050540160 Virchow PT8 2005 AMP, NAL, CIPL TPqnrS-2b 44 AMP, CIPL 
H060340262 Virginia 2006 SUL, TET, CIPL TPqnrS-1b 13.5 CIPL 

aDT, definitive phage type; PT, phage type.

bAMP, ampicillin (breakpoint final concentration 8 mg/L); LEX, cefalexin (16); RAD, cefradine (16); CXM, cefuroxime (16); CRO, ceftriaxone (1); CTX, cefotaxime (1); CHL, chloramphenicol (8); AMK, amikacin (4); GEN, gentamicin (4); NEO, neomycin (8); KAN, kanamycin (8); STR, streptomycin (16); SUL, sulphonamide (64); SPT, spectinomycin (64); TET, tetracyclines (8); TMP, trimethoprim (2); FUR, furazolidone (8); NAL, nalidixic acid (16); CIPL, low-level ciprofloxacin (MIC 0.25–1.0 mg/L).

Identification of qnrS1 in a Salmonella isolated from a child living on a farm, and in a poultry carcass isolate in Germany,10 suggests that the occurrence of qnr genes in isolates of animal origin warrants investigation. Since 1993, fluoroquinolones have been used for treatment of food production animals in the UK. Such use could lead to selection of strains carrying qnrS plasmids, which are then spread to humans through the food chain.

Mutation D87Y was identified in the gyrA QRDR of H043840529 and H050540160; both isolates were resistant to nalidixic acid and exhibited low-level resistance to ciprofloxacin. No mutations were identified in S197318, S206705, P557570 and H060340262; these isolates were susceptible to nalidixic acid, but exhibited low-level resistance to ciprofloxacin. In future, all enterobacterial isolates exhibiting this resistance type upon routine resistance phenotype testing within the HPA Laboratory of Enteric Pathogens will be screened for the presence of qnr genes.

Plasmid transfer by conjugation was successful for isolates P557570, H060340262, H043840529 and H050540160, whereas transformations were required to isolate qnrS1 plasmids from S197318 and S206705. Transferred qnrS1 plasmids were 13.5, 44 and >148 kb (Table 1). PCR-based Inc group testing found that TPqnrS-2a and -2b carry an IncN replicon. In this respect, pINF5, the qnrS plasmid identified in Germany, was hypothesized to have a pMUR050-like ancestor (an IncN plasmid).10 TPqnrS-1a and -1b, TPqnrS-3a and -3b and pAH0376 were negative for all 18 replicons.

Plasmid RFLP patterns are shown in Figure 1. TPqnrS-2a and -2b were indistinguishable and now referred to as TPqnrS-2. Salmonella RFLP patterns shared <44% genetic similarity with that of S. flexneri pAH0376. TPqnrS-2 is of similar mass to ~47 kb pAH0376, but RFLP patterns of these two plasmids shared only ~30% and ~44% similarity, respectively. Hybridization of a qnrS1 probe to PstI-digested plasmid DNA identified an ~16.8 kb fragment in TPqnrS-3a and -3b, an ~4.6 kb fragment in TPqnrS-1a and -1b and an ~3.9 kb fragment in pAH0376 and TPqnrS-2 (data not shown). These data suggest that qnrS1 on TPqnrS-2 may be located in a similar genetic environment to pAH0376 but different in TPqnrS-1a and -1b and TPqnrS-3a and -3b. The similarities between the digest patterns of TPqnrS-2 and TPqnrS-1b, but the difference in the mass of the qnrS1-hybridized fragments in these two plasmids, suggest that qnrS1 is situated in different genetic environments, but on plasmids that share a common ancestor.

Figure 1.

(a) Dendrogram showing PstI restriction digest profiles for qnrS-bearing plasmids. The molecular masses (in bp) of the fragments generated were calculated by comparison with a Lambda EcoRI/HindIII digest molecular weight ladder (ABgene, Surrey, UK). (b) Dendrogram showing HpaI restriction digest profiles for qnrS-bearing plasmids.

Figure 1.

(a) Dendrogram showing PstI restriction digest profiles for qnrS-bearing plasmids. The molecular masses (in bp) of the fragments generated were calculated by comparison with a Lambda EcoRI/HindIII digest molecular weight ladder (ABgene, Surrey, UK). (b) Dendrogram showing HpaI restriction digest profiles for qnrS-bearing plasmids.

Co-transferred resistances are detailed in Table 1. PCR and sequencing identified TEM-1 as responsible for the high-level ampicillin resistance transferred by TPqnrS-2a and -2b, and a group 9 CTX-M gene for the β-lactamase resistance transferred by TPqnrS-3b. The CTX-M was identified as either CTX-M-14/-18 (the same gene sequence has been given two names) or CTX-M-17 by PCR and sequencing using group 9-specific primers.19 Further identification was not possible, as the mutations that differentiate the two CTX-M genes are found within the binding site of the reverse primer. Co-transmission of resistance to fluoroquinolones and β-lactamases is clinically important, as co-selection of resistance by use of either agent may occur. ESBLs and fluoroquinolones are used for the treatment of serious Salmonella infections; fluoroquinolones are not recommended for children and growing adolescents; therefore, ESBLs may be the treatment of choice. Treatment failure after administration of fluoroquinolones to patients with Salmonella Typhi has been recorded in strains that exhibit decreased susceptibility; therefore, the spread of qnrS plasmids carrying ESBLs into Salmonella Typhi could have serious consequences for treatment of typhoid fever.

PFGE patterns of the two Stanley isolates had a Dice similarity coefficient of 87.5% and differed by three bands (data not shown). Plasmid RFLP and PCR data, together with PFGE typing, suggest these isolates may represent a clonal group in Thailand, which has persisted for at least 2 years, but S206705 has acquired a CTX-M gene at some stage. Many CTX-M genes are located near or within integrons or transposons, which could permit rapid dissemination and may explain the acquisition of blaCTX-M by TPqnrS-3b.

In summary, we report the first incidence of plasmid-mediated quinolone resistance in a S. enterica isolate from the UK. The presence of qnrS1 genes on different plasmid backbones, in Salmonella isolates of several serotypes and different species of Enterobacteriaceae from different countries, suggests successful dissemination of these plasmids or mobilization of the qnrS1 gene itself.

Acknowledgements

We would like to thank George Jacoby, Lahey Clinic, USA, for providing pMG252 and pMG298 and Mami Hata, Aichi Prefectural Institute of Public Health, Japan, and Myonsun Yoh, Research Institute for Microbial Diseases, Osaka University, Japan, for providing pAH0376. Part of this study was presented at the Forty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy (San Francisco, CA, USA, 2006; Poster C2-0088). This study was funded by the Department of Environment, Food and Rural Affairs, UK, project VM02136.

Transparency declarations

None to declare.

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