JAC Advance Access published online on March 13, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn115
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Original research |
Qnr-like pentapeptide repeat proteins in Gram-positive bacteria
1 Department of Microbiology, University of Seville, Seville, Spain 2 Service of Microbiology, University Hospital Virgen Macarena, Seville, Spain
* Corresponding author. Tel: +34-954-55-28-63; Fax: +34-954-37-74-13; E-mail: jmrodriguez{at}us.es
Received 21 November 2007; returned 18 January 2008; revised 7 February 2008; accepted 22 February 2008
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Abstract |
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Objectives: To study the role of Qnr-like pentapeptide repeat proteins (PRPs) from several Gram-positive species with quinolone resistance in vitro.
Methods: A PCR-based strategy was used to clone and express genes coding for Qnr-like PRPs in Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Clostridium perfringens, C. difficile, Bacillus cereus and B. subtilis in Escherichia coli DH10B. MIC values of nalidixic acid and fluoroquinolones were determined for reference strains and E. coli DH10B harbouring recombinant plasmids containing genes coding for PRPs.
Results: Amino acid identity of Qnr-like PRPs in Gram-positive strains compared with that of the plasmid-mediated quinolone resistance determinants QnrA1, QnrB1 and QnrS1 was in the range of 16% to 22%. Recombinant plasmids coding for Qnr-like PRPs conferred reduced susceptibility to fluoroquinolones (in the range of 0.016 to 0.064 mg/L for ciprofloxacin) and nalidixic acid (from 6 to 12 mg/L), depending on the antimicrobial agent and PRP. The PRP from B. subtilis showed no protective effect.
Conclusions: The PRPs analysed conferred a reduced susceptibility phenotype in E. coli; the data provide further evidence of the possible roles in quinolone resistance of PRPs from different Gram-positive species. These Gram-positive species may constitute a reservoir for Qnr-like quinolone resistance proteins.
Key Words: fluoroquinolones , resistance , plasmids
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Introduction |
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Fluoroquinolone resistance in bacteria mainly results from chromosome mutations. In 1998, the plasmid-mediated quinolone resistance determinant Qnr (later named QnrA) was reported.1 Later, distantly-related plasmid-mediated Qnr determinants (QnrS and QnrB) were described in Enterobacteriaceae.2 qnr genes have been reported worldwide in Enterobacteriaceae and confer reduced susceptibility to quinolones.2
QnrA is a pentapeptide repeat protein (PRP) that protects DNA gyrase and topoisomerase IV from inhibitory quinolone activity.3 Plasmid-mediated qnr genes confer quinolone resistance and increase the MIC of fluoroquinolones up to 32-fold.2 QnrB and QnrS, also belonging to the PRP family, share only 40% and 59% amino acid identity, respectively, with QnrA. In Gram-positive bacteria, just two PRPs from Enterococcus faecium and Mycobacterium tuberculosis, sharing only 20% amino acid identity with QnrA, have been described; these also confer reduced susceptibility to quinolones.4,5 These data suggest that other PRPs may play a role in quinolone resistance.
To gain further insights into the role in quinolone resistance of Qnr-like PRPs from different Gram-positive species, we performed a protein BLAST analysis and found that several PRPs in Enterococcus faecalis (AE016949), E. faecium (ZP_00604681), Listeria monocytogenes (EAL07413 [GenBank] ), Clostridium perfringens (NP_561876 [GenBank] ), Clostridium difficile (CAJ69589 [GenBank] ), Bacillus cereus (NP_831602 [GenBank] ) (considered a putative quinolone resistance protein) and Bacillus subtilis (CAB12929 [GenBank] ) shared different degrees of amino acid identity with plasmid-mediated Qnr determinants. We analysed the effect of these proteins on quinolone resistance by cloning their genes and studying their expression in Escherichia coli. Our results were compared with those obtained for plasmid-mediated Qnr determinants (QnrA, QnrS and QnrB).
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Bacterial strains and growth conditions
E. faecalis ATCC 29212, E. faecium ATCC 35667, L. monocytogenes ATCC 7644, C. perfringens ATCC 10388, C. difficile ATCC 9689, B. cereus ATCC 11778 and B. subtilis ATCC 12432 were used to determine MIC values and as templates for PCR amplification and cloning experiments. Bacterial strains were grown under standard conditions, using Columbia blood or Schaedler agar plates for aerobic and anaerobic species, respectively. E. coli DH10B was used as a recipient strain for cloning experiments.
PCR amplification and cloning assays
Genomic DNA was prepared using a standard procedure. A DNA fragment corresponding to the qnr-like gene coding for PRP was amplified by PCR using the primer pairs indicated in Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. The amplified fragments were then cloned into the chloramphenicol-resistant pPCR-Script vector (Stratagene, Cedar Creek, TX, USA) and sequenced. Cloning of PCR-amplified fragments from E. faecalis ATCC 29212, E. faecium ATCC 35667, L. monocytogenes ATCC 7644, C. perfringens ATCC 10388, C. difficile ATCC 9689, B. cereus ATCC 11778 and B. subtilis ATCC 12432 gave pEfsQnr, pEfmQnr, pLmQnr, pCpQnr, pCdQnr, pBcQnr and pBsQnr recombinant plasmids, respectively.
MIC values of nalidixic acid and fluoroquinolones for the various Gram-positive species and E. coli strains containing recombinant plasmids were determined using the Etest technique (AB Biodisk, Solna, Sweden). MICs were interpreted according to CLSI guidelines.6
Northern blot assays and RT–PCR
Total RNA was isolated using Tripure Isolation Reagent (Roche Diagnostics GmbH, Mannheim, Germany) and treated with RNase-free DNase I (Qiagen). Northern blot assays were carried out following standard protocols. RT–PCR was performed using the Titan One Tube RT–PCR System (Roche). See Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)] for primers and probes used.
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Results and discussion |
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MIC values of quinolones for reference strains and E. coli clones carrying recombinant plasmids are shown in Table 1. Once expressed in E. coli DH10B, the Qnr-like PRP conferred, in almost all cases (five or six out of seven PRPs, respectively), reduced susceptibility to fluoroquinolones (ranging from 0.016 to 0.064 mg/L for ciprofloxacin) and nalidixic acid (ranging from 6 to 12 mg/L) depending on the antimicrobial agent and the PRP (Table 1). MIC values were similar to those obtained for E. coli harbouring recombinant plasmids expressing qnrA1, qnrB1 and qnrS1 (Table 1). This was not the case for the BsQnr protein, which showed no effect against any quinolone or fluoroquinolone tested (1 mg/L and 0.003 mg/L for nalidixic acid and ciprofloxacin, respectively), or for the CdQnr protein, which showed no effect against nalidixic acid (1.5 mg/L) (Table 1). The Northern-blot assays verified that qnr-like genes were expressed in E. coli (data not shown). Interestingly, both BsQnr and CdQnr were also transcribed. No effect against quinolones in these two cases could be due to specific changes of conserved residues in the amino acid sequences of the proteins which affected their individual effect against quinolones (Figure 1), since both PRPs showed similar levels of amino acid identity when compared with the other proteins (Table 2).
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It has been suggested that the qnrA and qnrS genes found in Enterobacteriaceae plasmids could have been acquired from the chromosomal determinants of Shewanella algae and Vibrio splendidus, respectively.7,8 Whether similar events might occur in Gram-positive species has not yet been established.
The MIC values obtained indicate that chromosome-encoded Qnr-like determinants confer significantly reduced susceptibility to quinolones when expressed in E. coli. Arsène and Leclercq5 have shown that EfsQnr appears to take part in the intrinsic quinolone resistance of E. faecalis. Conventional RT–PCR showed transcription of the different PRPs analysed in their natural host (data not shown). Previous data about EfsQnr and MfpA proteins4,5 lead us to think that the PRPs of the species studied here may play a role in natural quinolone resistance, although the role in their native species is yet to be determined.
Sequences of qnr-like genes from the strains analysed in this study were almost identical (
99% amino acid identity) to those found in reference sequences in the NCBI database, with just 2–3 amino acid substitutions. Similar observations have been made when comparing sequences of the qnrA gene in S. algae and qnr-like genes in Vibrionaceae deposited in databases and other isolates.8,9
Amino acid identity of chromosome-encoded determinants in Gram-positive species ranged between 17% and 22% when compared with QnrA1. These percentages were similar for QnrB1 and QnrS1. When compared with EfsQnr, amino acid identity ranged between 25% and 32% (Table 2). All Qnr-like determinants belonged to the pentapeptide repeat family of proteins, as defined by the presence of repetitions in tandem of the consensus sequence [S,T,A,V][D,N][L,F][S,T,R][G].10 QnrA contains two domains of 11 and 32 units each, connected by a single glycine (G56).3 This glycine was not conserved in the PRPs from the Gram-positive species studied; serine, aspartic acid or arginine was found instead (Figure 1).
Alignment of proteins revealed the presence of four completely conserved residues (C72, G96, F114 and L159 with respect to the QnrA1 sequence). Among Gram-positive species, eight residues were conserved (Figure 1). These conserved positions may be important for the, currently unknown, physiological role of the proteins or for their effect against quinolones. Assays are in progress to clarify this.
Finally, this study emphasizes the fact that various PRPs present in clinically relevant Gram-positive genera (Enterococcus, Listeria, Clostridium or Bacillus), as well as in the previously described Gram-negative ones, Shewanellaceae or Vibrionaceae,8,9 might be reservoirs for Qnr-like determinants of quinolone resistance, and could participate in the natural resistance to fluoroquinolones of these species.
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This work was supported by the Ministerio de Sanidad y Consumo, the Instituto de Salud Carlos III, Spain (project PI060580), the Consejería de Innovación Ciencia y Empresa, Junta de Andalucía, Spain (P07-CTS-02908) and the Spanish Network for Research in Infectious Diseases, Spain (REIPI RD06/0008).
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Transparency declarations |
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None to declare.
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Supplementary data |
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Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org).
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References |
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1 . Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet (1998) 351:797–9.[CrossRef][Web of Science][Medline]
2 . Robicsek A, Jacoby GA, Hooper DC. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis (2006) 6:629–40.[CrossRef][Web of Science][Medline]
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Tran JH, Jacoby GA. Mechanism of plasmid-mediated quinolone resistance. Proc Natl Acad Sci USA (2002) 99:5638–42.
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Montero C, Mateu G, Rodriguez R, et al. Intrinsic resistance of Mycobacterium smegmatis to fluoroquinolones may be influenced by new pentapeptide protein MfpA. Antimicrob Agents Chemother (2001) 45:3387–92.
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Arsène S, Leclercq R. Role of a qnr-like gene in the intrinsic resistance of Enterococcus faecalis to fluoroquinolones. Antimicrob Agents Chemother (2007) 51:3254–8.
6 . Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15 (2005) Wayne, PA, USA: CLSI.
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Cattoir V, Poirel L, Mazel D, et al. Vibrio splendidus as the source of plasmid-mediated QnrS-like quinolone resistance determinants. Antimicrob Agents Chemother (2007) 51:2650–1.
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Poirel L, Rodriguez-Martinez JM, Mammeri H, et al. Origin of plasmid-mediated quinolone resistance determinant QnrA. Antimicrob Agents Chemother (2005) 49:3523–5.
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Poirel L, Liard A, Rodriguez-Martinez JM, et al. Vibrionaceae as a possible source of Qnr-like quinolone resistance determinants. J Antimicrob Chemother (2005) 56:1118–21.
10 . Vetting MW, Hegde SS, Fajardo JE, et al. Pentapeptide repeat proteins. Biochemistry (2006) 45:1–10.[CrossRef][Web of Science][Medline]
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