Plasmid-Mediated Quinolone Resistance; Interactions between Human, Animal, and Environmental Ecologies

Resistance to quinolones and fluoroquinolones is being increasingly reported among human but also veterinary isolates during the last two to three decades, very likely as a consequence of the large clinical usage of those antibiotics. Even if the principle mechanisms of resistance to quinolones are chromosome-encoded, due to modifications of molecular targets (DNA gyrase and topoisomerase IV), decreased outer-membrane permeability (porin defect), and overexpression of naturally occurring efflux, the emergence of plasmid-mediated quinolone resistance (PMQR) has been reported since 1998. Although these PMQR determinants confer low-level resistance to quinolones and/or fluoroquinolones, they are a favorable background for selection of additional chromosome-encoded quinolone resistance mechanisms. Different transferable mechanisms have been identified, corresponding to the production of Qnr proteins, of the aminoglycoside acetyltransferase AAC(6′)-Ib-cr, or of the QepA-type or OqxAB-type efflux pumps. Qnr proteins protect target enzymes (DNA gyrase and type IV topoisomerase) from quinolone inhibition. The AAC(6′)-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. Finally, the QepA and OqxAB efflux pumps extrude fluoroquinolones from the bacterial cell. A series of studies have identified the environment to be a reservoir of PMQR genes, with farm animals and aquatic habitats being significantly involved. In addition, the origin of the qnr genes has been identified, corresponding to the waterborne species Shewanella sp. Altogether, the recent observations suggest that the aquatic environment might constitute the original source of PMQR genes, that would secondly spread among animal or human isolates.


INTRODUCTION
Quinolones are fully synthetic and bactericidal antibacterial agents used widely in both human and veterinary medicine. The clinically available quinolones have been classified into several generations based of their spectrum of activity (Ball, 2000). The first generation quinolone (Q1G), nalidixic acid, has been discovered in 1962 (Lesher et al., 1962). Other Q1G, such as pipemidic acid and oxolinic acid, had been developed, the latter being used for in veterinary medicine. The quinolones of the second generation are made of addition of a fluorine atom at position C-6 to the quinolone nucleus, yielding to the fluoroquinolones (FQ; Paton and Reeves, 1988). The early FQ (e.g., norfloxacin, ofloxacin, pefloxacin, ciprofloxacin, or enrofloxacin) achieved higher serum levels and showed potent activity against Gram-negative bacteria, several Gram-positive bacteria (such as Staphylococcus aureus), and intracellular bacteria. In addition, ciprofloxacin is active against Pseudomonas aeruginosa. Newer FQ (third generation quinolones) were subsequently developed and presented increased activity toward Gram-positive bacteria, in particularly to Streptococcus pneumoniae (e.g., sparfloxacin, levofloxacin, or moxifloxacin), and potent activity against anaerobic bacteria (e.g., trovafloxacin, gatifloxacin, or gemifloxacin; Van Bambeke et al., 2005).
Even if the main factors leading to resistance to quinolones and FQ related to chromosomal mutations in the drug target genes, the discovery during the last decade of a series of plasmid-encoded resistance mechanisms has contributed to speculate about the origin and enhancing factors of that transferable resistance. In particular, the interplay between an environmental and animal source on one side, and the human clinical pathogens on the other side (in which the emergence of resistance to quinolones is a matter of fact) remains to be further explored and understood.
That review aims to present some of the current available data from which speculations can be established.

MECHANISM OF QUINOLONE ACTION
The targets of quinolone molecules are the type II topoisomerases: DNA gyrase (topoisomerase II) and DNA topoisomerase IV (Drlica and Zhao, 1997). As opposed to type I topoisomerases that transiently cleave one strand of the DNA double helix, type II topoisomerases break transiently both strands of a duplex and pass another double-helical segment through the break by ATP hydrolysis (Drlica and Zhao, 1997;Hawkey, 2003). The DNA gyrase introduces negative supercoils into DNA whereas topoisomerase IV exhibits a potent decatenation activity. Those enzymes are essential for bacterial growth by controlling the topological status of the chromosomal DNA to facilitate replication, transcription, recombination, and DNA repair (Drlica and Zhao, 1997;Hawkey, 2003). The DNA gyrase and the DNA topoisomerase IV are the main targets of quinolones in Gram-negatives and Grampositives, respectively. Quinolones inhibit the activity of type II topoisomerases by trapping these enzymes on DNA as drugenzyme-DNA complexes. Ternary complex formation is responsible for inhibition of bacterial growth (bacteriostatic action) by a rapid inhibition of DNA synthesis and a slower inhibition of RNA synthesis (Drlica and Zhao, 1997;Hawkey, 2003). Eventhough these drug-enzyme-DNA complexes block cell growth, they are not directly responsible for the lethal effect of quinolones. Indeed, bactericidal activity is due to the releasing of double-stranded DNA breaks from those complexes, but the detailed mechanism of action of quinolones still needs to be fully understood.

CHROMOSOME-ENCODED RESISTANCE
Resistance to quinolones in Enterobacteriaceae most commonly results from the accumulation of mutations primarily in DNA gyrase (GyrA) then in topoisomerase IV (ParC; Hooper, 2000;Ruiz, 2003;Hopkins et al., 2005;Jacoby, 2005). Alterations in GyrA of E. coli predominantly occur within the N-terminus of the protein in the so-called quinolone resistance determining region (QRDR) located between amino acids Ala67 and Gln106. Mutations appear most frequently at codons Ser83 and Asp87, which are located near the active sites of enzyme (Tyr122). In addition, quinolone resistance can be associated with a decreased membrane permeability and/or an overexpression of efflux pump systems (Hooper, 2000;Hopkins et al., 2005).

PLASMID-MEDIATED RESISTANCE
Although considered as impossible due to the plasmid curing effect of quinolones (Courvalin, 1990), plasmid-mediated quinolone resistance (PMQR) was first reported in 1998 from a Klebsiella pneumoniae isolate in the USA (Munshi et al., 1987). Indeed, a plasmid-mediated resistance to nalidixic acid in Shigella dysenteriae has been reported previously in 1987 (Munshi et al., 1987), but the reality of this phenomenon was later rejected (Courvalin, 1990). To date, several PMQR mechanisms have been identified: Qnr proteins, the aminoglycoside acetyltransferase AAC(6 )-Ib-cr, and the efflux pumps QepA and OqxAB.

Qnr structure and nomenclature
The first identified PMQR determinant corresponded to the Qnr protein, lately termed QnrA1 (Martinez-Martinez et al., 1998). The corresponding gene was identified on a broad-host range conjugative plasmid recovered from a ciprofloxacin-resistant K. pneumoniae isolate (Martinez-Martinez et al., 1998). QnrA1 is a 218-amino-acid protein that belongs to the pentapeptide repeat family, of which more than 500 members are known, distributed in prokaryotics and eukaryotics (Vetting et al., 2006). Those proteins are made of tandemly repeated amino acid sequences with a consensus sequence [S, T, A, or V] [D or N] [L or F] [S, T, or R] [G] (Vetting et al., 2006). Six other QnrA variants (QnrA2 to QnrA7) have been identified, and differ from QnrA1 by a few amino acid substitutions (Jacoby et al., 2008).
In addition, QnrVC-like proteins have been identified in Vibrio cholerae, sharing 57% amino acid identity with QnrA1 (Fonseca et al., 2008). Even if the qnrVC genes have been identified as acquired resistance genes, they are not plasmid-located, thus not considered as PMQR genes.

Mechanism of action
QnrA1 shares 20 and 19% amino acid identity with McbG and MfpA, respectively, two other members of the pentapeptide repeat family both involved in resistance to gyrase inhibitors (Cattoir and Nordmann, 2009). Qnr proteins may supplement resistance to quinolones due to altered quinolone target enzymes, efflux pump activation, or deficiencies in outer-membrane porins (Martinez-Martinez et al., 2003;Jeong et al., 2008). In addition, Qnr proteins facilitate selection of quinolone resistance mutants by raising the level at which they can be selected with a frequency more than 100-fold higher (Martinez-Martinez et al., 1998). The presence of Qnr determinants facilitates the selection of low-level of resistance to quinolones due to chromosome-encoded mechanisms. From a clinical point of view, Qnr determinants may increase the mutant prevention concentration (MPC) of ciprofloxacin by more than 10-fold, facilitating recovery of mutants with higher level of resistance to quinolones (Rodriguez-Martinez et al., 2007). Therefore, Qnr-positive isolates may be a favorable background for an in vivo-selection of additional chromosome-borne mechanism(s) of resistance to quinolones after treatment by fluoroquinolones (Poirel et al., 2006).

Epidemiology of Qnr determinants
Qnr in human clinical isolates. All types of Qnr determinants have been identified worldwide in many different enterobacterial species but mostly in K. pneumoniae, Enterobacter spp., E. coli, and Salmonella enterica from community and nosocomial isolates (Rodríguez-Martínez et al., 2011). Their overall prevalence may range from 0.2 to up to 94% depending on selection criteria of studied strains (resistance to ceftazidime, nalidixic acid, FQs,. . .; Strahilevitz et al., 2009;Rodríguez-Martínez et al., 2011). The prevalence of qnrB genes seems to be overall higher than that of the other qnr genes. However, the qnrS genes are very frequently identified in Salmonella sp., suggesting that they could represent a significant resistance trait along the food chain. For instance, a recent international survey (13 European countries) identified a qnrS gene in 10% of the Salmonella sp. collection (Veldman et al., 2011).
Very few studies have been performed to evaluate the prevalence of the qnrC and qnrD genes since those genes have been recently Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy identified. However, the qnrC has been identified from a Proteus mirabilis isolate from China, and its prevalence seems to be very low, at least in China . The qnrD gene has been identified in 22 out of 1215 Salmonella isolates obtained from different European countries, being either of human or animal isolates (Veldman et al., 2011).
Qnr in animal isolates. QnrS1 was first identified from a transferable plasmid carried by a clinical isolate of Shigella flexneri 2b as a source of a foodborne outbreak in Aichi prefecture, Japan (Hata et al., 2005). As described for the qnrA1 gene, the qnrS1 gene has been identified from several enterobacterial isolates (particularly in Salmonella spp.) in many countries. It has been identified in porcine E. coli (Szmolka et al., 2011) in Hungary, in equine E. coli in Czech Republic (Dolejska et al., 2011), and in poultry E. coli in China (Yue et al., 2011). The qnrS2 gene was identified in a single non-Typhi Salmonella clinical isolate from the USA (Gay et al., 2006). Finally, the qnrS3 variant has been identified in a single veterinary clinical E. coli isolate from China (GenBank accession no. EU077611).
In an interesting study including 1215 Salmonella and 333 E. coli isolates, six variants of qnrB were identified from 138 qnrB-positive isolates, most of them being obtained from turkeys (Veldman et al., 2011). The qnrD gene was identified in 22 Salmonella of eight different serovars, being mostly identified in Spain but also in Italy.
Whereas there is so far no report of qnrA-like genes in nonenterobacterial species, qnrB-and qnrS-like genes have been identified for instance in Pseudomonas fluorescens and Aeromonas spp. isolates, respectively (Ahmed et al., 2007;Cattoir et al., 2008b;Sanchez-Cespedes et al., 2008). Interestingly, qnrS and mostly qnrB genes were identified from zoo animals, mostly including reptiles (Ahmed et al., 2007).

Qnr determinants in aquatic environments.
Overall, the qnrStype genes seem to be the most commonly identified acquired qnr genes in the environment. They have been mainly identified from waterborne species, and in particular Aeromonas spp. The qnrS2 gene was identified from a mobilizable IncQ-related plasmid (pGNB2) isolated from an activated sludge bacterial community of a wastewater treatment plant in Germany (Bonemann et al., 2006), in two strains of Aeromonas spp. (Aeromonas punctata and A. media) isolates from the Seine river in France (Cattoir et al., 2007b), and lately in a single clinical Aeromonas veronii isolate from Spain (Sanchez-Cespedes et al., 2008). In Italy, a Citrobacter freundii strain producing the ESBL TEM-116 was recovered from a sewage effluent (Forcella et al., 2010). This ESBL gene was encoded on a plasmid that co-harbored the qnrB9 gene. This constitutes one of the few example showing the occurrence of a qnr gene from an enterobacterial isolate recovered from the environment.
The environmental species that have been found to carry qnr genes were mainly Aeromonas spp. or Vibrio spp. In China, an A. punctata strain recovered from a wastewater sample in the Shandong province carried the qnrVC4 gene on a plasmid (Xia et al., 2010). That strain was resistant to nalidixic acid but susceptible to fluoroquinolones.

Mobile genetic vehicles
All the qnr genes have been identified on plasmids that vary in size ranging from ca. 7 to 320 kb (Cattoir and Nordmann, 2009;Strahilevitz et al., 2009) Those plasmids, and especially the qnrA-and qnrB-positive ones, often harbor other antibiotic resistance genes conferring resistance to β-lactams, aminoglycosides, chloramphenicol, tetracycline, sulfonamides, trimethoprim, and rifampin.

The qnr genes originate from environmental species
By screening for a collection of 48 Gram-negative clinical and environmental bacterial species (Enterobacteriaceae, Aeromonadaceae, Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae, and Shewanellaceae), the origin of the qnrA gene was identified as being the chromosome of Shewanella algae (Poirel et al., 2005b). Indeed, three QnrA-like determinants (termed QnrA3, QnrA4, and QnrA5) have been identified in S. algae, and differ by a few amino acid substitutions from QnrA1. S. algae is widely distributed in aquatic environments and rarely involved in human infections. As opposed to what it has been described for the plasmid-mediated qnrA1 gene, the chromosomal qnrA-like genes were not associated with the ISCR1 element in the chromosome of S. algae (Poirel et al., 2005b). Finally, The G + C content (52%) of the qnrA-like of S. algae matched exactly that of the genome of S. algae (Poirel et al., 2005b).
It has been shown that Vibrio splendidus is a source of QnrSlike determinants since chromosomal-encoded Qnr-like proteins shared about 84 and 88% amino acid identity with the plasmidmediated determinants QnrS1 and QnrS2, respectively (Cattoir et al., 2007a). In addition, the G + C contents of qnrS-like genes from V. splendidus (ca. 45%) are close to those of qnrS1 and qnrS2 (ca. 44%). Although the exact progenitor species of the plasmid-encoded QnrS determinant remains unknown, the bacterial species should be closely related to V. splendidus and likely waterborne.
Recently, the progenitor of the qnrB-like genes was identified to be Citrobacter spp. which are enterobacterial species known to www.frontiersin.org be widely present in the aquatic environment, being either human commensal bacteria or opportunistic pathogens depending on the species (Jacoby et al., 2011).
Noteworthy, it has been shown that some bacterial species belonging to the Vibrionaceae family (such as Vibrio vulnificus, Vibrio parahaemolyticus, or Photobacterium profundum) also possess intrinsically chromosome-encoded Qnr-like determinants (sharing 40-67% identity with the plasmid-mediated Qnr determinants) and conferring resistance to quinolones (Poirel et al., 2005a). That means that those waterborne species may also constitute potential sources of emerging PMQR genes.
Several Qnr-like pentapeptide repeat proteins have been identified in the chromosome of Gram-positive bacteria (Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Clostridium perfringens, Clostridium difficile, Bacillus cereus, and Bacillus subtilis; Arsene and Leclercq, 2007;Rodriguez-Martinez et al., 2008). Amino acid sequences of these Qnr-like proteins are identical from 16 to 22% with the PMQR determinants QnrA1, QnrB1, and QnrS1 (Rodriguez-Martinez et al., 2008). Similarly, those Gram-positive species could also constitute a reservoir for Qnrlike although none of these genes has been yet identified as plasmid-located determinants.

AMINOGLYCOSIDE ACETYLTRANSFERASE AAC(6 )-Ib-cr
The AAC(6 )-Ib-cr enzyme is a PMQR determinant that has been discovered from qnrA-positive E. coli from Shanghai, China . The aac(6 )-Ib-cr (for ciprofloxacin resistance) gene encodes a variant of the widespread aminoglycoside acetyltransferase AAC(6 )-Ib usually responsible for resistance to kanamycin, tobramycin, and amikacin (Strahilevitz et al., 2009). This variant possesses two substitutions at codons 102 (Trp → Arg) and 179 (Asp → Tyr) compared to the wild-type AAC(6 )-Ib, both mutations seem to be required to confer reduced susceptibility to several FQ molecules . The protein AAC(6 )-Ib-cr is able to acetylate kanamycin, tobramycin, and amikacin, but also ciprofloxacin conferring slightly higher MIC values (twofold to fourfold increase). Nevertheless, it acetylates more efficiently aminoglycosides than ciprofloxacin. Since acetylation occurs at the amino nitrogen on the piperazinyl substituent, only FQs harboring an unsubstituted piperazinyl group (such as ciprofloxacin and norfloxacin) are substrates of AAC(6 )-Ib-cr . Although the aac(6 )-Ib-cr gene by itself confers low-level resistance to certain FQs, it may facilitate survival of target-site mutants with a 10-fold increase of their MPC (Cattoir and Nordmann, 2009).
The overall prevalence of aac(6 )-Ib-cr may range from 0.4 to up to 34% depending on the studied human clinical strains . This gene has been reported mostly from E. coli and K. pneumoniae clinical isolates. However, it has also been identified in Aeromonas spp. collected in 2006 from feces of zoo animals in Japan (Ahmed et al., 2007). Recently, it has been identified in Salmonella spp. recovered from chickens in Japan, and in E. coli of poultry origin in Spain or of pig origin in China Soufi et al., 2011;Du et al., 2012). Since this gene seems to be geographically widespread, stable over the time, and equally prevalent in ciprofloxacin-susceptible and -resistant strains , its significance remains debatable. Its occurrence could also result from human contamination, as suggested with a study from Gibson et al. (2010) identifying this gene in companion animals.
The aac(6 )-Ib-cr gene has been identified as a form a gene cassette into sul1-type class 1 integrons, and has been identified both among ESBL-positive and ESBL-negative enterobacterial isolates (Cattoir and Nordmann, 2009). Its occurrence in animals and in the environment is likely frequent, but extensive surveys are still required to better evaluate their prevalence in environmental habitats.
The occurrence of QepA among human clinical isolates seems to be quite limited according to the few studies that have been conducted on this subject worldwide. However, its occurrence in animals might be significant. A study performed on E. coli isolates from pigs in China showed that 28 (58.3%) out of 48 16S rRNA methylase RmtB-producing E. coli isolates were qepApositive suggesting a strong linkage between qepA and rmtB genes (Liu et al., 2008). RmtB confers resistance to all aminoglycosides (except streptomycin) by decreasing the affinity of the ribosome for the antibiotic after N7-methylation at the G1405 within the 16S rRNA (Perichon et al., 2007).
Other E. coli isolates from pigs in China have been reported as co-expressing the qepA, qnrS2, and aac(6 )-Ib-cr genes (Liu et al., 2008). This co-expression of several PMQR determinants may facilitate the selection of mutants under selective pressure of antimicrobial agents. QepA-producing enterobacterial isolates were also identified from pets in China (Deng et al., 2011). In Nigeria, an E. coli strain recovered from chicken co-harbored a qepA and a qnrB gene .
The natural reservoir of qepA remains unknown. However, it may be Actinomycetales species since QepA had significant amino acid identity with likely membrane transporters of the members of the order of Actinomycetales (such as Streptomyces globisporus, Streptomyces coelicolor, Nocardia farcinica, or Polaromonas spp.), and its high GC% content (72%) is compatible with this origin.

EFFLUX PUMP OqxAB
The OqxAB multidrug resistance mechanism was initially identified from E. coli strains recovered from swine manure (Hansen Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy et al., 2004). The identified plasmid harbored the oqxA and oqxB genes that are similar to genes encoding resistance-nodulationcell-division efflux systems. That plasmid conferred resistance to olaquindox that is a veterinary growth promoter. Then, it was shown to mediate resistance to other molecules, such as chloramphenicol, nalidixic acid, and ciprofloxacin (Hansen et al., 2007). In Denmark, a retrospective study showed that nine out of 156 E. coli strains isolated from pigs were positive for the oqxA gene (Hansen et al., 2005). Recently, a Chinese study showed that 39% of the E. coli isolates recovered from sows, piglets, weaners, and boars in swine farms, and chicken in chicken farms harbored the oqxAB gene (Zhao et al., 2010). An OqxAB-positive E. coli strain was also identified from a liver sample of a diseased chicken in China (Liu et al., 2008). Interestingly, another Chinese study reported a series of K. pneumoniae isolates in which the oqxAB genes were actually chromosomally located (Kim et al., 2009).

DISCUSSION
The discovery of a series of PMQR determinants within the last 10 years further raised out a novel issue regarding resistance to quinolones. Indeed, whereas such resistance was supposed to be only vertically transmitted, the occurrence of those PMQR encoding genes show that it may be also horizontally mediated. Noteworthy, and even if the first research interests focused on the impact and relevance of PMQR genes among human clinical isolates, subsequent studies rapidly showed that they were also of main concern in animal and environmental strains. Such observation raises out several questions: are there relationships between quinolones in the environment that are poorly biodegraded and the prevalence of those resistance mechanisms? Are those resistance mechanisms really new and emerging? Which is the extend of the interplay between the situation observed in the environment and the current clinical concerns?
The heavy use of quinolones in animals and in particular in fish farming might likely have played a role in the selection of some resistance mechanisms. This may have impacted the fauna itself, and as a consequence the environment through contamination of aquatic habitats, but that speculation remains debatable. A recent study showed that there was no correlation between the occurrence of FQ-resistant bacteria in aquatic environments and the FQ contamination in Vietnam and Thailand (Takasu et al., 2011). However, the authors designed their study by selecting FQ-resistant bacteria with high level of resistance (more than 16 mg/l), that is not a correct criteria when focusing on PMQR only conferring decreased susceptibility to FQ.
The fact that most if not all PMQR encoding genes originate from bacterial species that are naturally present in the environment, and in particular in the aquatic one, likely suggests that this latter may represent the main source of the problem. This is indeed probable that genetic events leading to the mobilization of the resistance gene from the natural reservoir (the progenitor or the donor) to the recipient (the target plasmid or the target strain) occur in those environments where the donor is numerous.

ACKNOWLEDGMENTS
This work was funded by a grant from the INSERM (U914).