Transferable mechanisms of quinolone resistance

International Journal of Antimicrobial Agents 40 (2012) 196–203
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Transferable mechanisms of quinolone resistance
Joaquim Ruiz a,b,∗ , Maria J. Pons a , Cláudia Gomes a
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
Quinolone resistance
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 [1], 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 [2]. 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been withdrawn from clinical practice following restriction of their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 [12]. 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 [15], 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 [20].
2. The history of transferable mechanisms of quinolone
Although the first definitively established TMQR was described
in 1998 [21], 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 [24]. 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
[23]. 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.
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
Chromosomally encoded Qnr-like proteins.
against E. coli and probably favours the development of quinolone
resistance in E. coli [25], 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 [20].
Currently described TMQRs (Table 2), usually encoded within
plasmids but also related to chromosomal structures [26], 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 [31]. 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 [33].
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 [34].
The presence of TMQRs in the absence of target mutations may
result in microorganisms presenting resistance to NAL and FQs [35]
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
[38]. Although a relevant role of efflux pumps cannot be ruled out
[39,40], this microorganism presents a chromosomally encoded
Qnr-like protein [41].
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 [21]. 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 [49]. This gene was named qnr (for
quinolone resistance) and is currently known as qnrA1 [50]. 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 [52], 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
Year of
Quinolone target protection
AAC(6 )-Ib-crc
Enzymatic inactivation
Slow growth
Efflux pumps
Exogenous exchange of DNA
Natural transformation
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 (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.
Not all of these Qnr-like proteins have been described in plasmids.
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) [26].
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] [56]. Although their original function remains unknown, one
possibility is their involvement in protection against natural DNA
gyrase inhibitors such as microcin B17 [57], 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 [63]. Similar results have been shown in AhQnr
(chromosomal Qnr-like protein from Aeromonas hydrophila) [64].
Xiong et al. [64] 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 [65]. A similar mechanism of action has been
proposed for other Qnr-like proteins such as MfpA in Mycobacterium tuberculosis [66], 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 [67]. 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 [63].
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 [74] 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 [46], 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
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 [50]). In addition, a repository
electronic database ( 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
ZP 05945497
orf A.baumannii
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 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.
[50], 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 [68].
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 [81].
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 [82]. 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
[50], suggesting their possible origin from an ancestor of the Vibrionaceae family [72]. Finally, Vibrio splendidus has been proposed as
one of the origins of the QnrS-like proteins [78].
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 [84]. 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://
Smqnr%20Web%20v2.htm) has been proposed to bring order to
the specific nomenclature of these alleles [87].
On studying the VPQnr, a chromosomally encoded Qnr-like protein of Vibrio parahaemolyticus (formerly VPA0095), Saga et al. [79]
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 [86]. 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
[89]. 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 [31].
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 [32]. 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 [34]. The
Table 3
Single amino acid substitutions negatively affecting the activity of Qnr proteins.
Qnr activityb
, D
, E
, D
C, V
C, V
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).
Only considering those substitutions affecting more than 4-fold the activity of
the Qnr proteins.
Other alterations described in the same position do not result in loss or gain of
activity (i.e. QnrA1, L38F, C92S, C115S; QnrC1, L38A).
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
[34]. 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 [91], 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 [92], was not an established phenomenon in bacteria until 2006. Thus, although first detected in
2003 during a study of TMQR in E. coli [55], 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
[27]. 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 [27]. 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 [93]. 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
J. Ruiz et al. / International Journal of Antimicrobial Agents 40 (2012) 196–203
Table 4
Characteristics of plasmid-encoded efflux pumps.
Efflux pump
Escherichia coli
E. coli
E. coli
Staphylococcus aureus
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.
Species in which the efflux pump has been detected.
Quinolones in which this information is available and in which the described effect is >2-fold.
Quinolones in which this information is available and in which the described effect is ≤2-fold.
Presence of the rmtB gene within the same plasmid.
Incompatibility group of the plasmid in which the efflux pump gene has been described.
First description.
Although no information is available, it is predicted that it will act similar to QepA1.
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 [93].
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) [27]. The
main role of the substitution at position 179 has been confirmed
by mechanistic studies [93]. 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
[28] and environmental microorganisms [96], 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 [25]. 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 [97], results in higher survival rates of E. coli grown
in minimal medium in the presence of CIP [22]. 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
[98], 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 [18]. 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 [99]. 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
OqxAB is a member of the resistance–nodulation–cell division
(RND) family of multidrug efflux pumps [101], 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 [106], 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 [109]. No information on its effect
against other quinolones has been reported.
On studying plasmids recovered from a wastewater bacterial
community, Szczepanowski et al. [103] 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 [110].
The qepA1 gene encodes a 14-transmembrane segment (14TMS) proton-dependent efflux pump belonging to the major
facilitator superfamily (MFS) [102], 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 [102].
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 [100], 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 [100]. 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 [100].
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 [28]. 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 [44]. 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 [113], 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
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
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. Concluding remarks
Currently, TMQRs are extensively described around the world.
Although they usually result in only a slight increase in the MICs
of quinolones, their effect is additive and their presence may facilitate the development of full quinolone resistance. Furthermore,
possible specific substitutions may enhance their activity. The
emergence of new related genes may continue in the next years,
whilst the possible adaptation of other enzymes, similar to what
occurred with AAC(6 )-Ib, is a potential risk.
The logistic support of Laura Puyol and Diana Barrios is acknowledged.
Funding: JR has a Miguel Servet Fellowship (Instituto de Salud
Carlos III, Spain).
Competing interests: None declared.
Ethical approval: Not required.
[1] Lesher GY, Froelich EJ, Gruett MD, Bailey JH, Brundage RP. 1,8-Naphthyridine
derivatives. A new class of chemotherapeutic agents. J Med Pharm Chem
[2] Mitscher LA. Bacterial topoisomerase inhibitors: quinolone and pyridone
antibacterial agents. Chem Rev 2005;105:559–92.
[3] Blum MD, Graham DJ, McCloskey CA. Temafloxacin syndrome: review of 95
cases. Clin Infect Dis 1994;18:946–50.
[4] Matthews MR, Caruso DM, Phillips BJ, Csontos LG. Fulminant toxic epidermal
necrolysis induced by trovafloxacin. Arch Intern Med 1999;159:2225.
[5] Norrby SR, Lietman PL. Safety and tolerability of fluoroquinolones. Drugs
1993;45(Suppl. 3):59–64.
[6] Acar JF, Goldstein FW. Trends in bacterial resistance to fluoroquinolones. Clin
Infect Dis 1997;24(Suppl. 1):S67–73.
J, Ruiz J, Carmona F, Nadal A, Gascón J. Salmonella
[7] Alonso D, Munoz
ovarian abscess following travel diarrhoea episode. Arch Gynecol Obstet
[8] Davis R, Markham A, Balfour JA. Ciprofloxacin. An updated review of its pharmacology, therapeutic efficacy and tolerability. Drugs 1996;51:1019–74.
[9] Ruiz J, Marco F, Oliveira I, Vila J, Gascón J. Trends in antimicrobial resistance levels among Campylobacter spp. causing traveler’s diarrhea. APMIS
[10] Anquetin G, Greiner J, Mahmoudi N, Santillana-Hayat M, Gozalbes R, Farhati
K, et al. Design, synthesis and activity against Toxoplasma gondii, Plasmodium
spp., and Mycobacterium tuberculosis of new 6-fluoroquinolones. Eur J Med
Chem 2006;41:1478–93.
J. Ruiz et al. / International Journal of Antimicrobial Agents 40 (2012) 196–203
[11] Romero IC, Saravia NG, Walker J. Selective action of fluoroquinolones against
intracellular amastigotes of Leishmania (Viannia) panamensis in vitro. J Parasitol 2005;91:1474–9.
[12] Pommier Y, Leo E, Zhang HL, Marchand C. DNA topoisomerases and
their poisoning by anticancer and antibacterial drugs. Chem Biol 2010;17:
[13] Endtz HP, Ruijs GJ, van Klingeren B, Jansen WH, van der Reyden T, Mouton RP.
Quinolone resistance in Campylobacter isolated from man and poultry following the introduction of fluoroquinolones in veterinary medicine. J Antimicrob
Chemother 1991;27:199–208.
[14] Millanao A, Barrientos H, Gomez C, Tomova A, Buschmann A, Dölz H, et al. Injudicious and excessive use of antibiotics: public health and salmon aquaculture
in Chile. Rev Med Chil 2009;139:107–18 [in Spanish].
[15] Collignon P, Powers JH, Chiller TM, Aidare-Kane A, Aarestrup FM. World
Health Organization ranking of antimicrobials according to their importance
in human medicine: a critical step for developing risk management strategies for the use of antimicrobials in food production animals. Clin Infect Dis
[16] Mensa L, Marco F, Vila J, Gascón J, Ruiz J. Quinolone resistance among
Shigella spp. isolated from travellers returning from India. Clin Microbiol
Infect 2008;14:279–81.
[17] Le Hello S, Hendriksen RS, Doublet B, Fisher I, Nielsen EM, Whichard
JM, et al. International spread of an epidemic population of Salmonella
enterica serotype Kentucky ST198 resistant to ciprofloxacin. J Infect Dis
[18] Ruiz J. Mechanisms of resistance to quinolones: target alterations,
decreased accumulation and DNA gyrase protection. J Antimicrob Chemother
[19] Ince D, Hooper DC. Quinolone resistance due to reduced target enzyme
expression. J Bacteriol 2003;185:6883–92.
[20] Courvalin P. Plasmid-mediated 4-quinolone resistance: a real or apparent
absence? Antimicrob Agents Chemother 1990;34:681–4.
[21] Martínez-Martínez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet 1998;351:797–9.
[22] Clerch B, Rivera E, Llagostera M. Identification of a pKM101 region which
confers a slow growth rate and interferes with susceptibility to quinolone in
Escherichia coli AB1157. J Bacteriol 1996;178:5568–72.
[23] Munshi MH, Sack DA, Haider K, Ahmed ZU, Rahaman MM, Morshed MG.
Plasmid-mediated resistance to nalidixic acid in Shigella dysenteriae type 1.
Lancet 1987;2:419–21.
[24] Panhotra BR, Desai B, Sharma PL. Nalidixic-acid-resistant Shigella dysenteriae
I. Lancet 1985;1:763.
[25] Dalhoff A, Matutat S, Ullmann U. Effect of quinolones against slowly growing
bacteria. Chemotherapy 1995;41:92–9.
[26] Ruiz E, Sáenz Y, Zarazaga M, Rocha-Gracia R, Martínez-Martínez L, Arlet G,
et al. qnr, aac(6 )-Ib-cr and qepA genes in Escherichia coli and Klebsiella spp.:
genetic environments and plasmid and chromosomal location. J Antimicrob
Chemother 2012;67:886–97.
[27] Robicsek A, Strahilevitz J, Jacoby GA, Macielag M, Abbanat D, Park CH, et al.
Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006;12:83–8.
[28] Liu JH, Deng YT, Zeng ZL, Gao JH, Chen L, Arakawa Y, et al. Coprevalence of plasmid-mediated quinolone resistance determinants QepA,
Qnr, and AAC(6 )-Ib-cr among 16S rRNA methylase RmtB-producing
Escherichia coli isolates from pigs. Antimicrob Agents Chemother 2008;52:
[29] Briales A, Rodríguez-Martínez JM, Velasco C, Díaz de Alba P, DomínguezHerrera J, Pachón J, et al. In vitro effect of qnrA1, qnrB1, and qnrS1
genes on fluoroquinolone activity against isogenic Escherichia coli isolates
with mutations in gyrA and parC. Antimicrob Agents Chemother 2011;55:
[30] Rodríguez-Martínez JM, Velasco C, Pascual A, García I, Martínez-Martínez
L. Correlation of quinolone resistance levels and differences in basal and
quinolone-induced expression from three qnrA-containing plasmids. Clin
Microbiol Infect 2006;12:440–5.
[31] Hegde SS, Vetting MW, Mitchenall LA, Maxwell A, Blanchard JS. Structural
and biochemical analysis of the pentapeptide repeat protein EfsQnr, a potent
DNA gyrase inhibitor. Antimicrob Agents Chemother 2011;55:110–7.
[32] Ruiz J, Gómez J, Navia MM, Ribera A, Sierra JM, Marco F, et al. High prevalence
of nalidixic acid resistant, ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae. Diagn Microbiol
Infect Dis 2002;42:257–61.
[33] Wain J, Hoa NT, Chinh NT, Vinh H, Everett MJ, Diep TS, et al. Quinoloneresistant Salmonella typhi in Viet Nam: molecular basis of resistance and
clinical response to treatment. Clin Infect Dis 1997;25:1404–10.
[34] Cesaro A, Bettoni RRD, Lascols C, Mérens A, Soussy CJ, Cambau E. Low selection
of topoisomerase mutants from strains of Escherichia coli harbouring plasmidborne qnr genes. J Antimicrob Chemother 2008;61:1007–15.
[35] Sato T, Yokota S, Uchida I, Okubo T, Ishihara K, Fujii N, et al. A
fluoroquinolone-resistant Escherichia coli clinical isolate without quinolone
resistance-determining region mutations found in Japan. Antimicrob Agents
Chemother 2011;55:3964–5.
[36] Gunell M, Webber MA, Kotilainen P, Lilly A, Caddick JM, Jalava J, et al. Mechanisms of resistance in nontyphoidal Salmonella enterica strains exhibiting a
nonclassical quinolone resistance phenotype. Antimicrob Agents Chemother
[37] de Toro M, Rojo-Bezares B, Vinué L, Undabeitia E, Torres C, Sáenz Y. In vivo
selection of aac(6 )-Ib-cr and mutations in the gyrA gene in a clinical qnrS1positive Salmonella enterica serovar Typhimurium DT104B strain recovered
after fluoroquinolone treatment. J Antimicrob Chemother 2010;65:1945–9.
[38] Ribera A, Doménech-Sanchez A, Ruiz J, Benedi VJ, Jimenez de Anta MT, Vila J.
Mutations in gyrA and parC QRDRs are not relevant for quinolone resistance
in epidemiological unrelated Stenotrophomonas maltophilia clinical isolates.
Microb Drug Resist 2002;8:245–51.
[39] Alonso A, Martínez JL. Cloning and characterization of SmeDEF, a novel
multidrug efflux pump of Stenotrophomonas maltophilia. Antimicrob Agents
Chemother 2000;44:3079–86.
[40] Ribera A, Jurado A, Ruiz J, Marco F, del Valle O, Mensa J, et al. In vitro activity of
clinafloxacin in comparison with other quinolones against Stenotrophomonas
maltophilia clinical isolates in the presence and absence of reserpine. Diagn
Microbiol Infect Dis 2002;42:123–8.
[41] Sánchez MB, Hernández A, Rodríguez-Martínez JM, Martínez-Martínez L,
Martínez JL. Predictive analysis of transmissible quinolone resistance indicates Stenotrophomonas maltophilia as potential source of a novel family of
Qnr determinants. BMC Microbiol 2008;8:148.
[42] Ahmed AM, Motoi Y, Sato M, Maruyama A, Watanabe H, Fukumoto Y, et al.
Zoo animals as reservoirs of Gram-negative bacteria harboring integrons and
antimicrobial resistance genes. Appl Environ Microbiol 2007;73:6686–90.
[43] Guo L, Zhang J, Xu C, Zhao Y, Ren T, Zhang B, et al. Molecular characterization
of fluoroquinolone resistance in Haemophilus parasuis isolated from pigs in
South China. J Antimicrob Chemother 2011;66:539–42.
[44] Nakaminami H, Noguchi N, Sasatsu M. Fluoroquinolone efflux by the plasmidmediated multidrug efflux pump QacB variant QacBIII in Staphylococcus
aureus. Antimicrob Agents Chemother 2010;54:4107–11.
[45] Tran QT, Nawaz MS, Deck J, Nguyen KT, Cerniglia CE. Plasmid-mediated
quinolone resistance in Pseudomonas putida isolates from imported shrimp.
Appl Environ Microbiol 2011;77:1885–7.
[46] Xia R, Guo X, Zhang Y, Xu H. qnrVC-like gene located in a novel complex class
1 integron harboring the ISCR1 element in an Aeromonas punctata strain from
an aquatic environment in Shandong Province, China. Antimicrob Agents
Chemother 2010;54:3471–4.
[47] Jacoby GA, Gacharna N, Black TA, Miller GH, Hooper DC. Temporal appearance of plasmid-mediated quinolone resistance genes. Antimicrob Agents
Chemother 2009;53:1665–6.
[48] Warburg G, Korem M, Robicsek A, Engelstein E, Moses AE, Block C, et al.
Changes in aac(6 )-Ib-cr prevalence and fluoroquinolone resistance in nosocomial isolates of Escherichia coli collected from 1991 through 2005.
Antimicrob Agents Chemother 2009;53:1268–70.
[49] Tran JH, Jacoby GA. Mechanism of plasmid-mediated quinolone resistance.
Proc Natl Acad Sci USA 2002;99:5638–42.
[50] Jacoby G, Cattoir V, Hooper D, Martínez-Martínez L, Nordmann P, Pascual A,
et al. qnr gene nomenclature. Antimicrob Agents Chemother 2008;52:2297–9.
[51] Quiroga MP, Andres P, Petroni A, Soler-Bistué AJC, Guerriero L, Vargas LJ, et al.
Complex class 1 integrons with diverse variable regions, including aac(6 )-Ibcr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial
isolates from Argentina. Antimicrob Agents Chemother 2007;51:4466–70.
[52] Jacoby GA, Chow N, Waites KB. Prevalence of plasmid-mediated quinolone
resistance. Antimicrob Agents Chemother 2003;47:559–62.
[53] Minarini LAR, Poirel L, Cattoir V, Darini ALC, Nordmann P. Plasmid-mediated
quinolone resistance determinants among enterobacterial isolates from outpatients in Brazil. J Antimicrob Chemother 2008;62:474–8.
[54] Robicsek A, Jacoby GA, Hooper DC. The worldwide emergence of plasmidmediated quinolone resistance. Lancet Infect Dis 2006;6:629–40.
[55] Wang M, Tran JH, Jacoby GA, Zhang Y, Wang F, Hooper DC. Plasmid-mediated
quinolone resistance in clinical isolates of Escherichia coli from Shanghai,
China. Antimicrob Agents Chemother 2003;47:2242–8.
[56] Vetting MW, Hegde SS, Fajardo JE, Fiser A, Roderick SL, Takiff HE, et al. Pentapeptide repeat proteins. Biochemistry 2006;45:1–10.
[57] Ellington MJ, Woodford N. Fluoroquinolone resistance and plasmid addiction systems: self-imposed selection pressure? J Antimicrob Chemother
[58] Garrido MC, Herrero M, Kolter R, Moreno F. The export of the DNA replication inhibitor microcin B17 provides immunity for the host cell. EMBO J
[59] Montero C, Mateu G, Rodriguez R, Takiff H. Intrinsic resistance of
Mycobacterium smegmatis to fluoroquinolones may be influenced by new pentapeptide protein MfpA. Antimicrob Agents Chemother 2001;45:3387–92.
[60] Chatterji M, Nagaraja V. GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase. EMBO Rep 2002;3:261–7.
[61] Chatterji M, Senqupta S, Nagaraja V. Chromosomally encoded gyrase inhibitor
GyrI protects Escherichia coli against DNA-damaging agents. Arch Microbiol
[62] Tran JH, Jacoby GA, Hooper DC. Interaction of the plasmid-encoded quinolone
resistance protein QnrA with Escherichia coli topoisomerase IV. Antimicrob
Agents Chemother 2005;49:3050–2.
[63] Vetting MW, Hegde SS, Wang M, Jacoby GA, Hooper DC, Blanchard JS. Structure of QnrB1, a plasmid-mediated fluoroquinolone resistance factor. J Biol
Chem 2011;286:25265–73.
[64] Xiong X, Bromley EH, Oelschlaeger P, Woolfson DN, Spencer J. Structural
insights into quinolone antibiotic resistance mediated by pentapeptide repeat
proteins: conserved surface loops direct the activity of a Qnr protein from a
Gram-negative bacterium. Nucleic Acids Res 2011;39:3917–27.
J. Ruiz et al. / International Journal of Antimicrobial Agents 40 (2012) 196–203
[65] Tran JH, Jacoby GA, Hooper DC. Interaction of the plasmid-encoded quinolone
resistance protein Qnr with Escherichia coli DNA gyrase. Antimicrob Agents
Chemother 2005;49:118–25.
[66] Hegde SS, Vetting MW, Roderick SL, Mitchenall LA, Maxwell A, Takiff HE, et al.
A fluoroquinolone resistance protein from Mycobacterium tuberculosis that
mimics DNA. Science 2005;308:1480–3.
[67] Kreuzer KN, Cozzarelli RN. Escherichia coli mutants thermosensitive for
deoxyribonucleic acid gyrase subunit A: effects on deoxyribonucleic
acid replication, transcription, and bacteriophage growth. J Bacteriol
[68] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary
distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731–9.
[69] Cavaco LM, Hasman H, Xia S, Aarestrup FM. qnrD, a novel gene conferring
transferable quinolone resistance in Salmonella enterica serovar Kentucky
and Bovismorbificans of human origin. Antimicrob Agents Chemother
[70] Hata M, Suzuki M, Matsumoto M, Takahashi M, Sato K, Ibe S, et al. Cloning of
a novel gene for quinolone resistance from a transferable plasmid in Shigella
flexneri 2b. Antimicrob Agents Chemother 2005;49:801–3.
[71] Jacoby GA, Walsh KE, Mills DM, Walker VJ, Oh H, Robicsek A, et al. qnrB,
another plasmid-mediated gene for quinolone resistance. Antimicrob Agents
Chemother 2006;50:1178–82.
[72] Wang M, Guo Q, Xu X, Wang X, Ye X, Wu S, et al. New plasmid-mediated
quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis.
Antimicrob Agents Chemother 2009;53:1892–7.
[73] Fonseca EL, Dos Santos Freitas F, Vieira VV, Vicente ACP. New qnr gene cassettes associated with superintegron repeats in Vibrio cholerae O1. Emerg
Infect Dis 2008;14:1129–31.
[74] Kim HB, Wang M, Ahmed S, Park CH, LaRocque RC, Faruque AS, et al. Transferable quinolone resistance in Vibrio cholerae. Antimicrob Agents Chemother
[75] Hjerde E, Lorentzen MS, Holden MT, Seeger K, Paulsen S, Bason N, et al. The
genome sequence of the fish pathogen Aliivibrio salmonicida strain LFI1238
shows extensive evidence of gene decay. BMC Genomics 2008;9:616.
[76] Poirel L, Liard A, Rodriguez-Martinez JM, Nordmann P. Vibrionaceae as a
possible source of Qnr-like quinolone resistance determinants. J Antimicrob
Chemother 2005;56:1118–21.
[77] Poirel L, Rodriguez-Martinez JM, Mammeri H, Liard A, Nordmann P. Origin
of plasmid-mediated quinolone resistance determinant QnrA. Antimicrob
Agents Chemother 2005;49:3523–5.
[78] Cattoir V, Poirel L, Mazel D, Soussy CJ, Nordmann P. Vibrio splendidus as the
source of plasmid-mediated QnrS-like quinolone resistance determinants.
Antimicrob Agents Chemother 2007;51:2650–1.
[79] Saga T, Kaku M, Onodera Y, Yamachika S, Sato K, Takase H. Vibrio parahaemolyticus chromosomal qnr homologue VPA0095: demonstration by
transformation with a mutated gene of its potential to reduce quinolone
susceptibility in Escherichia coli. Antimicrob Agents Chemother 2005;49:
[80] Rodríguez-Martínez JM, Velasco C, Briales A, García I, Conejo MC, Pascual A.
Qnr-like pentapeptide repeat proteins in Gram-positive bacteria. J Antimicrob
Chemother 2008;61:1240–3.
[81] Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, et al.
Environmental genome shotgun sequencing of the Sargasso Sea. Science
[82] Cheung TK, Chu YW, Chu MY, Ma CH, Yung RW, Kam KM. Plasmidmediated resistance to ciprofloxacin and cefotaxime in clinical isolates
of Salmonella enterica serotype Enteritidis in Hong Kong. J Antimicrob
Chemother 2005;56:586–9.
[83] Velasco C, Rodríguez-Martínez JM, Briales A, Díaz de Alba P, Calvo J, Pascual
A. Smaqnr, a new chromosome-encoded quinolone resistance determinant
in Serratia marcescens. J Antimicrob Chemother 2010;65:239–42.
[84] Jacoby GA, Griffin C, Hooper DC. Citrobacter spp. as a source of qnrB alleles.
Antimicrob Agents Chemother 2011;55:4979–84.
[85] Gordon NC, Wareham DW. Novel variants of the Smqnr family of quinolone
resistance genes in clinical isolates of Stenotrophomonas maltophilia. J Antimicrob Chemother 2010;65:483–9.
[86] Shimizu K, Kikuchi K, Sasaki T, Takahashi N, Ohtsuka M, Ono Y, et al. Smqnr,
a new chromosome-carried quinolone resistance gene in Stenotrophomonas
maltophilia. Antimicrob Agents Chemother 2008;52:3823–5.
[87] Wareham DW, Gordon NC, Shimizu K. Two new variants of and creation of
a repository for Stenotrophomonas maltophilia quinolone protection protein
(Smqnr) genes. Int J Antimicrob Agents 2011;37:89–90.
[88] Cattoir V, Poirel L, Nordmann P. In-vitro mutagenesis of qnrA and qnrS
genes and quinolone resistance in Escherichia coli. Clin Microbiol Infect
[89] Guo Q, Weng J, Xu X, Wang M, Wang X, Ye X, et al. A mutational analysis and
molecular dynamics simulation of quinolone resistance proteins QnrA1 and
QnrC from Proteus mirabilis. BMC Struct Biol 2010;10:33.
[90] Rodríguez-Martínez JM, Briales A, Velasco C, Conejo MC, Martínez-Martínez
L, Pascual A. Mutational analysis of quinolone resistance in the plasmidencoded pentapeptide repeat proteins QnrA, QnrB and QnrS. J Antimicrob
Chemother 2009;63:1128–34.
[91] Birmingham MC, Guarino R, Heller A, Wilton JH, Shah A, Hejmanowski L,
et al. Ciprofloxacin concentrations in lung tissue following a simple 400 mg
intravenous dose. J Antimicrob Chemother 1999;43(Suppl. A):43–8.
[92] Martens R, Wetzstein HG, Zadrazil F, Capelari M, Hoffmann P, Schmeer N.
Degradation of the fluoroquinolone enrofloxacin by wood-rotting fungi. Appl
Environ Microbiol 1996;62:4206–9.
[93] Vetting MW, Park CH, Hegde SS, Jacoby GA, Hooper DC, Blanchard JS.
Mechanistic and structural analysis of aminoglycoside N-acetyltransferase
AAC(6 )-Ib and its bifunctional, fluoroquinolone-active AAC(6 )-Ib-cr variant.
Biochemistry 2008;47:9825–35.
[94] Maurice F, Broutin I, Podglajen I, Benas P, Collatz E, Dardel F. Enzyme structural
plasticity and the emergence of broad-spectrum antibiotic resistance. EMBO
Rep 2008;9:344–9.
[95] Park CH, Robicsek A, Jacoby GA, Sahm D, Hooper DC. Prevalence in the United
States of aac(6 )-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother 2006;50:3953–5.
[96] Jung CM, Heinze TM, Strakosha R, Elkins CA, Sutherland JB. Acetylation of
fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated
from a municipal wastewater treatment plant. J Appl Microbiol 2009;106:
[97] McCann J, Spingarn NE, Kobori J, Ames BN. Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc Natl Acad Sci USA
[98] Winans SC, Walker GC. Identification of pKM101-encoded loci specifying
potentially lethal gene products. J Bacteriol 1985;161:417–24.
[99] Poole K. Efflux pumps as antimicrobial resistance mechanisms. Ann Med
[100] Cattoir V, Poirel L, Nordmann P. Plasmid-mediated quinolone resistance
pump QepA2 in an Escherichia coli isolate from France. Antimicrob Agents
Chemother 2008;52:3801–4.
[101] Hansen LH, Johannesen E, Burmølle M, Sørensen AH, Sørensen SJ. Plasmidencoded multidrug efflux pump conferring resistance to olaquindox in
Escherichia coli. Antimicrob Agents Chemother 2004;48:3332–7.
[102] Périchon B, Courvalin P, Galimand M. Transferable resistance to aminoglycosides by methylation of G1405 in 16S rRNA and to hydrophilic
fluoroquinolones by QepA-mediated efflux in Escherichia coli. Antimicrob
Agents Chemother 2007;51:2464–9.
[103] Szczepanowski R, Krahn I, Linke B, Goesmann A, Pülher A, Schlüter A. Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment
plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport
system. Microbiology 2004;150:3613–30.
[104] Yamane K, Wachino JI, Suzuki S, Kimura K, Shibata N, Kato H,
et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found
in an Escherichia coli clinical isolate. Antimicrob Agents Chemother
[105] Doi Y, Yokoyama K, Yamane K, Wachino J, Shibata N, Yagi T, et al.
Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring
high-level resistance to aminoglycosides. Antimicrob Agents Chemother
[106] Sørensen AH, Hansen LH, Johannesen E, Sørensen SJ. Conjugative plasmid conferring resistance to olaquindox. Antimicrob Agents Chemother
[107] Kim HB, Wang M, Park CH, Kim EC, Jacoby GA, Hooper DC. oqxAB encoding
a multidrug efflux pump in human clinical isolates of Enterobacteriaceae.
Antimicrob Agents Chemother 2009;53:3582–4.
[108] Zhao J, Chen Z, Chen S, Deng Y, Liu Y, Tian W, et al. Prevalence and
dissemination of oqxAB in Escherichia coli isolates from animals, farmworkers, and the environment. Antimicrob Agents Chemother 2010;54:
[109] Hansen LH, Jensen LB, Sørensen HI, Sørensen SJ. Substrate specificity of the
OqxAB multidrug resistance pump in Escherichia coli and selected enteric
bacteria. J Antimicrob Chemother 2007;60:145–7.
[110] Périchon B, Bogaerts P, Lambert T, Frangeul L, Courvalin P, Galimand M.
Sequence of conjugative plasmid pIP1206 mediating resistance to aminoglycosides by 16S rRNA methylation and to hydrophilic fluoroquinolones by
efflux. Antimicrob Agents Chemother 2008;52:2581–92.
[111] Yamane K, Wachino JI, Suzuki S, Arakawa Y. Plasmid-mediated qepA gene
among Escherichia coli clinical isolates from Japan. Antimicrob Agents
Chemother 2008;52:1564–6.
[112] Kim ES, Jeong JY, Choi SH, Lee SO, Kim SH, Kim MN, et al. Plasmid-mediated
fluoroquinolone efflux pump gene, qepA, in Escherichia coli clinical isolates in
Korea. Diagn Microbiol Infect Dis 2009;65:335–8.
[113] Paulsen IT, Brown MH, Littlejohn TG, Mitchell BA, Skurray RA. Multidrug
resistance proteins QacA and QacB from Staphylococcus aureus: membrane
topology and identification of residues involved in substrate specificity. Proc
Natl Acad Sci USA 1996;93:3630–5.
[114] Duesberg CB, Malhotra-Kumar S, Goossens H, McGee L, Klugman KP, Welte T,
et al. Interspecies recombination occurs frequently in quinolone resistancedetermining regions of clinical isolates of Streptococcus pyogenes. Antimicrob
Agents Chemother 2008;52:4191–3.
[115] Janoir C, Podglajen I, Kitzis MD, Poyart C, Gutmann L. In vitro exchange of
fluoroquinolone resistance determinants between Streptococcus pneumoniae
and viridans streptococci and genomic organization of the parE–parC region
in S. mitis. J Infect Dis 1999;180:555–8.
J, De La Campa AG. Horizontal trans[116] Ferrándiz MJ, Fenoll A, Linares
fer of parC and gyrA in fluoroquinolone-resistant clinical isolates of
Streptococcus pneumoniae. Antimicrob Agents Chemother 2000;44: