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Antimicrobial Agents and Chemotherapy, December 2006, p. 4220-4223, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00300-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Epidemiology of VIM-4 Metallo-ß-Lactamase-Producing Pseudomonas sp. Isolates in Hungary{triangledown}

Balázs Libisch,1* Mónika Muzslay,2 Mária Gacs,1 János Minárovits,3 Márta Knausz,4 Joseph Watine,5 Gábor Ternák,6 Éva Kenéz,7 Ildikó Kustos,8 László Rókusz,9 Klára Széles,10 Boglárka Balogh,1 and Miklós Füzi1

Department of Bacteriology, National Center for Epidemiology, 1097 Budapest, Hungary,1 Department of Molecular Epidemiology, National Center for Epidemiology, 1097 Budapest, Hungary,2 Microbiological Research Group, National Center for Epidemiology, 1529 Budapest, Hungary,3 Aladár Petz County Teaching Hospital, 9023 Gyor, Hungary,4 Laboratoire de Biologie Polyvalente, Hôpital Général, 12027 Rodez Cédex 9, France,5 Department of Infectious Diseases, Baranya County Hospital, 7623 Pécs, Hungary,6 Laboratory of Microbiology, Baranya County Hospital, 7623 Pécs, Hungary,7 Department of Medical Microbiology and Immunology, University of Pécs, 7643 Pécs, Hungary,8 Department of Internal Medicine, Central Military Hospital, 1553 Budapest, Hungary,9 Department of Epidemiology, National Public Health Service, 9024 Gyor, Hungary,10

Received 10 March 2006/ Returned for modification 24 May 2006/ Accepted 16 September 2006


   ABSTRACT
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VIM metallo-ß-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonizing 19 patients from seven hospitals in Hungary were characterized between October 2003 and November 2005. Macrorestriction analysis revealed the involvement of hospitals from three different towns in northwest Hungary in an outbreak caused by VIM-4-producing P. aeruginosa.


   TEXT
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The worldwide spread of acquired metallo-ß-lactamase (MBL)-producing gram-negative pathogens was observed in the past decade, with the blaVIM-type acquired MBL genes currently being the most prevalent in Europe (16, 23). The first VIM-producing Pseudomonas aeruginosa clinical isolates in Hungary (isolates PA396 and PA450) were characterized at the National Center for Epidemiology in 2003 (7). We established routine screening of carbapenem-resistant Pseudomonas sp. isolates provided by collaborating regional laboratories for acquired MBL genes. In 2005, MBL-positive isolates from six towns in Hungary were detected (Fig. 1). Our aim was to characterize these isolates and to examine the clonal relationships between them and between the major European multiresistant serotype O12 P. aeruginosa clone, clone P12 (4, 8, 10).


Figure 1
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  		FIG. 1. Geographic distribution of the VIM-positive P. aeruginosa clinical isolates in Hungary characterized in the present study. Small black circles indicate the cities. Budapest (the capital) is underlined. Mosonm., Mosonmagyaróvár. The serotype and PFGE subtype of the VIM-positive P. aeruginosa isolates characterized are shown under or next to the names of the cities.

 
The VIM-positive clinical and environmental isolates tested in this study are listed in Table 1, together with the previously published control isolates PA396 and PA450 (7). Isolates P12-Q and P12-E were from French patients "Q" and "E," respectively, who participated in a previous clone P12-related study (8). MICs were determined by the agar dilution method (1) for ß-lactam antibiotics and by the Etest (AB Biodisk, Solna, Sweden) for other antibiotics. The MBL Etest and the imipenem-EDTA, ceftazidime-EDTA, and cefepime-EDTA double-disk methods were used for phenotypic screening (21, 22).


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  		TABLE 1. Characteristics of VIM-producing Pseudomonas sp. isolates from Hungarya

 
blaVIM genes and class 1 integrons were detected by PCR (7). The variable regions of the integrons from isolates PA555 and MB197 were sequenced by using the following primers, together with those described previously (7): primer 197F (5'-AAT CGC TCA GTC GCC GAG-3'), primer 197R1 (5'-TAG TGC TTC TCC GTC GGG-3'), primer 197R2 (5'-AAT TCC GCA TTG CTG ATC G-3'), and primer 197R3 (5'-AGG TAT TGC TCC TGC ACT T-3'). Isolate PA555 was selected for full integron sequencing in 2003, as it was the first VIM-positive isolate from Pécs, Hungary, while isolate MB197 was selected as an invasive isolate from a cluster of clonally closely related VIM-positive isolates from northwest Hungary. For the other VIM-producing isolates, the integron structures were determined by PCR mapping and partial sequencing.

Pulsed-field gel electrophoresis (PFGE) was performed as described earlier (11), with modifications, and the patterns were interpreted by using Fingerprinting II Informatix software (Bio-Rad, Madrid, Spain). Pseudomonas aeruginosa antisera (Bio-Rad, Marnes-la-Coquette, France) were used for serotyping.

Conjugation experiments were carried out with strains Escherichia coli J5-3 Rifr and P. aeruginosa PAO4089Rp (6, 7) as the recipients. Transconjugants were selected on Mueller-Hinton agar plates containing 300 µg/ml rifampin and 32 µg/ml cefotaxime, 128 µg/ml piperacillin-tazobactam, or 128 µg/ml ticarcillin.

A total of 758 carbapenem-resistant Pseudomonas sp. isolates collected on a voluntary basis from 85 epidemiological settings in 42 towns in Hungary were screened for MBL production between October 2003 and November 2005. The settings are distributed in every geographical region of Hungary. Fifty P. aeruginosa isolates and one P. putida isolate (isolate PP524) proved to be positive by the phenotypic tests. Seven of these positive isolates from a Budapest hospital were not included in this study. PCRs with VIM- and integron-specific primers (7) gave positive results for all these isolates. The blaVIM genes of the isolates indicated by a superscript g in Table 1 were sequenced and were identified as blaVIM-4 in every case. P12-Q and P12-E were negative by the MBL phenotypic tests.

Antibiotic susceptibility values for representative VIM-positive isolates and isolate P12-Q are shown in Table 2. All isolates were multidrug resistant, and some of them were only sensitive to aztreonam. Isolate MB397 was panresistant to all antimicrobials tested.


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  		TABLE 2. Antibiotic MICs determined for VIM-producing Pseudomonas sp. isolates and isolate P12-Q

 
Two major groups could be identified among the VIM-positive P. aeruginosa isolates by serotyping and PFGE. Their geographical distribution is shown in Fig. 1. The first group comprised serotype O12 isolates from Budapest and Pécs (Table 1). These isolates were possibly related to each other, as determined by PFGE, and also to representative isolates of the major European P12 clone when a cutoff value of ≥80% was used (17-19). The O12 isolates from Budapest and Pécs carry an integron-borne blaVIM-4 gene with an identical 170-bp duplication in the last position, preceded by an aacA4 gene (Fig. 2, integrons a and b). An additional blaOXA cassette is present in the first position of integron a from Budapest (Table 1) (7).


Figure 2
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  		FIG. 2. Comparison of the schematic structures of the blaVIM-4-carrying class 1 integrons from Hungary. Open ellipses, attI1 site; black circles, 59-base elements; asterisks, blaVIM-4 cassettes with the 170-bp duplication; 5'-CS and 3'-CS, 5' and 3' conserved sequences, respectively. GenBank accession numbers and isolates harboring the integrons are as follows: (a) AY509609 and isolate PA396, (b) AY702100 and isolate PA555, (c) isolate MB387, (d) DQ357197 and isolate MB197.

 
The second group comprised all isolates from the Gyor, Sopron, and Mosonmagyaróvár hospitals. These serotype O11 isolates were identical or closely related to each other by PFGE by use of a Dice coefficient of ≥95% (Table 1) and carried a blaVIM-4 gene without the 170-bp duplication on an integron that also harbored a blaOXA-2 cassette. Two variants of this integron were identified (Fig. 2, integrons c and d), with the only difference being that an additional aacA7 cassette was present in the first position of integron c. We identified a carrier patient (patient P14) who was transferred between the intensive care units (ICUs) of the Gyor and Mosonmagyaróvár hospitals, providing an epidemiological link between them. Another patient (patient P13) with no related clinical history was identified as a carrier on admission to the hospital, suggesting the presence of VIM-positive strains in the community. These results demonstrate the involvement of institutions from three towns in an outbreak of VIM-4-producing P. aeruginosa. Our observations and the available literature (2, 20, 23) underscore the role of human carriers and the hospital environment as potential reservoirs for MBL-producing P. aeruginosa strains.

Mating-out assays were performed with the isolates underlined in Table 1. These experiments did not result in transconjugants under the experimental conditions applied, suggesting that the integrons are located either on the chromosome or on nonconjugative plasmids (12, 15). While integrons a, c, and d have so far been reported only from Hungary, class 1 integrons carrying the same gene cassettes in their variable regions as integron b were also identified in Poland and Belgium (3, 7, 9; P. Bogaerts, H. Rodriguez, C. Bauraing, A. Deplano, Y. Glupczynski, and M. J. Struelens, Abstr. 15th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P929, 2006).

The rates of imipenem resistance among P. aeruginosa clinical isolates in Hungary rose from 5.4% in 1996 to 13% in 2005 (5). The prevalence of VIM-positive P. aeruginosa isolates was estimated in the epidemiological settings from which the isolates were collected. On the basis of our experimental results and data from the National Bacteriological Surveillance database, we estimate that in 2004, VIM producers constituted about 0.4% and 0.05% of the imipenem-resistant isolates and all P. aeruginosa clinical isolates, respectively. In 2005, these values rose to about 6.5% and 0.8%, respectively. The observed increase could mainly be attributed to the clonal spread of the serotype O11 VIM-4-positive P. aeruginosa isolates in northwest Hungary.

This is the first report of an outbreak caused by acquired MBL-producing pathogens in Hungary. In hospitals in Greece, Italy, Korea, and Colombia, the dissemination of integrons carrying the blaVIM-1, blaVIM-2, or blaVIM-8 gene mostly occurred in serotype O11 and O12 clones of P. aeruginosa (2, 6, 14, 20). These studies, as well as ours, indicate that serotype O11 and O12 multiresistant clones of P. aeruginosa (13) play an important role in the dissemination of blaVIM through clonal spread but that other mechanisms, such as horizontal transfer, are also involved (3, 16, 23).

Nucleotide sequence accession numbers. The sequences of blaVIM harboring integrons were deposited in GenBank under accession numbers AY702100 and DQ357197.


   ACKNOWLEDGMENTS
 
This work was supported by grant T-08/186/2001 of the Hungarian Scientific Council for Health.

We thank L. Keresztes, L. Kereszturi, A. Kovács, A. Kvarda, K. Pintér, and J. Szentandrássy for their help.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bacteriology, National Center for Epidemiology, 1097 Budapest, Gyáli út 2-6, Hungary. Phone: 36 1 476 11 18. Fax: 36 1 476 1234. E-mail: libischb{at}oek.antsz.hu. Back

{triangledown} Published ahead of print on 25 September 2006. Back


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Antimicrobial Agents and Chemotherapy, December 2006, p. 4220-4223, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00300-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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