Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris Sud, 78 rue du Général Leclerc, 94275, K.-Bicêtre, France
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Abstract |
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Keywords: nalidixic acid , Qnr , fluoroquinolones
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Introduction |
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Mechanism of action of quinolones and of common bacterial resistance |
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Resistance to quinolones in Enterobacteriaceae most commonly arises stepwise as a result of mutation usually accumulating in the genes encoding primarily DNA gyrase and also topoisomerase IV. Decreased permeability by changes in the nature and amount of porins (in particular OmpF) or increased efflux by mutations in regulatory genes of chromosomally-encoded multidrug resistance pumps (Acr) or their regulatory systems (MarA, SoxS) may cause increments in quinolone resistance.1416
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Qnr family and QnrA-mediated resistance |
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Another QnrA determinant termed QnrA2 has been identified from a Klebsiella oxytoca isolate from China (GenBank accession number AY675584). QnrA2 differs from QnrA1 by a few amino acid substitutions (Figure 1).
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The detailed mode of action of Qnr determinants has been studied so far for QnrA only. QnrA binds to both subunits GyrA and GyrB of the gyrase at the early stages of interaction between gyrase and DNA.18 By lowering gyrase binding to DNA, QnrA reduces the amount of holoenzymeDNA targets for quinolone inhibition.17,18
Among other proteins of the pentapeptide family, there are two members of special interest in quinolone resistance. The first protein is McbG that protects bacteria which synthesize microcin B17 (MccB17) from self-inhibition.22 MccB17 is a post-transcriptional modified peptide of 3.1 kDa that blocks DNA replication.22 Like ciprofloxacin, this microcin is able to inhibit the activity of DNA gyrase and to stabilize the DNADNAgyrase complex in the presence of ATP.23,24 The self-immunity mechanism conferred by McbG requires products of other genes organized as an operon, mcbE and mcbF, for the expulsion of MccB17 from the cell.22 The second protein of the pentapeptide family is MfpA of Mycobacterium smegmatis that may contribute to quinolone resistance using efflux pumps.25 QnrA shares 19.6% and 18.9% amino acid identity with McbG and MfpA, respectively.
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Qnr-mediated quinolone resistance levels |
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Worldwide spread of Qnr determinants |
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In the pioneering study, a QnrA determinant was identified only from K. pneumoniae in Alabama12 and during a 6 month period in 1994 whereas it was not detected among 350 Gram-negative isolates that included strains producing reference plasmid-mediated cephalosporinases and clavulanic-acid expanded-spectrum ß-lactamases (ESBLs) and originating in 18 countries and 24 US states.28 After this initial prevalence survey, another study reported 11% QnrA-positive isolates among ciprofloxacin-resistant K. pneumoniae isolates from six US states collected from 1999 to 2002.33 A QnrA determinant was also identified in seven out of 17 E. cloacae isolates of variable susceptibility to ciprofloxacin and in two out of 20 ciprofloxacin-susceptible K. pneumoniae isolates from five US states.34
QnrA-like determinants in ciprofloxacin-resistant E. coli isolates collected from 2000 to 2002 were estimated to be 7.7% in Shanghai, China.32 A qnrA-like gene was detected in 11 out of 23 blaVEB-1-positive enterobacterial isolates (48%) from Bangkok, Thailand, collected in 1999 which were E. coli, K. pneumoniae and Enterobacter sakazakii, adding South East Asia to the list of regions in which QnrA determinants have spread.31 In addition, QnrA determinants were detected in E. coli isolates in South Korea.35
The qnrA gene was also detected in Europe and first in two out of 449 nalidixic-acid-resistant and non-duplicate enterobacterial isolates (0.5%) collected at the hospital Bicêtre (suburb of Paris, France) in 2003.27,31 The QnrA-positive isolates were E. coli and E. cloacae. In another European country, Germany, QnrA-positive Enterobacter spp. and Citrobacter freundii isolates were detected in four patients in two intensive care units among 703 cephalosporin-resistant or fluoroquinolone-resistant Enterobacteriaceae which were tested from 34 German intensive care units from 2000 to 2003.36 QnrA determinants have been identified in C. freundii, E. coli, Enterobacter amnigenus, E. cloacae and K. pneumoniae in the Netherlands.37
QnrA was also identified from C. freundii and E. cloacae in Turkey38 and from Providencia stuartii in Egypt.39
As indicated above whereas QnrS was identified from Japan only, the QnrB determinant was from India and the USA.20,21
None of the plasmid-mediated Qnr determinants have been identified so far in non-enterobacterial Gram-negative species (Pseudomonas aeruginosa, Acinetobacter baumannii, etc.), whereas several Qnr screening surveys included those types of isolates.28,31 However, it was shown that a plasmid-mediated QnrA determinant was able to be transferred to a P. aeruginosa reference strain by conjugation.12
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Association with expanded-spectrum ß-lactamases |
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QnrA determinants were also reported with plasmid-mediated cephalosporinases such as a blaFOX-5 gene in K. pneumoniae isolates from the USA.12,40
QnrB determinants were associated with the ESBL SHV-12 in several isolates.20
Association of antibiotic resistance genes may explain in part the frequent association between fluoroquinolone and expanded-spectrum cephalosporin resistance in Enterobacteriaceae.41 In addition, it raises the issue of the nature of antibiotic molecules that may select this co-resistance. We do not know if there is a special link between the two emerging mechanisms of resistance in Enterobacteriaceae, i.e. plasmid-mediated quinolone resistance and ESBL in community-acquired pathogens.
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Genetic vehicles |
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Another degree of mobility of qnrA-like genes has been identified since these genes are embedded in In4 family class 1 integrons, also known as complex sul1-type integrons.17,27,3133 These genetic structures possess duplicated qacE1 and sul1 genes that surround a sequence encoding Orf513 (Figure 2).42 This protein may act as a recombinase for mobilization of downstream-located antibiotic resistance genes. The qnrA gene was not associated with a 59-be element as a form of a gene cassette as found in common class 1 integrons. The definition of the CR1 conserved region (CR) established recently indicates that it consists of an orf513 gene that encodes a recombinase and a right-hand boundary that may act as a recombination cross-over site. It was shown that promoter sequences for expression of plasmid-encoded QnrA determinants overlap this CR1 element.27 Structural comparison of qnrA-positive integrons showed variability both in the upstream- and downstream-qnrA located DNA sequences (Figure 2). This suggests that the process that had led to qnrA gene insertion in the sul1-type integron may vary. An ampR gene involved in regulation of expression of the naturally-encoded cephalosporinase of Morganella morganii was located next to the qnrA gene in a sul1-type integron (Figure 2). However, no expanded-spectrum ß-lactamase gene was located inside any qnrA-positive sul1-type integrons (Figure 2). This observation indicates that co-localization of qnrA and expanded-spectrum ß-lactamase genes on the same plasmids probably results from unrelated genetic events.
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The genetic environment of the plasmid-encoded qnrB gene is unknown. However, the qnrS gene reported recently from Japan was not part of a sul1-type integron and not as a form of a gene cassette in a common class 1 integron.21 It was adjacent to a Tn3 transposon structure containing the ß-lactamase blaTEM-1 gene.21
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Origin of Qnr determinants |
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Positive results were obtained for Shewanella algae with identification of novel chromosome-encoded QnrA determinants termed QnrA3 to QnrA5 that differed by a few amino acid substitutions from the plasmid-mediated QnrA determinants (Figure 1).43 The G + C content (52%) of the qnrA-like genes of S. algae matches exactly that of the genome of S. algae.43 S. algae is a Gram-negative species belonging to the Shewanellaceae family that is widely distributed in marine and freshwater environments.44 S. algae is rarely involved in human infections, most being related to seawater exposure.45,46 The MIC of nalidixic acid was 2 mg/L and the MICs of the fluoroquinolones ciprofloxacin, ofloxacin, sparfloxacin and norfloxacin were 0.12, 0.5, 0.5 and 0.5 mg/L for S. algae strains, remaining in the susceptibility range according to NCCLS breakpoints.43 The CR1 element that provides promoter sequences for high-level expression of the plasmid-mediated qnrA gene in Enterobacteriaceae was not identified in S. algae.43 Since quinolones are also extensively used in animals and aquaculture,6,47 it is possible that subinhibitory concentrations of quinolones that are stable molecules in the environment48,49 may select for water-borne S. algae strains and enhance transfer of naturally occurring quinolone resistance determinants to Enterobacteriaceae. The aquatic environment has been shown to be a reservoir for antibiotic resistance genes and their transfer.50,51 In addition, whereas quinolones used in therapy are synthetic molecules, naturally-produced quinolones have been discovered recently52 that may also play a role in this horizontal transfer. In addition, it has been shown that quinolones induce an SOS repair system and antibiotic resistance gene transfer.53
Further work may also identify the reservoir of the distantly related QnrB and QnrS determinants that might also be psychrophilic bacterial species. Interestingly, several isolates from the US were found to produce both the QnrA and QnrB determinants20 suggesting that their progenitors may share an identical niche. Analysis of a qnrA-positive sul1-type integron from a Shanghai isolate that also contained an ampR gene (from M. morganii) indicated that construction of those sul1-type integrons may result from successive recombination events involving genes of unrelated bacterial origin. The role, if any, of those Qnr determinants in their natural hosts remains to be determined.
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Concluding remarks |
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By comparison with known flux of antibiotic resistance genes (such as narrow-spectrum penicillinase genes), it is possible that plasmid-mediated quinolone resistance determinants may be transferred to community-acquired Gram-negative bacterial species such as Neisseria spp. and Haemophilus spp. Current knowledge on Qnr determinants indicates that they are more diverse than previously expected. Their prevalence and the prevalence of their association with ESBL-encoding genes remain to be determined whereas Asian isolates seem already to be an important reservoir of Qnr determinants. Identification of qnrA genes embedded in integrons argues for their recent emergence in clinical isolates (rather than for their recent identification) since an increase in integron prevalence in multidrug-resistance in Enterobacteriaceae has been reported recently.54
The identification of the natural host, S. algae, as the source of plasmid-mediated QnrA determinants is an important step in discovering the location of this gene exchange (water-related environment, animals, etc.) and their enhancing factors. This may represent a unique opportunity for limiting the spread of these emerging antibiotic resistance determinants.
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Acknowledgements |
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