Letters to the Editor / International Journal of Antimicrobial Agents 40 (2012) 562– 573 571

F eterog(

algftrdIpbwI

sb6wPta

cCt(snoy

pbfipasEccsaitc

p(

[

[

[

[

[

ig. 1. Graphical distribution of Vibrio cholerae O1 biotype El Tor isolates based on hMAMA-PCR), by year.

ntimicrobial susceptibility results demonstrated that all of the iso-ates were sensitive to El Tor group V phage, resistant to classicalroup IV phage and resistant to polymyxin B, which concluded theact that all V. cholerae isolates incorporated in this study belongedo O1 biotype El Tor. The graphical presentation of MAMA-PCResults in Fig. 1 clustered V. cholerae isolates from Chennai, India,uring 1980–2010 into four periods: Period I, 1980–1984; Period

I, 1985–1991; Period III, 1992; and Period IV, 1993–2010. Theseeriods reflected predominance of the CT-B I variant, a mosaic com-ination both of CT-B I and II variants, appearance of CT-B III alongith the CT-B II variant, and uninterrupted prevalence of the CT-B

II variant, respectively.Among these 256 isolates 100 representative isolates were

elected to confirm the presence and absence of class 1 integrony PCR amplification of the 3′-CS. Of the 100 V. cholerae isolates,0 were found to be positive for 3′-CS; no 3′-CS amplificationas found within the 40 V. cholerae isolates from 1961–1969.

hage typing of each variant (CT-B I, II and III) showed a similaryping pattern dominated by type 27 and followed by types 24nd 26.

It was clearly found from these data that the influence of thelassical biotype still remained incessantly in the isolates fromhennai up to 1984. Interestingly, this influence of the classical bio-ype became sporadic with the appearance of typical El Tor variantsCT-B II) from the year 1961 and finally disappeared in 1991. Thistudy clarifies that V. cholerae O139 has changed the cholera sce-ario following its emergence in 1992 in Chennai. Since the onsetf O139 (in 1992), we found altered El Tor variants and from theear 1993 it was still continuing.

These findings are in accordance with previous reports [5] whichostulated that the classical cholera toxin gene has reappearedut its carrier has been El Tor, and this study showed that itsrst appearance was in 1966. MAMA-PCR and class 1 integron andhage typing results concluded that the genotypic variation, suchs polymorphic traits in ctxB genes, did not influence the antibioticusceptibility and phenotypic behaviour of V. cholerae O1 biotypel Tor strains. The pathogenic effect of El Tor vibrios harbouring thelassical ctxB gene is still an unsolved mystery but it is clear that aryptic change in the cholera scenario has taken place and the con-equences are still being evaluated from the viewpoint of clinicalppearance. This subtle genetic change in V. cholerae could resultn some alterations in clinical manifestations of cholera. In future,his observation will be helpful to understand the epidemiology ofholera in a more precise way.

Funding: All the research works of this manuscript have been

erformed in the National Institute of Cholera and Enteric DiseasesKolkata, India) with the support of institutional funding.

Competing interests: None declared.Ethical approval: Not required.

eneity of the ctxB gene determined by mismatch amplification mutation assay PCR

References

1] Safa A, Sultana J, Dac Cam P, Mwansa JC, Kong RY. Vibrio cholerae O1 hybrid ElTor strains, Asia and Africa. Emerg Infect Dis 2008;14:987–8.

2] Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB, Shimada T, et al.Emergence of novel strain of Vibrio cholerae with epidemic potential in southernand eastern India. Lancet 1993;341:703–4.

3] Morita M, Ohnishi M, Arakawa E, Bhuiyan NA, Nusrin S, Alam M, et al. Develop-ment and validation of a mismatch amplification mutation PCR assay to monitorthe dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor.Microbiol Immunol 2008;52:314–17.

4] Hall RM, Stokes HW. Integrons: novel DNA elements which capture genes bysite-specific recombination. Genetica 1993;90:115–32.

5] Ansaruzzaman M, Bhuiyan NA, Nair GB, Sack DA, Lucas M, Deen JL, et al.Cholera in Mozambique, variant of Vibrio cholerae. Emerg Infect Dis 2004;10:2057–9.

Mayukh DasTexas A&M University, College Station, TX, USA

Abhishek JaiswalSubharthi Pal

National Institute of Cholera and Enteric Diseases, Kolkata, India

Tushar Suvra BhowmickTexas A&M University, College Station, TX, USA

Amit GhoshNational Institute of Cholera and Enteric Diseases, Kolkata, India

A.K. GoelDefence Research and Development Establishment, Gwalior, India

B.L. Sarkar ∗

National Institute of Cholera and Enteric Diseases, Kolkata, India

∗ Corresponding author. Present address: Vibrio Phage ReferenceLaboratory, Division of Bacteriology, National Institute of Cholera

and Enteric Diseases, P-33, C. I. T. Road, Scheme XM, Beliaghata,Kolkata 700 010, India. Tel.: +91 33 2370 1176;

fax: +91 33 2370 5066.E-mail address: bl sarkar@hotmail.com (B.L. Sarkar)

3 August 2012doi:10.1016/j.ijantimicag.2012.08.005

Spread of the blaIMP-13 gene in French Pseudomonas aeruginosathrough sequence types ST621, ST308 and ST111

Sir,

IMP-13 is a class B metallo-�-lactamase (MBL) that was firstdescribed in a carbapenem-resistant clinical isolate of Pseudomonasaeruginosa in Italy in 2001 [1]. Since then, IMP-13-producing P.aeruginosa have been implicated in significant outbreaks in Italy

572 Letters to the Editor / International Journal of Antimicrobial Agents 40 (2012) 562– 573

Table 1Features of IMP-13 (or IMP-37)-producing Pseudomonas aeruginosa isolated in France.

Isolate Sample Date of isolation Location Serotype Genotype Sequence type MIC (mg/L)a oprD geneb

PIP IPM MEM ATM

6-Brest N/S 03/2006 Brest O12 A ST111 64 128 128 8 oprD-�TS (PP2)10-265 BAL 03/2010 Tours O11 C ST308 8 64 32 1 oprD-�TS (PA14)11-547 Catheter 05/2011 Chartres O4 B ST621 8 8 4 8 oprD-wt (PP2)11-655 Biliary fluid 07/2011 Villejuif O4 B ST621 64 128 128 16 oprD-�TS (PP2)11-688 Surgical scar 08/2011 Orléans O4 B ST621 24 4 2 16 oprD-wt (PP2)11-700 Vaginal secretion 08/2011 Tours O11 C ST308 8 128 64 1 oprD-�TS (PA14)11-723 Urine 09/2011 Chartres O4 B ST621 8 4 2 8 oprD-wt (PP2)11-799 Urine 11/2011 Chartres O4 B ST621 8 8 4 8 oprD-wt (PP2)11-810 Rectal swab 11/2011 Paris O4 B ST621 8 64 64 1 oprD-�TS (PP2)12-993 Rectal swab 04/2012 Paris O4 B ST621 24 64 64 16 oprD-�TS (PP2)

MIC, minimum inhibitory concentration; PIP, piperacillin; IPM, imipenem; MEM, meropenem; ATM, aztreonam; N/S, not specified; BAL, bronchoalveolar lavage.a MICs of ticarcillin and ceftazidime were >256 mg/L.

l termP

aadtts

wt(pnAm((idwc(T1faePao(iba(

trdrtSittcsa

b oprD-�TS, presence of a nucleotide substitution causing premature translationaP2 or PA14 (in parentheses).

nd have been detected sporadically in Romania, Austria, Belgiumnd Argentina [2,3]. In these isolates, the IMP-13 gene was mostlyescribed as part of the class 1 integron InPSG and was reported inhe same clonal sequence type (ST), i.e. ST621 [2,3]. Here we reporthe recent propagation in France of IMP-13-producing P. aeruginosatrains.

From March 2010 to April 2012, nine multidrug-resistant strainsith reduced susceptibility to carbapenems were analysed by

he French National Reference Centre for Antibiotic ResistanceBesanc on, France). These strains were isolated from clinical sam-les of nine patients admitted to six French hospitals located in twoeighbouring regions (Regions Centre and Paris-Ile-de-France).ll strains exhibited high levels of resistance to ticarcillin [mini-um inhibitory concentration (MIC) > 256 mg/L] and ceftazidime

MIC > 256 mg/L), whereas MICs of aztreonam were ≤16 mg/LTable 1). Susceptibility to carbapenems varied greatly among thesesolates. Two strains (11-688 and 11-723) displayed only a marginalecreased susceptibility to imipenem (MIC = 4 mg/L, comparedith 1 mg/L for reference strain PAO1) and were classified as ‘sus-

eptible’ according to Clinical and Laboratory Standards InstituteCLSI) and European Committee on Antimicrobial Susceptibilityesting (EUCAST) breakpoints, and two other strains (11-547 and1-799) fell into the ‘intermediate’ category (MIC = 8 mg/L). In theseour strains, DNA sequencing experiments revealed the presence of

functional porin OprD (the specific uptake pathway for carbapen-ms in P. aeruginosa) identical to that of previously described strainP2 [4]. The five remaining strains of the collection all showed

high-level resistance to imipenem (MIC ≥ 64 mg/L) as a resultf mutations generating premature stop codons in the oprD geneTable 1). Phenotypic tests recommended for the detection of MBLsn P. aeruginosa, such as the MBL Etest (bioMérieux), the com-ined meropenem ± dipicolinic acid disk test (Rosco Laboratories)nd the double-disk synergy test with meropenem/dipicolinic acidRosco Laboratories), were positive for all of the strains.

Genotyping of these strains as well as one representative ofhe Italian clone by pulsed-field gel electrophoresis (PFGE) usingestriction endonuclease DraI allowed the identification of twoistinct clones, named B and C, belonging to ST621 and ST308,espectively (Table 1). None of the nine French strains appearedo be clonally related to the IMP-13-positive clone Brest (clone A,T111) that had been involved in a nosocomial outbreak in Francen 2006. Seven of the isolates showed a pulsotype (B) identical tohat of the Italian clone [3]. These strains were found to contain

he same integron (InPSG) as the Italian clone, with the blaIMP-13assette located upstream of an aacA4 cassette. The two remainingtrains (10-265 and 11-700), which were isolated in the same cityfter a 17-month interval, belonged to a different pulsotype (clone

[

ination of protein OprD; wt, wild-type sequence identical to that of reference strain

C). In these bacteria, the blaIMP-13 gene was located downstreamfrom the aacA4 gene in a new class 1 integron named In771(GenBank accession no. JX131371). Of note, analysis of the geneticenvironment of blaIMP in the clone Brest revealed another newclass 1 integron, named In772 (GenBank accession no. JX131372),resembling In449 recently described in a strain of Pseudomonasmonteilii [5]. In772 differs from In449 by a deletion of 149 bpextending from the last 134 nucleotides of the blaIMP cassette tothe first 5 nucleotides of the aacA4 cassette. This genetic event wasresponsible for changes in the amino acid sequence of theIMP-13 protein (Pro244Gln, Asn246Thr and the additionof two amino acids, Ala–Ser, at its C-terminus). This lat-ter �-lactamase received a new variant number, IMP-37(http://www.lahey.org/Studies/). PCR experiments performedwith primers specific to tnpA gene from Tn5051 were negativein clones A and C, indicating that In772 and In771, respectively,are located on genetic structures different from that of clone B(in which InPSG is inserted in a Tn5051-like transposon). For eachstrain, efforts to transfer the resistance determinants by conju-gation were unsuccessful, suggesting a possible chromosomallocation of the blaIMP-13 genes.

Detection of IMP-13-producing P. aeruginosa with wild-typefunctional porin OprD may be a challenge for the microbiologist.Although such strains give positive results with commercial MBLdetection tests, they may be under-recognised and thus under-reported simply because they are susceptible to imipenem andconsidered as not eligible for MBL screening. The present studydemonstrates that the blaIMP-13 gene (or its variant blaIMP-37) isnow harboured by strains of P. aeruginosa belonging to differentlineages, which may accelerate its diffusion.

Acknowledgments

The authors are very grateful to Prof. Glupczynski (Brussels),Prof. Picard (Paris), Dr Tandé (Brest), Dr Haguenauer, Dr Toyer(Tours), Dr Mihaila (Villejuif), Dr Debroize (Luisant) and Dr Dosbaa(Paris) for providing clinical isolates.

Funding: This work was supported by the French Ministry ofHealth (InVS) and the Hospital of Besanc on (France).

Competing interests: None declared.Ethical approval: Not required.

References

1] Toleman MA, Biedenbach D, Bennett D, Jones RN, Walsh TR. Genetic charac-terization of a novel metallo-�-lactamase gene, blaIMP-13, harboured by a novelTn5051-type transposon disseminating carbapenemase genes in Europe: report

al of A

[

[

[

[

Letters to the Editor / International Journ

from the SENTRY worldwide antimicrobial surveillance programme. J Antimi-crob Chemother 2003;52:583–90.

2] Naas T, Bogaerts P, Kostyanev T, Cuzon G, Huang TD, Ozsu S, et al. Silent spreadof IMP-13-producing Pseudomonas aeruginosa belonging to sequence type 621in Belgium. J Antimicrob Chemother 2011;66:2178–9.

3] Santella G, Pollini S, Docquier JD, Mereuta AI, Gutkind G, Rossolini GM, et al.Intercontinental dissemination of IMP-13-producing Pseudomonas aeruginosabelonging in sequence type 621. J Clin Microbiol 2010;48:4342–3.

4] Epp SF, Köhler T, Plésiat P, Michéa-Hamzehpour M, Frey J, Pechère JC. C-terminalregion of Pseudomonas aeruginosa outer membrane porin OprD modulates sus-ceptibility to meropenem. Antimicrob Agents Chemother 2001;45:1780–7.

5] Bogaerts P, Bouchahrouf W, Lissoir B, Denis O, Glupczynski Y. IMP-13-producingPseudomonas monteilii recovered in a hospital environment. J AntimicrobChemother 2011;66:2434–5.

Damien Fournier ∗

Katy JeannotMarjorie Robert-Nicoud

Emeline MullerCentre National de Référence de la Résistance aux Antibiotiques,

Laboratoire de Bactériologie, Centre Hospitalier RégionalUniversitaire de Besanc on, 25030 Besanc on cedex, France

ntimicrobial Agents 40 (2012) 562– 573 573

Pascal CholleyLaboratoire d’Hygiène Hospitalière, Centre Hospitalier Régional

Universitaire de Besanc on, France

Nathalie van der Mee-MarquetService de Bactériologie et Hygiène, Réseau des Hygiénistes du

Centre, Centre Hospitalier Régional Universitaire de Tours, France

Patrick PlésiatCentre National de Référence de la Résistance aux Antibiotiques,

Laboratoire de Bactériologie, Centre Hospitalier RégionalUniversitaire de Besanc on, 25030 Besanc on cedex, France

∗ Corresponding author. Tel.: +33 3 81 66 81 96;fax: +33 3 81 66 89 14.

E-mail address: damien.fournier@univ-fcomte.fr (D. Fournier)

9 August 2012

doi:10.1016/j.ijantimicag.2012.08.006