JAC Advance Access originally published online on October 14, 2005
Journal of Antimicrobial Chemotherapy 2005 56(6):1118-1121; doi:10.1093/jac/dki371
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Vibrionaceae as a possible source of Qnr-like quinolone resistance determinants
1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 K.-Bicêtre Cedex, France; 2 University Hospital Virgen Macarena, University of Sevilla, Sevilla, Spain
* Corresponding author. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr
Received 19 May 2005; accepted 15 September 2005
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
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Objectives: To gain insight into the functionality of qnr-like genes of several bacterial species of Vibrionaceae that may encode quinolone resistance determinants.
Methods: A PCR-based strategy was used to obtain qnr-like genes of reference strains of Vibrio vulnificus CIP103196, Vibrio parahaemolyticus CIP71.2 and Photobacterium profundum CIP106289 that were sequenced, cloned and expressed in Escherichia coli. MICs of quinolones were determined for these reference strains and an E. coli reference strain harbouring recombinant plasmids.
Results: The Qnr-like proteins of these Vibrionaceae bacterial species shared 40–67% amino acid identity with the plasmid-mediated quinolone resistance determinants QnrA, QnrB and QnrS with a series of highly conserved residues. Once cloned in E. coli they conferred reduced susceptibility to quinolones.
Conclusions: This work provides further evidence that water-borne Vibrionaceae may constitute a reservoir for Qnr-like quinolone resistance determinants.
Keywords: plasmids , fluoroquinolones , pentapeptide repeats
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Quinolone resistance in Enterobacteriaceae results mostly from chromosomal mutations in genes coding for DNA gyrase (topoisomerase II), for efflux and outer membrane proteins or for their regulatory elements.1 A plasmid-mediated quinolone resistance determinant Qnr (later termed QnrA1) was reported in 1998 from Klebsiella pneumoniae first from the USA2 and then from different enterobacterial species from several continents.3
QnrA belongs to the pentapeptide repeat family and protects DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones.3 QnrA confers resistance to quinolones and increases MICs of fluoroquinolones up to 32-fold.3 Two distantly-related Qnr determinants have been identified recently. QnrB was from Citrobacter koseri, Escherichia coli, Enterobacter cloacae and K. pneumoniae from the USA and India4 whereas the QnrS determinant was from a Shigella flexneri isolate from Japan.5 QnrB and QnrS that belong also to the pentapeptide repeat family of proteins share 41% and 60% amino acid identity with QnrA, respectively.4,5
We have recently identified the origin of the plasmid-mediated qnrA gene in the chromosome of Shewanella algae.6 S. algae is a Gram-negative species belonging to the Shewanellaceae family that is widely distributed in marine and freshwater environments. It is rarely involved in human infections, mostly related to seawater exposure.6
To gain further insight into the origin of the other plasmid-mediated QnrB and QnrS determinants, we performed a BLAST analysis and identified that hypothetical proteins of Vibrio vulnificus (AA007889 [GenBank] ) and Vibrio parahaemolyticus (BAC61438 [GenBank] ) and Photobacterium profundum (CAG22829 [GenBank] ) shared significant similarity with plasmid-mediated Qnr determinants (but no Qnr homologue was identified in the genome of Vibrio cholerae). We analysed the functionality of those proteins by cloning their genes and by studying their expression in E. coli and compared the results with those obtained with an S. algae reference strain.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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S. algae CIP106454, V. vulnificus CIP103196, V. parahaemolyticus CIP71.2 and P. profundum CIP106289 were reference strains from the Institut Pasteur collection. They were used for MIC determinations and as whole-cell DNA templates for PCR and then cloning experiments. Culture conditions were standard using Trypticase soy-containing broth or plates (37°C, 24 h, aerobic atmosphere), except that 5% NaCl was added. In addition, significant growth of the P. profundum strain was obtained at 10°C for 72 h. E. coli reference strain TOP10 (Invitrogen, Life Technologies, Cergy-Pontoise) was used as the recipient strain in cloning experiments.
MICs of quinolones and fluoroquinolones for Shewanellaceae and Vibrionaceae strains and for E. coli TOP10 harbouring recombinant plasmids were determined using the Etest technique (AB Biodisk, Solna, Sweden). MICs were interpreted according to the guidelines of the CLSI.7
Whole-cell DNA of S. algae CIP106454, V. vulnificus CIP103196, V. parahaemolyticus CIP71.2 and P. profundum CIP106289 was extracted using a standard procedure, as described previously.8 A DNA fragment corresponding to the qnr-like gene of those strains was amplified by PCR using the following primer pairs: PreQnrVV1 (5'-TGGCGATTTAAGCCACTTG-3') and PreQnrVV2 (5'-TTCTTGGTCTAACGAGCTCG-3') amplified a 834 bp fragment containing the qnrVV gene of V. vulnificus; PreQnrVP1 (5'-TCTCGCTAAGGCTCGTAGC-3') and PreQnrVP2 (5'-TTCCTCGTCGAGGTTATTCG-3') amplified a 902 bp fragment containing the qnrVP gene of V. parahaemolyticus; PreQnrPP1 (5'-TTGTTAGGTGATAACCTTCGG-3') and PreQnrPP2 (5'-CAATATTGTTAACGGTGCGC-3') amplified a 752 bp fragment containing the qnrPP gene of P. profundum; and PreQnrAa (5'-GAAAGAGTTAGCACCCTCCC-3') and PreQnrAb (5'-CTAATCCGGCAGCACTATTA-3') amplified a 693 bp fragment containing the qnrA3 gene of S. algae (QnrA3 differed from QnrA1 by three amino acid substitutions).6 The amplified fragments were then cloned into the kanamycin-resistant pPCRBluntII-TOPO plasmid (Invitrogen). Selection of recombinant clones in E. coli was made using kanamycin (20 mg/L)-containing plates. Recombinant plasmids were purified with the Qiagen plasmid Midi kit (Qiagen, Courtaboeuf, France) and cloned DNA inserts of recombinant plasmids were sequenced on both strands, using an Applied Biosystems sequencer (ABI 3100). The nucleotide sequence and deduced protein sequence were analysed with software available at the National Center for Biotechnology Information web site (www.ncbi.nih.gov). Multiple protein sequence alignments were carried out with the program CLUSTALW (http://clustalw.genome.jp/).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Cloning of PCR-amplified fragments from S. algae CIP106454, V. vulnificus CIP103196, V. parahaemolyticus CIP71.2 and P. profundum CIP106289 gave recombinant plasmids pSA-1, pVV-1, pVP-1 and pPP-1, respectively. MICs of quinolones for the reference strains showed reduced susceptibility to quinolones (Table 1). Despite repeated attempts, MICs of quinolones for P. profundum CIP106289 could not be determined precisely due to its slow growth (10°C, 72 h). Based on the susceptibility to quinolones of reference strains, the presence of chromosome-encoded Qnr-like determinants could not be suspected. Once expressed in E. coli, recombinant plasmids with qnr-like genes conferred a significantly higher level of resistance to quinolones than to fluoroquinolones, as observed with the plasmid-mediated Qnr determinants in Enterobacteriaceae.3 The MIC values of these antibiotic molecules were similar whatever the qnr-like gene was, and similar to those obtained with the recombinant plasmid that expressed the QnrA3 determinant from S. algae CIP106454 (Table 1).
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A higher level of expression of the qnr-like genes leading to a higher level of resistance to quinolones may depend on the presence of genetic elements (common region, insertion sequences...), as observed for the plasmid-mediated qnrA gene in Enterobacteriaceae.8
Cloning and sequencing of qnr-like genes of V. vulnificus CIP103196, V. parahaemolyticus CIP71.2 and P. profundum CIP106289 identified Qnr-like determinants that were almost identical (no more than three amino acid substitutions) to those found by in silico analysis of the corresponding reference strains for which the entire genome sequence is available (data not shown). This result indicates a high degree of similarity of those sequences, as found for the qnrA-like genes in S. algae.6 For the sake of consistency, the Qnr-like homologue of Vibrio fischerii whose genome has been reported recently in the GenBank databases was added for sequence comparison (Figure 1a and b). Amino acid comparison showed that the chromosome-encoded determinants had amino acid identity that ranged from 40% to 75% (Figure 1b). In addition, these determinants, except for S. algae and QnrA that share 99% identity,6 had at the most 67% identity with the plasmid-encoded Qnr determinants (Figure 1b). The chromosome-encoded Qnr-like determinants all belonged to the pentapeptide repeat family of proteins, defined by the presence of repetitions in tandem of the pattern A(D/N)LXX, where X is any amino acid.3 Both the plasmid- and chromosome-encoded Qnr proteins were made of two domains of 11 and 32 units connected by a single glycine, as observed for the QnrA determinant3 (Figure 1a). A consensus sequence of the repeats of the Qnr family could be proposed as being A/C, D/N, L/F, S/R and G/R. Analysis of the genomic sequences that surrounded the qnr-like genes in V. vulnificus, V. parahaemolyticus and P. profundum identified unrelated DNA sequences encoding putative proteins (outer membrane proteins...) but no insertion sequence or genetic structures known to be able to mobilize resistance genes were identified (data not shown).
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Since this work was in progress, another Qnr determinant of reference strain V. parahaemolyticus VPA0095 was cloned and expressed in E. coli.9 Interestingly, the authors reported a Qnr-like mutant protein obtained during a cloning process that had a cysteine to tyrosine change at position 115.9 This amino acid substitution conferred a higher level of resistance to quinolones and fluoroquinolones.9 Since the chromosome- and plasmid-mediated Qnr determinants have a cysteine residue at position 115 (Figure 1a), further studies may evaluate the role of change at that position for conferring clinically-significant resistance to fluoroquinolones.
This work underlines that water-borne Gram-negative bacterial species such as Shewanellaceae and now Vibrionaceae may be a reservoir for Qnr-like quinolone resistance determinants. The origin of the plasmid-encoded QnrB and QnrS determinants still remains to be discovered. Their reservoir might be other psychrophilic bacterial species of the aquatic environment10 such as Aeromonadaceae and Plesiomonas. Finally, knowledge of DNA sequences of these qnr-like determinants of Vibrionaceae will be helpful to design screening surveys for identifying novel plasmid-mediated qnr-like genes in particularly in Enterobacteriaceae that would be similar to those of Vibrionaceae.
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Acknowledgements |
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This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539) Université Paris XI, France and by a grant from the European Community (6th PCRD, LSHM-CT-2003-503335). L. P. is a researcher from the INSERM (Paris, France) and J. -M. R. -M. was a recipient of a travel grant from the Spanish Society for Clinical Microbiology and Infectious Diseases in 2004. We also thank A. Pascual for constant support of J.-M. R.-M. and G. A. Jacoby for sharing with us the QnrB sequence.
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References |
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Saga T, Kaku M, Onodera Y et al. Vibrio parahaemolyticus chromosomal homologue VPA0095: demonstration by transformation with a mutated gene of its potential to reduce quinolone susceptibility in Escherichia coli. Antimicrob Agents Chemother 2005; 49: 2144–5.
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