Expanded-spectrum [beta]-lactamase and plasmid-mediated quinolone resistance.

To the Editor: The emergence of plasmid-mediated, and thus transferable, quinolone resistance determinants has been recently discovered (1) and shown to involve the pentapeptide repeat protein Qnr, which interacts with DNA gyrase and topoisomerase IV to prevent quinolone inhibition (2,3). Qnr determinants confer resistance to nalidixic acid and reduced susceptibility to fluoroquinolones (3). They have been identified worldwide in a variety of enterobacterial species and were often associated to expanded-spectrum [beta]3-lactamases (ESBLs) (2). The association between the ESBL VEB-1 and the QnrA1 determinants was reported (4). Because plasmid co-localization of QnrA and VEB-1 encoding genes has been reported repeatedly from scattered clonally-unrelated enterobacterial isolates, our objective was to use replicon typing to trace a possible dissemination of a common plasmid worldwide.

The [bla.sub.VEB-1]- and/or qnrA-positive plasmids that have been included in the study were from 17 isolates previously described in detail (3-8) (Table). Escheriehia coli transconjugants (Tc) were obtained for 14 of 17 clinical isolates, allowing an accurate replicon typing since original clinical isolates might harbor several plasmids. They were collected from 1999 to 2005, from patients hospitalized in different parts of the world (Table). The 13 [bla.sub.VEB-1]-positive isolates were from 5 countries (France, Turkey, Algeria, Thailand, and Canada), scattered on 4 continents. Among them, the Providencia stuartii and Proteus mirabilis isolates from Algeria were negative for qnrA1. In addition, 4 [bla.sub.VEB-1]-negative but qnrA1-positive isolates recovered from France and Australia were also included in the study.

PCR-based replicon typing (PBRT), which recognizes FIA, FIB, FIE, HI1, HI2, Il-IT[gamma], L/M, N, P, W, T, A/C, K, B/O, X, Y, and FII replicons (9), was applied to type the resistance plasmids from all the strains. Amplicons were confirmed by DNA sequencing and used as probes in hybridization experiments on purified plasmids (data not shown).

PBRT results showed that the 13 [bla.sub.VEB-1]-positive plasmids (including 11 qnrAl-positive) belonged to the IncA/C incompatibility group. DNA sequencing identified the A/[C.sub.2] replicon variant (European Molecular Biology Laboratory no. AM087198) in all these plasmids (Table). Plasmids of this type were recently identified in the United States and in Italy carrying the AmpC-type cephalosporinase CMY2-encoding gene (10). In 2 strains (E. coli TcGOC and Citrobacter freundii LUT), the IncA/[C.sub.2] plasmids were associated with additional replicons, which suggests the presence of multiple plasmids or fusions between plasmids of different backbones. By contrast, all the 4 [bla.sub.VEB-1]-negative isolates but qnrA1-positive were negative for the A/C replicon, except transconjugant TcK147; however, sequencing identified an A/[C.sub.1]-type replicon in that strain. These results indicated that the genes encoding QnrA1 and VEB-1, when identified concomitantly in a given isolate, were always located on plasmids belonging to the same IncA/[C.sub.2]-incompatibility group that may vary in size and digestion pattern (Table; unpub, data). In addition, we showed that plasmids carrying the [bla.sub.VEB-1] gene but lacking qnrA1 were also of the IncA/[C.sub.2] type (Table). Plasmids that were [bla.sub.VEB-1]-negative but qnrAl-positive were of distinct replicon types, thus suggesting independent acquisition of the qnrA1 gene on different plasmids. It is remarkable that since VEB-1 is apparently always encoded by IncA/[C.sub.2] plasmids, when genes for QnrA1 and VEB-1 are found together, they also occur on IncA/[C.sub.2] plasmids.

Thus, evidence here shows that the IncA/[C.sub.2] plasmid is the main vehicle of the [bla.sub.VEB-1] gene worldwide, on which the qnrA1 gene may be added. The possibility that both [bla.sub.VEB-1] and qnrA1 genes may be identified on a single genetic structure in several isolates has been recently shown with their identification within the same sull-type integron (6).

Because results of these experiments provided a good marker for tracing [bla.sub.VEB-1]-positive plasmids, and taking in account the property of A/C-type plasmids to have a broad range of hosts (note: this has not been demonstrated for the specific A/[C.sub.2] subgroup), we tried to amplify the A/[C.sub.2] replicon in a collection of 15 [bla.sub.VEB-1]-positive and clonally unrelated Pseudomonas aeruginosa isolates from France, Thailand, India, and Kuwait. The [bla.sub.VEB-1] gene was supposed to be chromosome-encoded in those isolates. PCR failed to give any positive results, confirming the absence of an IncA/C-type plasmid and also ruling out the hypothesis of IncA/[C.sub.2]-type plasmid co-integration at the origin of [bla.sub.VEB-1] acquisition in P. aeruginosa.

The spread of plasmids carrying a large array of resistance genes among Enterobacteriaceae is of concern since this provides a convenient genetic mechanism for a given strain to become panresistant to antimicrobial drugs. In particular, the recent identification of the Qnr determinants has shown that plasmids may provide resistance (or at least reduced susceptibility) to quinolones and fluoroquinolones, whereas they are already known to carry resistance to [beta]-lactams, aminoglycosides, chloramphenicol, tetracycline, rifampin, sulfonamides, and disinfectants, pQR1 (4) or pl (6) are examples of well-characterized plasmids that mediate multidrug resistance by carrying [bla.sub.VEB-1] and qnrA1, together with aminoglycoside resistance genes aadB, aacA1, and aadA1, chloramphenicol resistance gene cmlA, rifampin resistance gene arr2, disinfectant resistance gene qacI, and sulfonamides resistance gene sul1.

Our study showed that the IncA/ [C.sub.2]-type plasmids may be the source of such worldwide dissemination. It means that 1 plasmid scaffold has brought the same (or at least very similar) multidrug resistance to multiple enterobacterial species in different continents.

Acknowledgments

We thank S. Bernabeu for technical assistance.

This work was funded by grants from the European Community (6th PCRD, LSHM-CT-2005-018705).

References

(1.) Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet. 1998;351:797-9.

(2.) Robicsek A, Jacoby GA, Hooper DC. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis. 2006;6:629-40.

(3.) Nordmann P, Poirel L. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J Antimicrob Chemother. 2005;56:463-9.

(4.) Poirel L, Van De Loo M, Mammeri H, Nordmann P. Association of plasmid-mediated quinolone resistance with extended-spectrum [beta]3-lactamase VEB-1. Antimicrob Agents Chemother. 2005;49:3091-4.

(5.) Girlich D, Poirel L, Leelaporn A, Karim A, Tribuddharat C, Fennewald M, et al. Molecular epidemiology of the integronlocated VEB-1 extended-spectrum [beta]3-lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand. J Clin Microbiol. 2001;39:175-82.

(6.) Poirel L, Pitout JD, Calvo L, Rodriguez-Martinez JM, Church D, Nordmann P. In vivo selection of fluoroquinolone-resistant Escherichia coli isolates expressing plasmid-mediated quinolone resistance and expanded-spectrum [beta]3-lactamase. Antimicrob Agents Chemother. 2006;50:1525-7.

(7.) Rodriguez-Martinez JM, Poirel L, Pascual A, Nordmann P. Plasmid-mediated quinolone resistance in Australia. Microb Drug Resist. 2006; 12:99-102.

(8.) Poirel L, Leviandier C, Nordmann P. Prevalence and genetic analysis of plasmid-mediated quinolone resistance determinants QnrA and QnrS in Enterobacteriaceae in a French University Hospital. Antimicrob Agents Chemother. 2006;50:3992-7.

(9.) Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219-28.

(10.) Carattoli A, Miriagou V, Bertini A, Loli A, Colinon C, Villa L, et al. Replicon typing of plasmids encoding resistance to newer [beta]-lactams. Emerg Infect Dis. 2006; 12:1145-8.

Address for correspondence: Laurent Poirel, Service de Bacteriologie-Virologie, Hopital de Bicetre, 78 Rue du General Leclerc, 94275 Le Kremlin-Bicetre CEDEX, France; email: [e-mail]

Laurent Poirel, * Laura Villa, ([dagger]) Alessia Bertini, ([dagger]) Johann D. Pitout, ([double dagger]) Patrice Nordmann, * and Alessandra Carattoli ([dagger])

* Hopital de Bicetre, le Kremlin-Bicetre, France; ([dagger]) Istituto Superiore di Sanita, Rome, Italy; and ([double dagger]) University of Calgary, Calgary, Alberta, Canada

Table. Features of the VEB-1- or QnrA-positive isolates used in
this study *

                                                         Year of
Straint (Ref.)           Sp. of origin       Country     isolation

Escherichia coli            E. coli         Thailand       1999
TcE1 (5)
E. coli TcE4 (5)            E. coli         Thailand       1999
E. coli TcE5 (5)            E. coli         Thailand       1999
E. coli TcE7 (5)            E. coli         Thailand       1999
E. coli TcE8 (5)            E. coli         Thailand       1999
E. coli TcEl6 (5)           E. coli         Thailand       1999
E. coli TcEl8 (5)           E. coli         Thailand       1999
E. coli Tc(pl) (5)          E. coli          Canada        2000
E. coli Tc(pQR1)            E. coli          France        2003
(4)
E. coli Tc(GOC) (4)       Enterobacter       France        2003
                            cloacae
Citrobacter freundii      C. freundii        Turkey        2004
LUT (3)
Providencia stuartii      P. stuartii        Algeria       2004
15 (this study)
E. coli TcMAA (this         Proteus          Algeria       2004
study)                     mirabilis
E. coli TcK147 (7)         Klebsiella       Australia      2002
                           pneumoniae
E. cloacae Al (8)          E. cloacae        France        2004
E. coli TcA2 (8)          Enterobacter       France        2005
                           aerogenes
E. coli TcA3 (8)         K. pneumoniae       France        2005

                            Plasmid
Straint (Ref.)             size (kb)          ESBL        QnrA1

Escherichia coli              160             VEB-1         +
TcE1 (5)
E. coli TcE4 (5)              150             VEB-1         +
E. coli TcE5 (5)              150             VEB-1         +
E. coli TcE7 (5)              150             VEB-1         +
E. coli TcE8 (5)              150             VEB-1         +
E. coli TcEl6 (5)             140             VEB-1         +
E. coli TcEl8 (5)             180             VEB-1         +
E. coli Tc(pl) (5)            180             VEB-1         +
E. coli Tc(pQR1)              180             VEB-1         +
(4)
E. coli Tc(GOC) (4)           190             VEB-1         +
Citrobacter freundii           ND             VEB-1         +
LUT (3)
Providencia stuartii           ND             VEB-1         -
15 (this study)
E. coli TcMAA (this           190             VEB-1         -
study)
E. coli TcK147 (7)            160            SHV-12         +
E. cloacae Al (8)              75            SHV-12         +
E. coli TcA2 (8)              150            SHV-12         +
E. coli TcA3 (8)               40               -           +

                                                    Resistance
Straint (Ref.)              Replicon         markers ([double dagger])

Escherichia coli          A/[C.sub.2]            NAL, K, SSS, C,
TcE1 (5)                                               RA
E. coli TcE4 (5)          A/[C.sub.2]            NAL, K, TM, SSS,
                                                      C, RA
E. coli TcE5 (5)          A/[C.sub.2]            NAL, K, TM, SSS,
                                                       SXT
E. coli TcE7 (5)          A/[C.sub.2]            NAL, K, TM, SSS,
                                                      C, RA
E. coli TcE8 (5)          A/[C.sub.2]            NAL, K, TM, SSS,
                                                       TE
E, coli TcEl6 (5)         A/[C.sub.2]            NAL, K, TM, SSS,
                                                       RA
E, coli TcEl8 (5)         A/[C.sub.2]              NAL, K, SSS,
                                                      C, RA
E. coli Tc(pl) (5)        A/[C.sub.2]              NAL, K, SSS,
                                                      C, RA
E. coli Tc(pQR1)          A/[C.sub.2]              NAL, K, SSS,
(4)                                                 C, RA, SXT
E. coli Tc(GOC) (4)     A/[C.sub.2], FIB            NAL, K, TM,
                                                      SSS, C
Citrobacter freundii    A/[C.sub.2], FIB,              NA
LUT (3)                        K
Providencia stuartii      A/[C.sub.2]                  NA
15 (this study)
E. coli TcMAA (this       A/[C.sub.2]               K, TM, SSS,
study)                                                C, SXT
E. coli TcK147 (7)      HI2, A/[C.sub.1]            NAL, K, TM, C,
                               P                     TE, SXT
E. cloacae Al (8)             HI2                       NA
E. coli TcA2 (8)              FII                  NAL, K, TM, TE

E. coli TcA3 (8)             11, K               NAL, K, TM, C, TE

Ref., reference; ESBL, expanded-spectrum [beta]-lactamase;
NAL, nalidixic acid; K, kanamycin; SSS, sulfonamides;
C, chloramphenicol; RA, rifampin; TM, tobramycin; SXT,
trimethoprim-sulfamethoxazole; TE, tetracycline; ND, not determinable;
NA, not applicable.

([dagger]) indicates that this is a transconjugant or a transformant.

([double dagger]) Non-[beta]-lactam-associated markers.