1 Microbiology Division, Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong SAR, China; 2 Infection Control Branch, Centre for Health Protection, Department of Health, Hong Kong SAR, China
Received 22 April 2005; returned 6 June 2005; revised 19 June 2005; accepted 20 June 2005
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Abstract |
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Methods: Three Salmonella Enteritidis isolates from blood culture and one from stool were collected due to their increased resistance to ciprofloxacin and cefotaxime. PFGE analysis was used to investigate their genetic relatedness. Conjugation experiments were employed to show if the genetic determinants involved were plasmid-mediated. MICs of various antimicrobials were determined by VITEK 2 and Etest. Based on the susceptibility and conjugation experiment results, previously described PCR methods were employed to detect sequences homologous to qnr and blaCTX-M suspected to be involved in the reduced susceptibility to ciprofloxacin and cefotaxime, respectively.
Results: PFGE analysis showed that the four Salmonella isolates were clonally unrelated. The presence of a qnr-like gene and the CTX-M allele blaCTX-M-14 on four different transferable plasmids harboured by the four isolates was confirmed.
Conclusions: This is the first report of transferable fluoroquinolone resistance due to a new qnr allele, which appeared to be linked to blaCTX-M-14, in isolates of Salmonella Enteritidis in Hong Kong.
Keywords: Qnr , CTX-M-14 , extended-spectrum ß-lactamases , ESBLs
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Introduction |
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Materials and methods |
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Four isolates of Salmonella enterica serotype Enteritidis resistant to ciprofloxacin and cefotaxime according to NCCLS (now CLSI) guidelines5 were obtained from three blood cultures and one stool specimen between July and October 2003 in a regional hospital in Hong Kong. Whilst the blood culture isolates were obtained from three hospitalized patients (Isolate-1 to -3), within 4 weeks, the stool isolate (Isolate-4) was from a person who suffered from diarrhoea and attended the Accident and Emergency Department 9 weeks after the last blood culture isolate (Table 1).
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MICs of various ß-lactams and fluoroquinolones were determined by the VITEK 2 system (bioMérieux, Marcy l'Étoile, France) using the AST-N017 card, and the Etest (AB Biodisk, Solna, Sweden) was used to detect low level reduction in ciprofloxacin susceptibility.
PFGE
PFGE was performed as described elsewhere using CHEF Mapper (Bio-Rad).6 Fingerprints were generated by restriction endonucleases XbaI or BlnI. Thirty concurrently isolated Salmonella Enteritidis were also analysed for baseline comparison.
Conjugation experiments
Conjugation experiments using a chloramphenicol-resistant Escherichia coli strain HB101 as recipient were performed as previously described.7 Transconjugants were selected on Luria-Bertani (LB) agar plates containing chloramphenicol (32 mg/L) plus cefotaxime (16 mg/L).
Plasmid analysis
Plasmids were purified from the transconjugants by an alkaline-lysis method8 and electrophoresed in 1% (w/v) agarose after restriction digestion by EcoRI or NotI for size estimation and pattern comparison.
PCR amplification and DNA sequencing
Sequences encoding the CTX-M-type ß-lactamase and Qnr peptide were detected by published PCR methods. The primer pair M9U and M9L, designed by Saladin et al.,9 was used for detecting alleles of the blaCTX-M-9 cluster within the CTX-M gene family (expected amplicon size = 869 bp). For qnr-like sequences, the primers QP1 and QP2, designed by Jacoby et al.,10 were used (expected amplicon size = 543 bp). Two primers Qnr-F (5'-ATGGATATTATTGATAAAGTTTTTCAGC-3') and Qnr-R (5'-CTAATCCGGCAGCACTATGACTCCCAAG-3') were designed to obtain the entire gene (657 bp). The quinolone resistance determining region (QRDR) of the gyrase subunit A gene (gyrA) was examined as previously described.11
Nucleotide accession number
The nucleotide sequence of the qnr allele described in this work has been deposited in GenBank/EMBL databases with the assigned accession number AY906856.
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Results and discussion |
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Since the three blood culture isolates and the one stool isolate were obtained within a period of 13 weeks, the initial concern was that the four multidrug-resistant (MDR) Salmonella Enteritidis infections shared a common origin. PFGE was applied to the four isolates together with 30 concurrently isolated but epidemiologically unrelated Salmonella Enteritidis for background comparison. All the fingerprints generated using XbaI or BlnI had an average of 12 bands with band size ranging from 20 to 1500 kb. The four MDR isolates were found to have different XbaI fingerprints (>2 bands difference) and similar differences were also seen with their BlnI fingerprints. These levels of variation were even higher than that among the 30 concurrently isolated Salmonella Enteritidis suggesting that the four isolates were not clonally related, hence the sources of the infections were not likely to be common. Nonetheless, the unusual increased resistance to ciprofloxacin and cefotaxime prompted our further investigation.
Conjugation experiments and susceptibility tests
Conjugation experiments showed that the cefotaxime-resistant phenotype in all four Salmonella isolates was transferable, hence the resistance determinants involved were probably plasmid-mediated. All four salmonellae and their derived transconjugants exhibited classical CTX-M ß-lactamase phenotypes, i.e. resistant to cefotaxime (MIC 64 mg/L) but susceptible to ceftazidime (MIC
16 mg/L), and their susceptibilities to cefoxitin and imipenem suggested the absence of AmpC ß-lactamase and carbapenemase (Table 1). Interestingly, it was discovered that the reduced susceptibilities to ciprofloxacin, norfloxacin and ofloxacin of the four Salmonella isolates were co-transferred with the cefotaxime-resistant/ceftazidime-susceptible phenotype to the four transconjugants (Table 1). This observation suggested that the genetic determinants conferring resistance to these three fluoroquinolones were also present in the transconjugated plasmids.
PCR and DNA sequencing of qnr and blaCTX-M
Based on the MIC and conjugation experiment results, it was speculated that sequences encoding CTX-M-type ß-lactamase and Qnr peptide may be present in the four Salmonella plasmids.
Indeed, the primer pair M9U and M9L designed for detecting alleles of the blaCTX-M-9 cluster within the CTX-M gene family produced amplicons of the expected size (869 bp) from the crude cell lysates and extracted plasmids of all four isolates and their derived transconjugants.9 Sequencing subsequently revealed that all four Salmonella isolates and their transconjugants possess the previously described CTX-M allele blaCTX-M-14.9 For qnr-like sequences, the primers QP1 and QP2 also produced amplicons (543 bp) from the crude cell lysates and extracted plasmids of all four Salmonella isolates and their derived transconjugants.10 After DNA sequencing showed that the amplicons have homology to the first qnr gene to be described (AY070235),10 two more primers were designed based on the published sequence for amplification of the entire qnr gene. An identical open reading frame (ORF) of 657 bp was subsequently identified in all four Salmonella isolates and their transconjugants, which has 95.4% DNA sequence identity to the first qnr gene to be discovered (AY070235). This qnr-like sequence theoretically translates into a protein with 218 residues and differs by three residues to each of the two previously described Qnr proteins in K. pneumoniae (AY070235) and Klebsiella oxytoca (AY675584) (Figure 1).
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Mutations in the gyrA QRDR and active efflux pumps are known to be major mechanisms of fluoroquinolone resistance in Salmonella.11,12 In an attempt to assess how the plasmid-mediated qnr gene contributed to the fluoroquinolone resistance compared with the gyrA QRDR mutations, the gyrA QRDRs of the four Salmonella isolates were sequenced. All the four gyrA QRDR sequences showed the same point mutation (GACGGC) which led to an amino acid change at residue 87 (Asp
Gly), but no mutation at another hotspot Ser-83.11 This mutation alone was thought to be enough for conferring high-level ciprofloxacin resistance to Salmonella.11 Conversely, when the Salmonella Qnr ORF was cloned into an expression vector and transformed into E. coli (Top 10F'), it conferred only low level protection (MIC = 0.094 mg/L by Etest) against ciprofloxacin to the host (MIC = 0.003 mg/L). In contrast, these levels of protection against ciprofloxacin (MIC = 4 mg/L) and other fluoroquinolones were much higher in the transconjugants derived from the four Salmonella isolates when compared with the recipient strain (MIC = 0.064 mg/L) (Table 1). One speculation was that sequences other than the qnr gene present on the four wild-type plasmids, for instance a promoter that intensifies the qnr expression, may be responsible for the increased protection. However, it is likely that the genetic background differences, such as their active efflux capacities, between the strains of E. coli as plasmid recipients may have attributed to these resistance level differences. In fact, it can be seen that the HB101 strain was intrinsically resistant to nalidixic acid and it was not due to gyrA QRDR mutations (data not shown). Therefore, only after the level of involvement of active efflux pumps has also been determined in the four MDR salmonellae and their transconjugants, can the participation of Qnr in the overall fluoroquinolone resistance be assessed more precisely. Also noteworthy is that, although all the four plasmids transconjugated from the four Salmonella isolates possessed qnr and blaCTX-M-14, they were found to be structurally different. Plasmids purified from the four transconjugants exhibited differences in size and also in restriction patterns generated by single as well as double digestion by the endonucleases EcoRI and NotI (data not shown) suggesting that the dissemination of the resistance determinants relied on mobile genetic elements such as integrons rather than proliferation of a single resistance plasmid. Further characterization of the four plasmids and regions flanking the resistance genes should help to address some of these problems.
Qnr-based fluoroquinolone resistance opens up the possibility of large-scale reduction in clinical usefulness of this important class of antimicrobial, especially so when a common enteric pathogen is involved. Nonetheless, the exact level of involvement of Qnr per se in overall fluoroquinolone resistance compared with mutations in the QRDR of gyrase or topoisomerase IV subunit genes and active efflux pumps is yet to be fully established. In fact, some reports suggested Qnr confers only low-level resistance, and its main contribution is to facilitate development of QRDR mutations.1,10 More worrying is perhaps the apparent linkage between qnr and blaCTX-M, and this has already been reported previously.13
In summary, we have reported the first transferable fluoroquinolone resistance due to a new qnr allele that appeared to be linked to blaCTX-M-14 in isolates of Salmonella Enteritidis in Hong Kong.
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Note added in proof |
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References |
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