International Journal of Antimicrobial Agents 40 (2012) 196–203 Contents lists available at SciVerse ScienceDirect International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag Review Transferable mechanisms of quinolone resistance Joaquim Ruiz a,b,∗ , Maria J. Pons a , Cláudia Gomes a a b Centre de Recerca en Salut Internacional de Barcelona (CRESIB), Hospital Clinic, Universitat de Barcelona, C/Rosselló 149-153, Barcelona, Spain CIBER Epidemiología y Salud Pública (CIBERESP), Spain a r t i c l e i n f o Keywords: Quinolone resistance Qnr Qep OqxAB AAC(6 )-Ib-cr Natural transformation a b s t r a c t Quinolones were introduced into clinical practice in the late 1960s. Although quinolone resistance was described early, no transferable mechanism of quinolone resistance (TMQR) was conﬁrmed until 1998. To date, ﬁve different TMQRs have been described in the literature, including target protection (Qnr), quinolone modiﬁcation (AAC(6 )-Ib-cr), plasmid-encoded efﬂux systems (e.g. QepA or OqxAB, amongst others), effect on bacterial growth rates and natural transformation. Although TMQRs usually only result in a slight increase in the minimum inhibitory concentrations of quinolones, they possess an additive effect and may facilitate the acquisition of full quinolone resistance. The emergence of new related genes may continue in the next years. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. 1. Introduction Nalidixic acid (NAL) was the ﬁrst quinolone-derived agent demonstrating antibacterial activity as well as useful clinical parameters , being followed in the 1970s by novel compounds of this family such as pipemidic acid, although the clinical indication for these quinolones remained limited to urinary tract infections. The posterior addition of a ﬂuorine atom at position 6 of the quinolone molecule greatly enhanced its activity. Since then, a large series of molecules of this family has been synthesised and several have been introduced into clinical practice, with ciproﬂoxacin (CIP) being the most representative . However, some quinolones have been withdrawn from clinical practice following restriction of their use owing to secondary effects, including some deaths [3–5]. Fluoroquinolones (FQs) have been extensively used to treat a great variety of bacterial infections [6–9]. Moreover, some FQs possess activity against eukaryotic targets and have been studied as a possible novel antiparasitic treatment [10,11] or explored as potential antineoplastic agents . Finally, quinolones have been extensively used in veterinary practice [13,14]. However, the World Health Organization (WHO) currently considers FQs to be critically important antimicrobials, proposing very restricted use in veterinary practice , and a number of countries such as those of the European Union have forbidden some related uses (i.e. use as growth promoters). ∗ Corresponding author. Present address: Centre de Recerca en Salut Internacional de Barcelona (CRESIB), Hospital Clínic, Ediﬁci CEK, Planta 1, C/Rosselló 149-153, 08036 Barcelona, Spain. Tel.: +34 932 275 400x4547; fax: +34 932 279 853. E-mail address: [email protected] (J. Ruiz). This high level of use results in the selection and spread of quinolone-resistant microorganisms [13,16,17]. For a long time, the mechanisms of quinolone resistance described were exclusively chromosomal (Table 1), including speciﬁc amino acid substitutions in the quinolone targets (DNA gyrase and topoisomerase IV), decreased quinolone uptake into bacteria owing to either alterations in the outer membrane protein composition or to overexpression of efﬂux pumps, and alterations in the expression levels of the quinolone targets [18,19]. None the less, the absence of transferable mechanisms of quinolone resistance (TMQRs) has long been of note and considered to be unlikely as quinolones are fully synthetic drugs . 2. The history of transferable mechanisms of quinolone resistance Although the ﬁrst deﬁnitively established TMQR was described in 1998 , various articles published prior to this year presented similar results [22–24]. However, these results were not conﬁrmed, or further studies showed possible data misinterpretation. In 1985, conjugation of NAL resistance was reported with a frequency of 10−6 –10−7 , but the transfer of a plasmid was not established and ethidium bromide was unable to revert the resistance in the transconjugants . Then a mechanism of resistance to NAL carried on a transposon was proposed. The presence of mutations in the quinolones targets was not analysed and no further data have been found in the literature. Acquisition of NAL resistance in an Escherichia coli K12 recipient together with the transfer of a plasmid of 20 MDa was described during a Shigella dysenteriae type 1 outbreak in Bangladesh . Selection of the transconjugants was performed in minimal medium, which minimises the bactericidal effect of quinolones 0924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2012.02.011 J. Ruiz et al. / International Journal of Antimicrobial Agents 40 (2012) 196–203 Table 1 Chromosomally encoded mechanisms of quinolone resistance. Target alterations Efﬂux pumps Outer membrane alterations Lower target expression levels Target protectiona a Chromosomally encoded Qnr-like proteins. against E. coli and probably favours the development of quinolone resistance in E. coli , and no analysis was undertaken of the possible selection of spontaneous NAL-resistant recipient E. coli mutants by the development of mutations in the quinolone targets or decreased intracellular uptake. After re-inspection of the data, the presence of spontaneous NAL-resistant mutants of the recipient E. coli rather than authentic transconjugants was conﬁrmed . Currently described TMQRs (Table 2), usually encoded within plasmids but also related to chromosomal structures , only produce a low level of resistance to quinolones, usually below the considered resistance breakpoints. However, concomitant presence of two or more of these mechanisms in the same microorganism should be taken into account because of their additive action, with an increase in the minimum inhibitory concentrations (MICs) to quinolones [27–29]. Similarly, different basal levels of expression of some of these mechanisms have been described, such as QnrA or QepA determinants, which may result in different levels of resistance to quinolones [28,30]. However, on cloning the EfsQnr (Qnr-like protein from Enterococcus faecalis) into E. coli it was observed that its overexpression strongly affects the viability of host bacteria . Although a speciﬁc toxic effect of EfsQnr on the E. coli recipient cannot be excluded, this observation suggests a possible inverse relationship between the Qnr protection level against quinolone action and noxious effects on bacterial viability. The main relevance of decreased susceptibility to FQs lies in the fact that the microorganisms may develop full resistance to those antimicrobials more easily [21,27,32], and treatment with quinolones in this setting may result in therapeutic failure . However, a recent report has shown that the presence of Qnr determinants may result in a lower selection of mutations in the genes encoding the quinolone targets but in higher values of the mutant prevention concentration (MPC) of FQs . 197 The presence of TMQRs in the absence of target mutations may result in microorganisms presenting resistance to NAL and FQs  or in isolates exhibiting susceptibility to NAL but intermediate or full resistance to FQs [36,37]. Interestingly, in Stenotrophomonas maltophilia the NAL-susceptible, FQ-resistant phenotype, is usual . Although a relevant role of efﬂux pumps cannot be ruled out [39,40], this microorganism presents a chromosomally encoded Qnr-like protein . Although extensively sought in Enterobacteriaceae, TMQRs have also been described in microorganisms belonging to other genera, such as Aeromonas spp., Haemophilus parasuis, Pseudomonas spp. or Staphylococcus aureus [42–46]. Retrospective studies have shown that the oldest isolates in which a qnr-like gene has been detected were collected in 1988 (one Citrobacter freundii and one Klebsiella pneumoniae from the USA and Argentina, respectively), whilst the aac(6 )-Ib-cr gene was detected in an isolate of E. coli recovered in 1998 in Israel [47,48]. Future studies will probably detect that older isolates carried these or other TMQRs. 3. Transferable mechanisms of quinolone resistance 3.1. Quinolone target protection During a study aimed at establishing the presence of extendedspectrum ␤-lactamases encoded on conjugative plasmids in Enterobacteriaceae, the transfer of a quinolone resistance determinant able to confer low-level resistance to some quinolones was detected . This study also showed that strains carrying the aforementioned plasmid acquired quinolone resistance more easily . Further studies identiﬁed the gene encoding the protein responsible for this phenomenon . This gene was named qnr (for quinolone resistance) and is currently known as qnrA1 . Similar to other Qnr-encoding genes (not qnrS type), the qnrA1 gene is located within a complex class 1 integron environment upstream from the ﬁrst qacE1–sul1 [49,51]. Although a low prevalence of Qnr determinants was shown in early studies , further studies have shown that the qnr genes are distributed worldwide, being present in different microorganisms (mainly Enterobacteriaceae) and with a prevalence that varies from area to area [39,53–55]. Table 2 Currently described transferable mechanisms of quinolone resistance (TMQRs). Plasmid encoded Generic mechanism Speciﬁc mechanism Alleles/genesa,b Year of description Quinolone target protection QnrA QnrS QnrB QnrC QnrD QnrVC AAC(6 )-Ib-crc pKM101 QepA-like OqxAB QacBIII Others 7 6 48 1 1 10 1 1998 2005 2006 2009 2009 2008 2006 1996 2007 2003 2010 2004 Enzymatic inactivation Slow growth Efﬂux pumps Exogenous exchange of DNA Natural transformation a 2 1 1 1 GyrA/ParC One QnrS (GenBank accession no. AEG47318) and one QnrB (GenBank accession no. ABO93588) allele, although fully sequenced in the GenBank database, were not present in http://www.lahey.org/qnrStudies (last accessed 9 January 2011). Similarly, no QnrVC is present in the aforementioned internet repository; GenBank accession nos. for possible members of this Qnr family are presented in Fig. 1. b Not all of these Qnr-like proteins have been described in plasmids. c The presence of two subvariants has been proposed [aac(6 )-Ib-crA and aac(6 )-Ib-crC], deﬁned on the basis of a mute change in the amino acid codon encoding R102 (AGG or CGG) . 198 J. Ruiz et al. / International Journal of Antimicrobial Agents 40 (2012) 196–203 The Qnr proteins are pentapeptide-repeat proteins (PRPs) characterised by a tandem ﬁve amino acid repeat with the recurrent motif [Ser, Thr, Ala or Val] [Asp or Asn] [Leu or Phe] [Ser, Thr or Arg] [Gly] . Although their original function remains unknown, one possibility is their involvement in protection against natural DNA gyrase inhibitors such as microcin B17 , like other members of the PRP family such as McbG or MfpA proteins [58,59] or other unrelated proteins such as GyrI [60,61]. The Qnr proteins interact with DNA gyrase and topoisomerase IV, hindering the action of the quinolones and minimising their inhibitory effect as has been shown by different in vitro studies [49,62]. Crystallography studies have shown that Qnr proteins are dimers and that, in general, each one folds into a right-handed ␤-helix (like other PRPs) with nine complete coils [31,63,64]. In plasmid-encoded and some chromosomally encoded Qnr proteins, two non-canonical PRP sequences interrupt the threading of pentapeptides into the ␤-helical fold, producing outward projecting loops (loop A and loop B) that interrupt the regularity of the PRP surface [63,64]. In QnrB1, deletion of loop B results in the lack of a protective effect, whereas deletion of loop A drastically reduces the protective effect . Similar results have been shown in AhQnr (chromosomal Qnr-like protein from Aeromonas hydrophila) . Xiong et al.  proposed that AhQnr might locate in the gyrase structure interacting by electrostatic charges, whilst loop A interacts with the GyrA ‘tower’ and loop B with GyrB (TOPRIM domain). Thus, Qnr proteins acting as a DNA mimic might reduce the amount of the holoenzyme–DNA complex, minimising the possibility of the quinolones cleaving this structure and producing DNA breakage . A similar mechanism of action has been proposed for other Qnr-like proteins such as MfpA in Mycobacterium tuberculosis , in accordance with the ‘poison’ theory that proposes stable barriers to the replication or transcription processes associated with the action of quinolones on the complex holoenzyme–DNA, which would be directly responsible for the killing activity of quinolones . Alternatively, it has been proposed that Qnr proteins act by binding to and destabilising the complex topoisomerase–quinolone–DNA, favouring the regeneration of a catalytically active form of the topoisomerase . Five families of Qnr proteins have been established according to their DNA homology, each one comprising one or more alleles (sequence possessing at least one difference at the amino acid level): QnrA (7 alleles); QnrB (48 alleles); QnrS (6 alleles); QnrC (1 allele); and QnrD (1 allele) (Table 2; Fig. 1) [50,53,69–72]. In addition, the QnrVC family (10 alleles) has recently been described [46,73,74]. QnrVC alleles are transferable  and some of them have been reported to be encoded within an integron environment, such as QnrVC4 (protein GenBank accession no. ADI55014) or an unnamed ORF (DNA GenBank accession no. GU944730). Thus, QnrVC4 has been described within a complex class 1 integron located in a large non-conjugative plasmid in Aeromonas caviae , whilst the unnamed ORF was described within an integron environment in Acinetobacter baumannii. Moreover, different Qnr-like proteins have been identiﬁed as part of the chromosome of different Gram-positive or Gram-negative microorganisms [50,75–77]. Exponential growth of the Qnr-related genes described has led to a proposal to bring order to the current nomenclature of these genes (for more information see ). In addition, a repository electronic database (http://www.lahey.org/qnrStudies) has been proposed to maintain order in the nomenclature of the currently described and future newly detected Qnr determinants. Despite this proposal, a high number of complete or partial qnr genes are present in GenBank with a very confused and non-standard nomenclature. As mentioned previously, some proteins showing homology with Qnr proteins, able to confer low levels of quinolone resistance QnrB2 ABO93588 QnrB20 QnrB13 QnrB24 QnrB29 QnrB23 QnrB30 QnrB15 QnrB14 QnrB32 QnrB9 QnrB7 QnrB41 QnrB16 QnrB42 QnrB1 QnrB6 QnrB3 QnrB18 QnrB17 QnrB31 QnrB19 QnrB5 QnrB10 QnrB36 QnrB40 QnrB28 QnrB33 QnrB27 QnrB8 QnrB21 QnrB25 QnrB35 QnrB38 QnrB4 QnrB22 QnrB37 QnrB12 QnrB11 QnrB34 QnrD QnrA4 QnrA3 QnrA5 QnrA6 QnrA7 QnrA1 QnrA2 QnrS3 QnrS1 QnrS4 QnrS5 AEG74318 QnrS2 QnrC ZP 05945497 QnrVC1 QnrVC3 orf A.baumannii EGQ95960.1 QnrVC4 AEM62764.1 0.05 Fig. 1. Phylogenetic relationship amongst the qnr genes. Six qnr gene families have been included in the phylogenetic tree, including qnrA, qnrB, qnrS, qnrC, qnrD and qnrVC. The selected sequences for qnrA, qnrB, qnrS, qnrC and qnrD are those present on the webpage http://www.lahey.org/qnrStudies in which a GenBank accession no. is indicated. In addition, the protein sequences ABO93588 (QnrB-like) and AEG47318 (QnrS-like), both present in GenBank fulﬁlling the criteria deﬁned by Jacoby et al. , have been added. In addition, seven protein sequences present in GenBank have been selected related to qnrVC1 following the aforementioned criteria. The distancebased tree was generated using p-distance with the neighbour-joining method with MEGA version . J. Ruiz et al. / International Journal of Antimicrobial Agents 40 (2012) 196–203 when cloned in E. coli or other bacteria, have been identiﬁed within the chromophore of either Gram-positive or Gram-negative microorganisms [76–80], the latter mainly associated with aquatic environments. In addition, qnr-like genes have been detected during the course of metagenomic studies . It has been proposed that some Qnr family proteins derived from chromosomal ancestors present in waterborne Gram-negative microorganisms such as Shewanellaceae or Vibrionaceae [76–79]. In fact, four chromosomally encoded proteins of Shewanella algae and one of Shewanella putrefaciens present >70% homology with members of the QnrA family [50,77], thereby being considered as QnrA family members (QnrA2, S. putrefaciens; and QnrA3, QnrA4, QnrA5 and QnrA7, S. algae). To date, QnrA2 and QnrA3 have also been detected within plasmids in other microorganisms such as Klebsiella oxytoca or Salmonella enterica serotype Enteritidis isolates . QnrC presents an amino acid similarity with VcQnr1 (QnrVC1) of >70%, which might allow these Qnr proteins to be classiﬁed within a common family as different alleles of the same gene , suggesting their possible origin from an ancestor of the Vibrionaceae family . Finally, Vibrio splendidus has been proposed as one of the origins of the QnrS-like proteins . QnrB-related proteins have also been detected in seawater metagenome analysis, and chromosomally encoded in S. maltophilia as well as in some Enterobacteriaceae such as Serratia proteamaculans, Serratia marcescens or Citrobacter spp. [41,81,83,84], this latter genus being proposed as the original source of QnrB proteins . The high similarity between QnrD and QnrB-related proteins suggests that the aforementioned chromosomally encoded QnrB-related proteins might also possess a close phylogenetic relationship with QnrD. Finally, at present no Grampositive plasmid-derived qnr gene family has been described. A high intraspecies variability has been detected amongst SmQnr of S. maltophilia [41,85–87]. At least 47 different alleles are currently present in GenBank, and an Internet repository (http:// www.icms.qmul.ac.uk/centres/immunologyandinfectiousdisease/ Smqnr%20Web%20v2.htm) has been proposed to bring order to the speciﬁc nomenclature of these alleles . On studying the VPQnr, a chromosomally encoded Qnr-like protein of Vibrio parahaemolyticus (formerly VPA0095), Saga et al.  showed that a single substitution (C115 → Y) was able to produce a signiﬁcant increase in quinolone resistance levels when cloned and expressed in E. coli. Similar results have been published regarding different MIC levels of quinolones that appear to be associated with the presence of allelic variants of the SmQnr . Following this evidence, some authors have analysed the effect of point mutations in members of the QnrA, QnrB, QnrC and QnrS families [88–90], showing the presence of speciﬁc amino acid substitutions or deletions resulting in partial or full loss of activity of the Qnr proteins (Table 3). Although most of these alterations are not located within loop A or loop B, this loss of activity of Qnr proteins has been associated with alterations in hydrophobicity and protein conformation . On the other hand, it was observed that the QnrS1 substitution D185 → Y conferred slightly higher levels of resistance than parental proteins [i.e. four-fold for CIP, moxiﬂoxacin (MOX) and levoﬂoxacin (LVX)], demonstrating the potential risk of possible future in vivo selection of more active Qnr proteins. However, the aforementioned possible inverse relationship between protection level and viability should be taken into account as a possible limitation to this selection . Qnr determinants have been related to an enhanced facility to develop full resistance to quinolones [21,29], in agreement with the fact that decreased resistance levels to FQs favour the acquisition of full resistance to these agents . However, it has been reported that despite this enhanced facility to develop higher levels of quinolone resistance, E. coli isolates harbouring a qnr determinant present a low selection of topoisomerase mutations . The 199 Table 3 Single amino acid substitutions negatively affecting the activity of Qnr proteins. Positiona Wt 13 38c 56 72 92c 96 97 114 115c 116 117 153 159 185d 188 F L G C C G A F C S A S L D D Qnr activityb QnrA1 QnrB1 S P , D Y Y D , E Y Y D QnrC1 QnrS1 R Y , D Y Y D Y D D Y D C, V P C, V P D D Y D V a Numbering refers to QnrA1, QnrC1 and QnrS1 positions. QnrB1 transcription starts three amino acids later, thus it is necessary to subtract three from all positions stated in the ﬁrst column (i.e. G56 in QnrA corresponds to G53 in QnrB1). b Only considering those substitutions affecting more than 4-fold the activity of the Qnr proteins. c Other alterations described in the same position do not result in loss or gain of activity (i.e. QnrA1, L38F, C92S, C115S; QnrC1, L38A). d The same amino acid substitution in QnrS1 results in enhanced activity. authors suggest that this phenomenon might be related to both the action of the Qnr proteins, making it difﬁcult for quinolones to interact with their speciﬁc targets, and the presence of other mechanisms of resistance that might be selected in qnr-carrying isolates. It should be taken into account that in the aforementioned study, the antibiotic concentration present in the selective plates used to obtain the mutants was established according to the MIC of the microorganisms, thereby being signiﬁcantly higher (>10-fold) in the plates in which isolates carrying qnr genes were analysed . In fact, the same authors noted that the MPC of the selected FQ was even higher in strains carrying a qnr gene (range of MPC of CIP, 2–4 mg/L; range of MPC of MOX, 2–8 mg/L) than in those not carrying the gene (MPC of CIP, 0.12 mg/L; MPC of MOX, 0.5 mg/L). These MPC values of the strains carrying a qnr gene were higher than the antibiotic concentrations that might be reached in serum following a therapeutic dose , showing the relevance of these genes in clinical practice. 3.2. Enzymatic inactivation Enzymatic inactivation of quinolones, although observed in some fungi several years ago , was not an established phenomenon in bacteria until 2006. Thus, although ﬁrst detected in 2003 during a study of TMQR in E. coli , it was not until 2006 that it was deﬁnitively established that an aminoglycoside-modifying enzyme, a variant of AAC(6 )-Ib named AAC(6 )-Ib-cr, conferred low-level resistance to some quinolones such as CIP and norﬂoxacin (NOR), retaining its ability to inactivate aminoglycosides . This AAC(6 )-Ib variant presented two amino acid substitutions (W102 → R and D179 → Y) related to the ability to inactivate the aforementioned quinolones by means of an N-acetylation of its piperazinyl amine . No effect of AAC(6 )-Ib-cr has been observed on quinolones with an unsubstituted piperazinyl group. The interaction between AAC(6 )-Ib-cr and its substrates differs according to whether they are aminoglycosides or FQs . When this enzyme acts on aminoglycosides, the interactions between AAC(6 )-Ib-cr and these antimicrobial agents are made by numerous hydrogen-bonding interactions. However, when acting on quinolones, the interactions appear to be related to several stacking interactions. A difference has been established in the optimum pH 200 J. Ruiz et al. / International Journal of Antimicrobial Agents 40 (2012) 196–203 Table 4 Characteristics of plasmid-encoded efﬂux pumps. Efﬂux pump Speciesa Quinolones b OqxAB QepA1 QepA2 pRSB101h QacBIII Escherichia coli E. coli E. coli N/I Staphylococcus aureus Inhibitable rmtBd Ince Yearf Ref. N/I CCCP, NMR, PA␤N N/Ig N/I N/I Yes Yes No No N/I IncX1 IncFI IncFI N/I N/I 2003 2007 2008 2004 2010 [106,107] [102,104]    c Substrates Non-substrates NAL, FMQ, CIP, NOR CIP, NOR CIP, NOR NAL, NOR NOR, CIP N/I NAL, MOX, LVX, PFLX, SPFX, GFLX NAL, OFX, MOX, LVX, PFLX, GFLX N/I LVX NAL, nalidixic acid; FMQ, ﬂumequine; CIP, ciproﬂoxacin; NOR, norﬂoxacin; MOX, moxiﬂoxacin; LVX, levoﬂoxacin; PFLX, peﬂoxacin; SPFX, sparﬂoxacin; GFLX, gatiﬂoxacin; OFX, oﬂoxacin; N/I, no information; CCCP, carbonyl cyanide m-chlorophenylhydrazone; NMR, 1-(1-naphtylmethyl)-piperazine; PA␤N, Phe-Arg-␤-naphthylamide. a Species in which the efﬂux pump has been detected. b Quinolones in which this information is available and in which the described effect is >2-fold. c Quinolones in which this information is available and in which the described effect is ≤2-fold. d Presence of the rmtB gene within the same plasmid. e Incompatibility group of the plasmid in which the efﬂux pump gene has been described. f First description. g Although no information is available, it is predicted that it will act similar to QepA1. h Efﬂux pump located within pRSB101 plasmid that has not yet received a formal name. levels for the activity of AAC(6 )-Ib-cr according to its substrates. Thus, the optimum pH for the activity of AAC(6 )-Ib-cr when it acts on aminoglycosides is 6.1, whereas it is 7.7 when acting on FQs . Analysis of the two amino acid substitutions by themselves showed that the single presence of the D179 → Y substitution appears to confer a slight increase in CIP resistance level (less than that present in isolates presenting both substitutions) . The main role of the substitution at position 179 has been conﬁrmed by mechanistic studies . Thus, Y179 would be involved in an increase in the afﬁnity of AAC(6 )-Ib-cr for FQs, interacting with them, whilst R102 plays a role as a stabiliser of the interactions. Another proposed interaction model has also suggested a direct role in the interactions between Y179 and FQs, whilst involving the R102 in an interaction with the ﬂuoroquinolone carboxylate . AAC(6 )-Ib-cr has been described worldwide, either plasmid or chromosomally encoded [26,37,48,95], including farm animal  and environmental microorganisms , showing its great potential to be disseminated. Moreover, it is possible that further adaptations of this or other enzymes able to modify antibacterial agents would be more efﬁcient as a mechanism of quinolone resistance. 3.3. Slow growth In some microorganisms such as E. coli, slow growth has been related to alterations in quinolone activity . Although the most frequent reports of this phenomenon have been observed when the microorganisms are grown in minimal medium or in the presence of speciﬁc mutations in the promoter region of the topoisomerase IV-encoding genes [19,25], some plasmids affecting the duplication rates may affect microorganism susceptibility levels. In 1996, it was described that the presence of the plasmid pKM101, belonging to the IncN incompatibility group and used in the Ames test , results in higher survival rates of E. coli grown in minimal medium in the presence of CIP . The authors disregarded the possible role of the mucAB genes, involved in the error-prone repair of DNA, as being responsible for these enhanced survival rates and the presence of a slow-growth phenotype. This slow-growth phenotype was associated with a 2.2 kb region of the plasmid containing the korB, traL, korA and traM genes and was suppressed (as were higher survival rates) in minimal medium in the presence of adenine and hypoxanthine. Curiously, in starvation conditions, E. coli strains carrying the pKM101 plasmid showed lower rates of survival in the presence of CIP. The authors hypothesised about the role of the region between the korB and korA genes, but the exact mechanism of action remains unknown. Although this slow-growth phenotype (resulting in small-colony formation) has been described by other authors , no other report has been found in the literature to provide more accurate information. 3.4. Plasmid-encoded efﬂux pumps Efﬂux pumps are a well-established quinolone resistance mechanism . Different families of efﬂux pumps are able to pump out quinolones possessing different afﬁnities, affecting in a different manner the corresponding ﬁnal MIC levels and thus having different clinical relevance . Although usually encoded in the bacterial chromophore, a few different plasmid-encoded efﬂux pumps able to pump out quinolones have been described (Tables 2 and 4) [100–104]. Interestingly, most of the plasmids carrying these efﬂux pumps have a close relationship with the farm world, suggesting that present or past use of antimicrobial agents in veterinary practice may not be excluded as a possible selection factor, and the association between these efﬂux pumps and the presence of the rmtB gene, encoding an unusual mechanism of resistance (target methylation) to aminoglycosides, is frequent [28,105]. OqxAB is a member of the resistance–nodulation–cell division (RND) family of multidrug efﬂux pumps , encoded on pOLA52, a plasmid belonging to the IncX1 incompatibility group, possessing a high similarity (99%) with a putative chromosomally encoded efﬂux pump system of K. pneumoniae (GenBank accession nos. ABR 76475 and ABR 76476). Although ﬁrst described in farm animals as involved in the development of resistance to olaquindox, a quinoxaline-di-N-oxide used as a growth promoter in pigs , it has been detected in humans both in pathogenic and commensal microorganisms, with its presence amongst farm workers in China being of note [107,108]. Further studies have shown that OqxAB is able to pump out NAL, ﬂumequine, CIP and NOR, increasing the MICs to these agents 8-, 32-, 32- and 64-fold, respectively . No information on its effect against other quinolones has been reported. On studying plasmids recovered from a wastewater bacterial community, Szczepanowski et al.  described within a plasmid (pRSB101) the presence of a tripartite multidrug efﬂux pump composed by a transcriptional regulator and a membrane fusion protein belonging to an RND-type efﬂux system with homology of ca. 40% to a putative efﬂux system of Geobacter sulfurreducens (GenBank accession nos. NP 952005 and NP 952003) and a permease and an ATPase belonging to an ATP-binding cassette (ABC)-type efﬂux pump also presenting great homology (59% and 45%, respectively) with the same G. sulfurreducens system (GenBank accession J. Ruiz et al. / International Journal of Antimicrobial Agents 40 (2012) 196–203 nos. N 95002 and N 952001). This system lacks an outer membrane component (downstream from the ﬁrst gene of the system, only the 3 -end region of an oprM-related gene remains). Thus, the authors hypothesised that this system is completed by a heterologous host-encoded outer membrane protein. Although no analysis of the possible presence of quinolone target alterations was performed, when the vector was cloned into E. coli the MICs of NAL and CIP were 80 mg/L and 0.5 mg/L, respectively, and when the efﬂux system was subcloned into a pBluescript-II-KS, the MICs of NAL and CIP rose to 550 mg/L and 1.25 mg/L, respectively. QepA1 (from quinolone efﬂux pump) was described in 2007, encoded within a conjugative plasmid present in E. coli clinical isolates [102,104]. The qepA1 gene was carried on an IncF1 plasmid (pIP1206) possessing high homology with pRSB107, a plasmid isolated from a sewage treatment plant together with the aforementioned pRSB101 . The qepA1 gene encodes a 14-transmembrane segment (14TMS) proton-dependent efﬂux pump belonging to the major facilitator superfamily (MFS) , inhibitable by the presence of different efﬂux pump inhibitors, with hydrophilic quinolones such as CIP and NOR as substrates, but with a slight or no effect on more hydrophobic quinolones such as NAL, LVX or MOX, amongst others (Table 4), or different unrelated antimicrobial agents such as erythromycin, chloramphenicol, tetracycline or rifampicin. Thus, when transferred to E. coli, an increase from 2fold (NAL, sparﬂoxacin, lomeﬂoxacin) to 64-fold (CIP) is produced in the MICs of different quinolones [102,104]. However, as with other TMQRs, by itself its effect on the susceptibility levels of the affected quinolones is below the established resistance breakpoints. Although on analysing the G + C content (72%) of the qepA1 gene, the actinomycetes group has been suggested as the original source of this gene, at present the origin of this efﬂux pump currently remains unknown . QepA2, another QepA-like efﬂux pump, that has been described in a mobilisable non-conjugative plasmid of 90 kb (pQep) belonging to the IncF1 incompatibility group , differs in 2 of the 511 amino acids with respect to QepA1, presenting Gly in position 99 and Ile in position 134. Despite these differences, the spectrum of QepA2 substrates (hydrophilic quinolones) is similar to that of QepA1. The main differences between QepA1 and QepA2 lie in their genetic environment. Thus, whilst the rmtB gene is located within the same plasmid as the qepA1 gene, it remains absent in the plasmid that carries the qepA2 gene . Another difference is the presence of qepA2 located between two copies of the ISCR3C element, instead of an association with IS26 elements that has been related to QepA1. It has been proposed that this ISCR-like element is involved in the processes of mobilisation of the qepA2 gene . QepA1 and QepA2 probably act as a factor favouring the development of full resistance to CIP or NOR, increasing the frequency of selection of resistant mutants. However, their lack of effect over other quinolones probably does not affect their speciﬁc ability to select resistant mutants. Although the prevalence of the QepA-like efﬂux pumps appears to be very low, with different survey studies reporting frequencies ranging from 0.3% to 0.8% [100–112], higher frequencies have been reported in samples from animal origins . None the less, the real relevance and extension of these efﬂux pumps remains unknown, requiring lengthy studies to obtain valid conclusions. Plasmid-encoded efﬂux pumps able to extrude quinolones have also been described in S. aureus . Thus, on analysing the variability of the plasmid-encoded efﬂux pumps QacA and QacB, two 14-TMS proton-dependent efﬂux pumps belonging to the MFS family , amongst meticillin-resistant S. aureus (MRSA), it was observed that a speciﬁc allele of QacB (QacBIII) was able to pump out CIP and NOR but not LVX. Mutagenesis studies showed that this ability was related to the presence of a speciﬁc glutamic acid 201 at position 320. In addition, these results also suggest a possible slight effect of QacA on the MIC of the same quinolones. Further studies to evaluate its effect on other quinolones and to identify other possible alleles of QacA/QacB able to extrude quinolones are needed. 3.5. Natural transformation Acquisition of quinolone resistance mediated by natural transformation of DNA fragments of gyrA or parC genes carrying amino acid substitutions in the quinolone resistance-related positions has been described in different species of the genus Streptococcus, such as Streptococcus pneumoniae, different viridans streptococci, Streptococcus dysgalactiae or Streptococcus pyogenes [114–116]. In vitro studies have shown that resistance could be transferred either from DNA from viridans streptococci to S. pneumoniae or vice versa, as well as amongst S. dysgalactiae and S. pyogenes. The frequencies of transformation ranged from 10−3 to <10−7 in correlation with the homologies of their quinolone resistancedetermining regions (QRDRs). The relevance of this mechanism of resistance both lies in the speciﬁc frequencies of transformation as well as in the incidence of quinolone resistance amongst donor isolates. 4. 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