Molecular Epidemiology of Acquired-Metallo--Lactamase-Producing Bacteria in Poland
http://www.100md.com
《抗菌试剂及化学方法》
ABSTRACT
We have analyzed 40 metallo--lactamase (MBL)-producing isolates of Pseudomonas aeruginosa (n = 38), Pseudomonas putida (n = 1), and Acinetobacter genospecies 3 (n = 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced -lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The blaVIM genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained blaVIM-2 and two of which contained blaVIM-4. The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.
INTRODUCTION
The class B metallo--lactamases (MBLs), which hydrolyze penicillins, cephalosporins, and carbapenems and are not inhibited by site-directed -lactam inhibitors (3), have become a problem with potentially disastrous consequences for therapy of bacterial infections in future (16, 34, 37). The acquired MBL variants first appeared at the end of the 1980s in Pseudomonas aeruginosa in Japan (35), and since the mid-1990s, they have been observed in many countries all over the world. So far P. aeruginosa has remained the major producer of these enzymes; however, their genes effectively diffuse into other gram-negative bacteria as well. Of the four evolutionary families of acquired MBLs discerned until now, the IMP- and VIM-type -lactamases occur most widely, with the latter ones clearly dominating in Europe. MBL genes usually reside within class 1 integrons of various compositions of gene cassettes, which may be located in either chromosomal or plasmid DNA (16, 34, 37). The growing prevalence of MBL producers in nosocomial bacterial populations has been attributed to their independent emergence, clonal spread, and/or horizontal transfer of MBL determinants (4, 6, 7, 14, 21, 23, 26, 27, 30, 31, 36).
The first report on MBL-producing bacteria in Poland appeared in 2003 and concerned a single P. aeruginosa isolate identified within the SENTRY surveillance project. It was recovered in 2001 in a hospital in Zabrze and carried the blaVIM-2 gene located in a unique class 1 integron together with the aminoglycoside resistance gene aacA4 (33). The next report described a group of P. aeruginosa isolates from a large pediatric center in Warsaw, identified in 1998 to 2001. They all carried another class 1 integron, with aacA4 and blaVIM-4 gene cassettes, which was very similar to a blaVIM-1-containing integron from Greece (20). In this study, we have analyzed a wider group of MBL-producing isolates from numerous Polish hospitals.
MATERIALS AND METHODS
Clinical isolates. The study was carried out on 40 imipenem-nonsusceptible clinical isolates, including 38 P. aeruginosa isolates, 1 Pseudomonas putida isolate, and 1 Acinetobacter genospecies 3 isolate, recovered in 2000 to 2004 in 17 hospitals in 12 Polish cities (Table 1 and Fig. 1). All but three isolates from 2001 to 2004 were identified as putative MBL producers during routine diagnostics in clinical microbiology laboratories and sent to the National Institute of Public Health (NIPH) in Warsaw for confirmation of the detection results. The laboratories were among those which in the end of 2001 or the beginning of 2002 started to perform disk synergy tests for MBL production (1), encouraged to do so by the NIPH and according to its recommendations (11). Three isolates from 2003 from hospitals in Bydgoszcz (center BGII) and Cracow (KR) were collected during the NIPH survey of antimicrobial susceptibility of P. aeruginosa in Poland (M. Gniadkowski et al., unpublished) (for details see Discussion). Four P. aeruginosa isolates from 2000 from one of hospitals in Wrocaw (WRII) were distinguished by the retrospective analysis of the collection of bacterial isolates maintained in this center. Groups of more than 1 isolate were identified in six hospitals, including WRII (10 isolates) and centers in Gdask (GD; six isolates) and Zabrze (ZA; six isolates). The isolates were recovered from a variety of clinical specimens, mostly urine (n = 13), sputum (n = 10), and blood (n = 6). Taxonomic identification was performed using the ATB ID32GN test (bioMerieux, Charbonnieres-les-Bains, France), and, in the case of the Acinetobacter isolate, it was supplemented by the genetic ARDRA analysis (32).
The P. aeruginosa isolate 2751 from the hospital in Zabrze was sent in 2001 by the NIPH to the coordinating center of the SENTRY Antimicrobial Surveillance Program and was subsequently analyzed by Walsh et al. under the SENTRY strain designation 81-11963A (33). It was included in this study as a positive control in MBL and blaVIM gene detection and for epidemiological purposes.
MBL detection. The screening for MBL production was carried out with Etest MBL strips (AB Biodisk, Solna, Sweden) and by two variants of the synergy disk test, with EDTA and 2-mercaptopropionic acid as MBL inhibitors (1). Additionally, MBL detection was confirmed by the assay for imipenem hydrolytic activity in bacterial sonicates, as described by Cardoso et al. (4), in a Power Wave X spectrophotometer (Bio-Tek Instruments, Winooski, VT).
Antimicrobial susceptibility testing. MICs of various antimicrobials were evaluated by the agar dilution method, according to the Clinical and Laboratory Standards Institute (CLSI [formerly NCCLS]) recommendations (5). In -lactam-inhibitor combinations, the constant concentrations of clavulanate and tazobactam were 2 and 4 μg/ml, respectively. P. aeruginosa ATCC 27853, Escherichia coli ATCC 25922, and E. coli ATCC 35218 were used as reference strains.
PFGE typing. The pulsed-field gel electrophoresis (PFGE) analysis was performed as described by Grundmann et al. (10), using the XbaI restriction enzyme (MBI Fermentas, Vilnius, Lithuania) and a contour-clamped homogeneous electric field DRIII PFGE system (Bio-Rad, Hercules, Calif.). The PFGE results were interpreted according to Tenover et al. (29).
PCR detection and identification of blaVIM-type genes. Total bacterial DNAs were purified with the Genomic DNA Prep Plus kit (A & A Biotechnology, Gdask, Poland) and screened by PCR for the presence of genes coding for VIM-type -lactamases (blaVIM genes). Two pairs of primers were used (Table 2). Primers VIM-1L and VIM-1R were specific for blaVIM-1-related genes (blaVIM-1, blaVIM-4, blaVIM-5, and blaVIM-11a), whereas primers VIM-2L and VIM-2R were designed to amplify blaVIM-2-related genes (blaVIM-2, blaVIM-3, blaVIM-6, blaVIM-8, blaVIM-9, blaVIM-10, and blaVIM-11b) (34). The PCR conditions used were those described previously (8).
For sequencing of the blaVIM coding regions, the VIM-1L/VIM-1R and VIM-2L/VIM-2R PCR products were purified with the QIAquick PCR purification kit (QIAGEN, Hilden, Germany). The amplicons were directly sequenced on both DNA strands using sets of blaVIM-4- or blaVIM-2-specific primers and an ABI PRISM 310 sequencer (Applied Biosystems, Foster City, Calif.).
PCR detection of integrons. The PCR detection of class 1 integrons was performed with primers INT1F and INT1R specific for the intI1 gene (Table 2) (12).
PCR-RFLP analysis and sequencing of integronic variable regions. Variable regions of class 1 integrons containing the blaVIM gene cassettes were studied by the PCR-restriction fragment length polymorphism (RFLP) approach. The regions were amplified in two parts, 5' and 3', with primers specific for blaVIM genes and integronic conserved segments (5'-CS or 3'-CS) in each pair (Table 2) (15). The 5' parts of the all regions were amplified with primers 5CS and VIM178C, whereas the 3' parts were amplified with primers VIM-1L or VIM-2L (for blaVIM-4- or blaVIM-2-containing regions, respectively) and primer 3CS. In the case of the P. aeruginosa isolates from centers in Wrocaw, Jelenia Gora, and Pozna, the reverse primer used was INT/3CS (specific for the qacE1 gene) (25). The resulting amplicons were digested with the MboI restriction endonuclease (MBI Fermentas) and electrophoresed in 2% agarose gels (NuSieve 3:1; Cambrex Bio Science, Rockland, ME).
Sequencing of the 5' and 3' parts of the integronic variable regions was started with primers used for amplification of these fragments and continued with consecutive primers designed according to the gradually accumulated sequence data.
Studies of the location of the blaVIM-containing integrons. Undigested total DNAs of the isolates were electrophoresed by PFGE, blotted onto a Hybond-N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom), and hybridized with a probe specific for blaVIM genes. The probe was the 0.8-kb PCR product, obtained with primers VIM-2L and VIM-2R and total DNA of isolate P. aeruginosa 188 (blaVIM-2) as a template. Following exposure, the probe was washed out from the membrane, which was then rehybridized with the 16S-23S rRNA gene probe, obtained in a PCR as previously described (8). Probe labeling, hybridization, and signal detection were performed with the ECL Random-Prime Labeling and Detection system (Amersham Pharmacia Biotech).
The imipenem resistance conjugal transfer was carried out by the filter-mating procedure (28), with P. aeruginosa PAO1161 (leu r–) (2) resistant to rifampin as a recipient. Transconjugants were selected on Trypticase soy agar (Oxoid, Basingstoke, United Kingdom) plates supplemented with rifampin (300 μg/ml; Polfa Tarchomin, Warsaw, Poland) and imipenem (8 μg/ml; Merck Sharp & Dohme, Rahway, N.J.).
Sequence data analysis. All sequence data determined in this work were analyzed with the use of the Lasergene Version 6 software (DNASTAR, Madison, WI).
Nucleotide sequence accession numbers. The nucleotide sequences of the integronic variable regions determined in this work will appear in the EMBL database under the following accession numbers: AM087404 for the isolate P. aeruginosa 188, AM087405 for P. aeruginosa 804, AM087406 for P. aeruginosa 1262, AM087407 for P. aeruginosa 1885, AM087408 for P. aeruginosa 1956, AM087409 for P. aeruginosa 1266, AM087410 for P. aeruginosa 1264, and AM087411 for P. aeruginosa 2611.
ESULTS
MBL detection. The imipenem-nonsusceptible isolates were tested for the presence of MBLs by several methods (Etest, disk synergy tests, and the spectrophotometric assay). All of the methods yielded positive results for all of the 40 study isolates.
PFGE typing. The 38 P. aeruginosa isolates were typed by PFGE. Eight different PFGE types, designated from A to H, were distinguished among the isolates (Table 1). Five of the types, A, B, C, D, and F, characterized more than one isolate, and all groups of several isolates from a single center were classified into a single PFGE type. Isolates of PFGE types A, B, and C were identified in several centers each, including hospitals in different cities, which, however, were always located close to each other, usually in specific geographic regions (Fig. 1). The PFGE types B and C were split further into four subtypes each.
Identification of blaVIM genes. All of the study isolates were found to be positive in PCRs for the presence of blaVIM genes. Four P. aeruginosa isolates of the PFGE subtype B4 from Gdask and a unique P. aeruginosa isolate from Katowice (PFGE type E) produced amplicons of the expected size with primers specific for the blaVIM-1-related genes. For all of the remaining isolates, the amplicons were yielded by PCR specific for blaVIM-2-related genes. DNA sequencing, carried out for isolates representing all PFGE types and subtypes, revealed that the amplicons were consistent with either blaVIM-4 or blaVIM-2 genes, respectively.
Detection of integrons. PCR specific for the integrase gene intI1 revealed the presence of class 1 integrons in all study isolates. Positive PCR results with the forward primer INT1F and reverse primer VIM-1R or VIM-2R indicated that the blaVIM-4 and blaVIM-2 genes resided inside this type of element (results not shown).
PCR-RFLP of the variable regions of the blaVIM-containing integrons. The PCR-RFLP analysis of the variable regions of the blaVIM-containing class 1 integrons was performed for all study isolates. It revealed the presence of eight different MboI restriction patterns of the amplified DNA, which corresponded to eight variants of the integronic gene cassette arrays (variants a to h). In general, the distribution of the integron polymorphs correlated well with the P. aeruginosa PFGE types and/or the geographic origins of all isolates (Table 1 and Fig. 1).
Structure of the cassette arrays with blaVIM genes. Eight isolates representing all of the integron polymorphs identified were selected for sequencing the entire variable regions (Table 1). The array structures comprised from one to four gene cassettes, and the genes with known functions besides blaVIM-2 or blaVIM-4 were exclusively those coding for aminoglycoside-modifying enzymes. Only polymorph h from P. aeruginosa from Cracow (isolate 1885) consisted of a sole blaVIM gene cassette, blaVIM-2, and over the entire sequenced fragment, it was identical to the integron In56 from P. aeruginosa COL-1 from France (22). The same blaVIM-2 cassette was also found in polymorphs b and c from the isolates from the East Pomerania region and in variant f from Mazovia (Fig. 1). Gene cassette arrays b and c (isolates 1262 and 1266, respectively) differed from each other only by the presence of the aacC1 cassette between blaVIM-2 and aphA15 in polymorph c. The aacC1 gene demonstrated five mutations (one amino acid substitution, Glu142Asp) when compared to aacC1 in the blaVIM-2-containing integron In58 from P. aeruginosa RON-1 from France (21). On the other hand, the aphA15 gene cassette, present both in variants b and c, was identical to that often observed in blaVIM-1- containing integrons in Italy (13, 26, 30). In the integron polymorph f (isolate 804), the blaVIM-2 cassette was accompanied by aadB, identical to that described in In34 (19) and a fused gene, aadA6/aadA10. Its 806-bp-long aadA6 part was almost identical to that from a blaVIM-4-containing integron from Greece (GenBank accession no. AY460181), whereas the 19-bp-long 3' end fully matched the aadA10 sequence from the integron in plasmid R388-R151 (18).
Sequencing of the a-type polymorph from P. aeruginosa 1956 from Zabrze (the Upper Silesia region) confirmed the structure obtained earlier by Walsh et al. for isolate 2751. blaVIM-2 was followed by the aacA4 gene cassette in this integron, and the 59-base element (59-be) of blaVIM-2 was unusually short (19 bp) (33). The aacA4 cassette was identical to that identified in In58 (21), and the two integrons might have been related to each other (33). Polymorph e from the isolates from Lower Silesia/Great Poland (isolate 188) contained the same genes but in the opposite configuration. The blaVIM-2 cassette was unique by the fact that its 59-be was almost completely deleted and the qacE1 gene directly followed the inverse core site. aacA4 was identical to those in In58 (21) and polymorph a. The same composition of gene cassettes was identified in In72 from a P. aeruginosa isolate from Italy (17); however, four point mutations in aacA4 and the 59-be deletion in blaVIM-2 rather excluded the close relatedness between polymorph e and In72.
Similarly to all but one of the blaVIM-2-containing integrons, the two variants with blaVIM-4 were found to be of unique cassette compositions. The blaVIM-4 cassettes were identical to each other and to that originally described by Pournaras et al. (24). The aacA4 gene cassette which followed blaVIM-4 in polymorph d from the isolates from East Pomerania (isolate 1264) was specific and contained a single mutation (amino acid substitution Asp164Val) compared to those in variants a and e and in In58 (21). Polymorph g from a single P. aeruginosa isolate (isolate 2611) from Katowice (Upper Silesia) possessed aacA7, aadA6, and orfD cassettes behind blaVIM-4. It differed from the integron identified in Greece only by the presence of aacA7 (GenBank accession no. AY460181).
Location of the blaVIM gene-containing integrons. The undigested total DNA of the isolates was separated by PFGE and hybridized separately with the blaVIM and rRNA gene probes. Each isolate was characterized by a DNA band of the same migration and the highest molecular weight (MW) observed which hybridized with the rRNA gene probe and most probably represented the chromosomal DNA. Some isolates produced additional DNA bands of faster migration, and these could correspond to plasmid molecules of highly diverse MWs. In the case of the Acinetobacter isolate from Upper Silesia (integron variant a) and all of the P. aeruginosa and P. putida isolates from Mazovia (integron variant f), the blaVIM probe hybridized only to the bands of faster electrophoretic migration. For all of the remaining isolates, the signal was detected at the putative chromosomal band (results not shown).
The representative isolates of Acinetobacter (isolate 446), P. aeruginosa (isolates 900, 1381, and 2248), and P. putida (isolate 2596) with the probable plasmidic location of the blaVIM-2-containing integrons were subjected to the imipenem resistance transfer experiment. Transconjugants with the MBL-positive phenotype were produced by the Mazovian isolates P. aeruginosa 900 and 1381 and P. putida 2596, all of which carried integron variant f.
Antimicrobial susceptibility. The MIC patterns that characterized the study isolates were similar to those usually observed for MBL producers (16, 34, 37) (Table 3). In general, the MICs of penicillins, cephalosporins, and carbapenems were significantly increased. Almost all of the isolates were resistant to gentamicin and nonsusceptible to tobramycin and ciprofloxacin.
anges of the -lactam MICs were very wide among the isolates, which was well exemplified by the MICs of piperacillin, ceftazidime, and imipenem (MICs, 8 to 512 μg/ml) or meropenem (MICs, 1 to 512 μg/ml). This diversity could even be observed within particular clones of P. aeruginosa with the same blaVIM integron variant, as it was the case of VIM-2-producing isolates of PFGE types B and C.
DISCUSSION
In 1996, a 6-month survey of antimicrobial susceptibility of P. aeruginosa clinical isolates was performed in 30 hospitals in 26 Polish cities, and it demonstrated that 11.6% and 8.6% of 674 isolates collected were nonsusceptible to imipenem and meropenem, respectively (9). The retrospective analysis revealed that none of these organisms produced MBL (Gniadkowski et al., unpublished). A similar study was conducted in 2003 in 26 centers in 23 cities, the majority of which (23 hospitals) were participants in the 1996 survey. The prevalences of nonsusceptibility to imipenem and meropenem among 621 study isolates were determined to be 15.5% and 12.9%, respectively (Gniadkowski et al., unpublished). Three isolates, included in this report, were identified as MBL producers (3.1% of the imipenem-nonsusceptible organisms). All of these observations suggested that resistance to carbapenems in nosocomial populations of P. aeruginosa in Poland has been increasing in recent years at a moderate rate and that MBLs have played a rather marginal role in it compared to the situation in other countries (34). Nevertheless, the occurrence of MBL producers in a growing number of hospitals as documented by Walsh et al. (33) and this work, and by Patzer et al. [20; J. A. Patzer, M. A. Toleman, A. Grzesik, D. Dzieranowska, and T. R. Walsh, Abstr. 15th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P412, 2005; Clin. Microbiol. Infect. 11(Suppl. 2):100, 2005], even if sporadic in the majority of cases, indicated clearly that MBLs have started to constitute a countrywide problem. (Only three of the hospitals from which the study isolates were derived participated in the P. aeruginosa surveys mentioned above).
Both the PFGE typing of the study P. aeruginosa isolates and the analysis of the variable regions of blaVIM-containing integrons revealed their specific geographic distribution, which indicated that MBL-mediated resistance determinants had emerged several times in separate areas of Poland. Of the eight different polymorphs of the integronic gene cassette arrays, two variants were most likely related to each other (b and c in P. aeruginosa in East Pomerania); therefore, the emergence of the whole set should be assigned to at least seven independent selection events in five regions. If the data collected by others (20; Patzer et al., 15th Eur. Congr. Clin. Microbiol. Infect. Dis.) are also considered, the number of such selection events in Poland may have exceeded the number of 10. The comparison of the gene cassette sequences and configurations with those observed in other countries suggested that most of these integrons were assembled in Poland from existing bacterial populations as opposed to being imported from foreign environments. The probable exception was polymorph h with the sole blaVIM-2 cassette, which was identical to the original blaVIM-2-containing integron In56 from France (22). It is also possible that polymorphs a and g had either common ancestors with or were directly derived from integrons identified in France (In58) (21, 33) and Greece (AY460181), respectively.
Some of the blaVIM integron variants have started to disseminate in local populations of gram-negative nonfermenters, mostly the blaVIM-2-containing variants a, b/c, and e and f, as well as polymorph d with blaVIM-4. The dissemination of variants a and f in the regions of Upper Silesia and Mazovia, respectively, may be attributed to both clonal spread of producer strains and the DNA horizontal transfer. The clonal spread was illustrated well by "fresh" outbreaks of P. aeruginosa in the hospitals in Pock (integron variant f) and Zabrze (variant a). On the other hand, the presence of variant f in P. aeruginosa isolates of three distinct PFGE types and in P. putida from four Mazovian hospitals (in Pock and Warsaw) most probably resulted from the plasmid transfer, as demonstrated by the results of the mating experiment and the fact that at least two of these isolates shared plasmids of the same size (results not shown). Similarly, the integron variant a could have been exchanged between the P. aeruginosa clone from Zabrze and Acinetobacter from Bielsko-Biaa, though only in the Acinetobacter isolate it was located on a plasmid. The combination of clonal spread and DNA horizontal transfer in dissemination of MBL producers has been documented several times until now, although usually at the level of a single center (14, 23). An essential role of the horizontal gene transfer in wider-scale epidemiology of MBLs has been either postulated or demonstrated: e.g., in Japan (27), Taiwan (36), and Italy (30).
The multicenter clonal dissemination occurred in the case of P. aeruginosa of PFGE types B (integron variants b/c or d) and C (variant e) in East Pomerania and Lower Silesia/Great Poland, respectively. In both cases, some level of diversity of PFGE patterns of the isolates indicated the microevolution commenced in the epidemic P. aeruginosa clones. The situation of the PFGE type B clone was complicated by the alternative presence of integrons of two different origins in its isolates, either with blaVIM-2 (b/c) or blaVIM-4 (d). All of the data suggested that the clone had been spread and diversified in hospitals of East Pomerania before the emergence of blaVIM-carrying integrons and that later it continued the dissemination. The clonal spread of MBL producers, though usually at the single-center level, has been reported in a number of papers so far and has been postulated to be the major mechanism of diffusion of these organisms (4, 6, 31). Recently, Riccio et al. described epidemiology of VIM-1-producing P. aeruginosa in Northern Italy, documenting independent introductions of blaVIM-1-containing integrons into the epidemic clonal complex of P. aeruginosa spread a priori in hospitals of the region (26).
Although the number of countries in which clinical isolates with acquired MBLs have been reported is high (16, 34), this work is one of the relatively few studies that show the molecular epidemiology of MBL-producing bacteria on a regional or countrywide scale (7, 26, 27, 30). The fact that it has consisted of five separate regional situations so far indicates the early stage of the dissemination of these organisms in Poland. Already striking are the number and diversity of the phenomena observed, such as the independent selection of several resistance determinants; their horizontal transmission, including the interspecies transfer, clonal spread of producer strains on a single center, and regional scale; parallel penetration of a bacterial clone by two different integron types with MBL genes; and evolution of an integron. This work may be a good starting point for the future precise monitoring of MBL producers in the country.
ACKNOWLEDGMENTS
We thank A. Bliniuk, K. Filczak, B. Findysz-Dylg, E. Gospodarek, B. Jaworska-Bach, J. Kdzierska, E. Kozera, D. Krawiecka, A. Mol, K. Pawlik, W. Piechota, W. wistun, and A. Wrobel who kindly provided the clinical isolates and B. Bukowska for her technical support. We are also thankful to G. Jagura-Burdzy for the P. aeruginosa PAO1161 strain and J. Empel for critical reading of the manuscript.
This work was partially financed by grant no. 6 PCRD LSHM-CT-2003-503-335 from the European Commission.
EFERENCES
Arakawa, Y., N. Shibata, K. Shibayama, H. Kurokawa, T. Yagi, H. Fujiwara, and M. Goto. 2000. Convenient test for screening metallo--lactamase-producing gram-negative bacteria by using thiol compounds. J. Clin. Microbiol. 38:40-43.
Bartosik, A. A., K. Lasocki, J. Mierzejewska, C. M. Thomas, and G. Jagura-Burdzy. 2004. ParB of Pseudomonas aeruginosa: interactions with its partner ParA and its target parS and specific effects on bacterial growth. J. Bacteriol. 186:6983-6998.
Bush, K., G. A. Jacoby, and A. A. Medeiros. 1995. A functional classification scheme for -lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-1233.
Cardoso, O., R. Leito, A. Figueiredo, J. C. Sousa, A. Duarte, and L. Peixe. 2002. Metallo--lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal. Microb. Drug Resist. 8:93-97.
Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing: 15th informational supplement, M100-S15. CLSI, Wayne, Pa.
Cornaglia, G., A. Mazzariol, L. Lauretti, G. M. Rossolini, and R. Fontana. 2000. Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-1, a novel transferable metallo--lactamase. Clin. Infect. Dis. 31:1119-1125.
Giakkoupi, P., G. Petrikkos, L. S. Tzouvelekis, S. Tsonas, The WHONET GREECE Study Group, N. J. Legakis, and A. C. Vatopoulos. 2003. Spread of integron-associated VIM-type metallo--lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals. J. Clin. Microbiol. 41:822-825.
Gniadkowski, M., I. Schneider, A. Paucha, R. Jungwirth, B. Mikiewicz, and A. Bauernfeind. 1998. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing -lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrob. Agents Chemother. 42:827-832.
Gniadkowski, M., A. Skoczyska, J. Fiett, K. Trzciski, and W. Hryniewicz. 1998. Susceptibility of Pseudomonas aeruginosa isolated from hospital infections to antibiotics. Pol. Merk. Lek. 30:346-350. (In Polish.)
Grundmann, H., C. Schneider, D. Hartung, F. D. Daschner, and T. L. Pitt. 1995. Discriminatory power of three DNA-based typing techniques for Pseudomonas aeruginosa. J. Clin. Microbiol. 33:528-534.
Hryniewicz, W., A. Sulikowska, K. Szczypa, J. Krzyszto-Russjan, and M. Gniadkowski. 2002. Recommendations for susceptibility testing to antimicrobial agents of selected bacterial species. Mikrobiologia Medycyna 32:12-39. (In Polish.)
Koeleman, J. G. M., J. Stoof, M. W. Van Der Bijl, C. M. J. E. Vandenbroucke- Grauls, and P. H. M. Savelkoul. 2001. Identification of epidemic strains of Acinetobacter baumannii by integrase gene PCR. J. Clin. Microbiol. 39:8-13.
Lauretti, L., M. L. Riccio, A. Mazzariol, G. Cornaglia, G. Amicosante, R. Fontana, and G. M. Rossolini. 1999. Cloning and characterization of blaVIM, a new integron-borne metallo--lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 43:1584-1590.
Lee, K., J. B. Lim, J. H. Yum, D. Yong, Y. Chong, J. M. Kim, and D. M. Livermore. 2002. blaVIM-2 cassette-containing novel integrons in metallo--lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital. Antimicrob. Agents Chemother. 46:1053-1058.
Levesque, C., L. Piche, C. Larose, and P. H. Roy. 1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39:185-191.
Nordmann, P., and L. Poirel. 2002. Emerging carbapenemases in Gram-negative aerobes. Clin. Microbiol. Infect. 8:321-331.
Pallecchi, L., M. L. Riccio, J. D. Docquier, R. Fontana, and G. M. Rossolini. 2001. Molecular heterogeneity of bla(VIM-2)-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-beta-lactamase. FEMS Microbiol. Lett. 195:145-150.
Partridge, S. R., C. M. Collis, and R. M. Hall. 2002. Class 1 integron containing a new gene cassette, aadA10, associated with Tn1404 from R151. Antimicrob. Agents Chemother. 46:2400-2408.
Partridge, S. R., and R. M. Hall. 2003. In34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob. Agents Chemother. 47:342-349.
Patzer, J., M. A. Toleman, L. M. Deshpande, W. Kamiska, D. Dzieranowska, P. M. Bennett, R. N. Jones, and T. R. Walsh. 2004. Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998-2001). J. Antimicrob. Chemother. 53:451-456.
Poirel, L., T. Lambert, S. Türkoglü, E. Ronco, J.-L. Gaillard, and P. Nordmann. 2001. Characterization of class 1 integrons from Pseudomonas aeruginosa that contain the blaVIM-2 carbapenem-hydrolyzing -lactamase gene and of two novel aminoglycoside resistance gene cassettes. Antimicrob. Agents Chemother. 45:546-552.
Poirel, L., T. Naas, D. Nicolas, L. Collet, S. Bellais, J.-D. Cavallo, and P. Nordmann. 2000. Characterization of VIM-2, a carbapenem-hydrolyzing metallo--lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. 44:891-897.
Pournaras, S., M. Maniati, E. Petinaki, L. S. Tzouvelekis, A. Tsakris, N. J. Legakis, and A. N. Maniatis. 2003. Hospital outbreak of multiple clones of Pseudomonas aeruginosa carrying the unrelated metallo--lactamase gene variants blaVIM-2 and blaVIM-4. J. Antimicrob. Chemother. 51:1409-1411.
Pournaras, S., A. Tsakris, M. Maniati, L. S. Tzouvelekis, and A. N. Maniatis. 2002. Novel variant (blaVIM-4) of the metallo--lactamase gene blaVIM-1 in a clinical strain of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 46:4026-4028
iccio, M. L., N. Franceschini, L. Boschi, B. Caravelli, G. Cornaglia, R. Fontana, G. Amicosante, and G. M. Rossolini. 2000. Characterization of the metallo--lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of blaIMP allelic variants carried by gene cassettes of different phylogeny. Antimicrob. Agents Chemother. 44:1229-1235.
iccio, M. L., L. Pallecchi, J.-D. Docquier, S. Cresti, M. R. Catania, L. Pagani, C. Lagatolla, G. Cornaglia, R. Fontana, and G. M. Rossolini. 2005. Clonal relatedness and conserved integron sequences in epidemiologically unrelated Pseudomonas aeruginosa strains producing the VIM-1 metallo--lactamase from different Italian hospitals. Antimicrob. Agents Chemother. 49:104-110
Senda, K., Y. Arakawa, K. Nakashima, H. Ito, S. Ichiyama, K. Shimokata, N. Kato, and M. Ohta. 1996. Multifocal outbreaks of metallo--lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum -lactams, including carbapenems. Antimicrob. Agents Chemother. 40:349-353.
Stuart-Keil, K. G., A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen. 1998. Plasmids responsible for horizontal transfer of naphthalene catabolism genes between bacteria at a coal tar-contaminated site are homologous to pDTG1 from Pseudomonas putida NCIB 9816-4. Appl. Environ. Microbiol. 64:3633-3640.
Tenover, F. C., R. D. Arbeit, V. R. Goering, P. A. Mickelsen, B. E. Murray, D. H. Pershing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.
Toleman, M. A., D. Biedenbach, D. M. Bennett, R. N. Jones, and T. R. Walsh. 2005. Italian metallo--lactamases: a national problem Report from the SENTRY Antimicrobial Surveillance Programme. J. Antimicrob. Chemother. 55:61-70.
Tsakris, A., S. Pournaras, N. Woodford, M.-F. I. Palepou, G. S. Babini, J. Douboyas, and D. M. Livermore. 2000. Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece. J. Clin. Microbiol. 38:1290-1292.
Vaneechoutte, M., L. Dijkshoorn, I. Tjernberg, A. Elaichouni, P. de Vos, G. Claeys, and G. Verschraegen. 1995. Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 33:11-15.
Walsh, T. R., M. A. Toleman, W. Hryniewicz, P. M. Bennett, and R. N. Jones. 2003. Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from the SENTRY Antimicrobial Surveillance Program. J. Antimicrob. Chemother. 52:116-119.
Walsh, T. R., M. A. Toleman, L. Poirel, and P. Nordmann. 2005. Metallo--lactamases: the quiet before the storm Clin. Microbiol. Rev. 18:306-325.
Watanabe, M., S. Iyobe, M. Inoue, and S. Mitsuhashi. 1991. Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35:147-151.
Yan, J.-J., P.-R. Hsueh, W.-C. Ko, K.-T. Luh, S.-H. Tsai, H.-M. Wu, and J.-J. Wu. 2001. Metallo--lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme. Antimicrob. Agents Chemother. 45:2224-2228.
Zhiyong, Z., L. Xiaoju, and G. Yanyu. 2003. Metallo--lactamases of non-fermenting Gram-negative bacteria. Rev. Med. Microbiol. 14:79-93.
National Institute of Public Health, 00-725 Warsaw, Poland
1 Department of Microbiology, Medical University in Wrocaw, 50-368 Wrocaw, Poland
2 Institute of Microbiology, Wrocaw University, 51-148 Wrocaw, Poland
3 Department of Clinical Microbiology, Public Hospital No. 1, 80-211 Gdask, Poland4(Janusz Fiett, Anna Barani)
We have analyzed 40 metallo--lactamase (MBL)-producing isolates of Pseudomonas aeruginosa (n = 38), Pseudomonas putida (n = 1), and Acinetobacter genospecies 3 (n = 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced -lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The blaVIM genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained blaVIM-2 and two of which contained blaVIM-4. The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.
INTRODUCTION
The class B metallo--lactamases (MBLs), which hydrolyze penicillins, cephalosporins, and carbapenems and are not inhibited by site-directed -lactam inhibitors (3), have become a problem with potentially disastrous consequences for therapy of bacterial infections in future (16, 34, 37). The acquired MBL variants first appeared at the end of the 1980s in Pseudomonas aeruginosa in Japan (35), and since the mid-1990s, they have been observed in many countries all over the world. So far P. aeruginosa has remained the major producer of these enzymes; however, their genes effectively diffuse into other gram-negative bacteria as well. Of the four evolutionary families of acquired MBLs discerned until now, the IMP- and VIM-type -lactamases occur most widely, with the latter ones clearly dominating in Europe. MBL genes usually reside within class 1 integrons of various compositions of gene cassettes, which may be located in either chromosomal or plasmid DNA (16, 34, 37). The growing prevalence of MBL producers in nosocomial bacterial populations has been attributed to their independent emergence, clonal spread, and/or horizontal transfer of MBL determinants (4, 6, 7, 14, 21, 23, 26, 27, 30, 31, 36).
The first report on MBL-producing bacteria in Poland appeared in 2003 and concerned a single P. aeruginosa isolate identified within the SENTRY surveillance project. It was recovered in 2001 in a hospital in Zabrze and carried the blaVIM-2 gene located in a unique class 1 integron together with the aminoglycoside resistance gene aacA4 (33). The next report described a group of P. aeruginosa isolates from a large pediatric center in Warsaw, identified in 1998 to 2001. They all carried another class 1 integron, with aacA4 and blaVIM-4 gene cassettes, which was very similar to a blaVIM-1-containing integron from Greece (20). In this study, we have analyzed a wider group of MBL-producing isolates from numerous Polish hospitals.
MATERIALS AND METHODS
Clinical isolates. The study was carried out on 40 imipenem-nonsusceptible clinical isolates, including 38 P. aeruginosa isolates, 1 Pseudomonas putida isolate, and 1 Acinetobacter genospecies 3 isolate, recovered in 2000 to 2004 in 17 hospitals in 12 Polish cities (Table 1 and Fig. 1). All but three isolates from 2001 to 2004 were identified as putative MBL producers during routine diagnostics in clinical microbiology laboratories and sent to the National Institute of Public Health (NIPH) in Warsaw for confirmation of the detection results. The laboratories were among those which in the end of 2001 or the beginning of 2002 started to perform disk synergy tests for MBL production (1), encouraged to do so by the NIPH and according to its recommendations (11). Three isolates from 2003 from hospitals in Bydgoszcz (center BGII) and Cracow (KR) were collected during the NIPH survey of antimicrobial susceptibility of P. aeruginosa in Poland (M. Gniadkowski et al., unpublished) (for details see Discussion). Four P. aeruginosa isolates from 2000 from one of hospitals in Wrocaw (WRII) were distinguished by the retrospective analysis of the collection of bacterial isolates maintained in this center. Groups of more than 1 isolate were identified in six hospitals, including WRII (10 isolates) and centers in Gdask (GD; six isolates) and Zabrze (ZA; six isolates). The isolates were recovered from a variety of clinical specimens, mostly urine (n = 13), sputum (n = 10), and blood (n = 6). Taxonomic identification was performed using the ATB ID32GN test (bioMerieux, Charbonnieres-les-Bains, France), and, in the case of the Acinetobacter isolate, it was supplemented by the genetic ARDRA analysis (32).
The P. aeruginosa isolate 2751 from the hospital in Zabrze was sent in 2001 by the NIPH to the coordinating center of the SENTRY Antimicrobial Surveillance Program and was subsequently analyzed by Walsh et al. under the SENTRY strain designation 81-11963A (33). It was included in this study as a positive control in MBL and blaVIM gene detection and for epidemiological purposes.
MBL detection. The screening for MBL production was carried out with Etest MBL strips (AB Biodisk, Solna, Sweden) and by two variants of the synergy disk test, with EDTA and 2-mercaptopropionic acid as MBL inhibitors (1). Additionally, MBL detection was confirmed by the assay for imipenem hydrolytic activity in bacterial sonicates, as described by Cardoso et al. (4), in a Power Wave X spectrophotometer (Bio-Tek Instruments, Winooski, VT).
Antimicrobial susceptibility testing. MICs of various antimicrobials were evaluated by the agar dilution method, according to the Clinical and Laboratory Standards Institute (CLSI [formerly NCCLS]) recommendations (5). In -lactam-inhibitor combinations, the constant concentrations of clavulanate and tazobactam were 2 and 4 μg/ml, respectively. P. aeruginosa ATCC 27853, Escherichia coli ATCC 25922, and E. coli ATCC 35218 were used as reference strains.
PFGE typing. The pulsed-field gel electrophoresis (PFGE) analysis was performed as described by Grundmann et al. (10), using the XbaI restriction enzyme (MBI Fermentas, Vilnius, Lithuania) and a contour-clamped homogeneous electric field DRIII PFGE system (Bio-Rad, Hercules, Calif.). The PFGE results were interpreted according to Tenover et al. (29).
PCR detection and identification of blaVIM-type genes. Total bacterial DNAs were purified with the Genomic DNA Prep Plus kit (A & A Biotechnology, Gdask, Poland) and screened by PCR for the presence of genes coding for VIM-type -lactamases (blaVIM genes). Two pairs of primers were used (Table 2). Primers VIM-1L and VIM-1R were specific for blaVIM-1-related genes (blaVIM-1, blaVIM-4, blaVIM-5, and blaVIM-11a), whereas primers VIM-2L and VIM-2R were designed to amplify blaVIM-2-related genes (blaVIM-2, blaVIM-3, blaVIM-6, blaVIM-8, blaVIM-9, blaVIM-10, and blaVIM-11b) (34). The PCR conditions used were those described previously (8).
For sequencing of the blaVIM coding regions, the VIM-1L/VIM-1R and VIM-2L/VIM-2R PCR products were purified with the QIAquick PCR purification kit (QIAGEN, Hilden, Germany). The amplicons were directly sequenced on both DNA strands using sets of blaVIM-4- or blaVIM-2-specific primers and an ABI PRISM 310 sequencer (Applied Biosystems, Foster City, Calif.).
PCR detection of integrons. The PCR detection of class 1 integrons was performed with primers INT1F and INT1R specific for the intI1 gene (Table 2) (12).
PCR-RFLP analysis and sequencing of integronic variable regions. Variable regions of class 1 integrons containing the blaVIM gene cassettes were studied by the PCR-restriction fragment length polymorphism (RFLP) approach. The regions were amplified in two parts, 5' and 3', with primers specific for blaVIM genes and integronic conserved segments (5'-CS or 3'-CS) in each pair (Table 2) (15). The 5' parts of the all regions were amplified with primers 5CS and VIM178C, whereas the 3' parts were amplified with primers VIM-1L or VIM-2L (for blaVIM-4- or blaVIM-2-containing regions, respectively) and primer 3CS. In the case of the P. aeruginosa isolates from centers in Wrocaw, Jelenia Gora, and Pozna, the reverse primer used was INT/3CS (specific for the qacE1 gene) (25). The resulting amplicons were digested with the MboI restriction endonuclease (MBI Fermentas) and electrophoresed in 2% agarose gels (NuSieve 3:1; Cambrex Bio Science, Rockland, ME).
Sequencing of the 5' and 3' parts of the integronic variable regions was started with primers used for amplification of these fragments and continued with consecutive primers designed according to the gradually accumulated sequence data.
Studies of the location of the blaVIM-containing integrons. Undigested total DNAs of the isolates were electrophoresed by PFGE, blotted onto a Hybond-N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom), and hybridized with a probe specific for blaVIM genes. The probe was the 0.8-kb PCR product, obtained with primers VIM-2L and VIM-2R and total DNA of isolate P. aeruginosa 188 (blaVIM-2) as a template. Following exposure, the probe was washed out from the membrane, which was then rehybridized with the 16S-23S rRNA gene probe, obtained in a PCR as previously described (8). Probe labeling, hybridization, and signal detection were performed with the ECL Random-Prime Labeling and Detection system (Amersham Pharmacia Biotech).
The imipenem resistance conjugal transfer was carried out by the filter-mating procedure (28), with P. aeruginosa PAO1161 (leu r–) (2) resistant to rifampin as a recipient. Transconjugants were selected on Trypticase soy agar (Oxoid, Basingstoke, United Kingdom) plates supplemented with rifampin (300 μg/ml; Polfa Tarchomin, Warsaw, Poland) and imipenem (8 μg/ml; Merck Sharp & Dohme, Rahway, N.J.).
Sequence data analysis. All sequence data determined in this work were analyzed with the use of the Lasergene Version 6 software (DNASTAR, Madison, WI).
Nucleotide sequence accession numbers. The nucleotide sequences of the integronic variable regions determined in this work will appear in the EMBL database under the following accession numbers: AM087404 for the isolate P. aeruginosa 188, AM087405 for P. aeruginosa 804, AM087406 for P. aeruginosa 1262, AM087407 for P. aeruginosa 1885, AM087408 for P. aeruginosa 1956, AM087409 for P. aeruginosa 1266, AM087410 for P. aeruginosa 1264, and AM087411 for P. aeruginosa 2611.
ESULTS
MBL detection. The imipenem-nonsusceptible isolates were tested for the presence of MBLs by several methods (Etest, disk synergy tests, and the spectrophotometric assay). All of the methods yielded positive results for all of the 40 study isolates.
PFGE typing. The 38 P. aeruginosa isolates were typed by PFGE. Eight different PFGE types, designated from A to H, were distinguished among the isolates (Table 1). Five of the types, A, B, C, D, and F, characterized more than one isolate, and all groups of several isolates from a single center were classified into a single PFGE type. Isolates of PFGE types A, B, and C were identified in several centers each, including hospitals in different cities, which, however, were always located close to each other, usually in specific geographic regions (Fig. 1). The PFGE types B and C were split further into four subtypes each.
Identification of blaVIM genes. All of the study isolates were found to be positive in PCRs for the presence of blaVIM genes. Four P. aeruginosa isolates of the PFGE subtype B4 from Gdask and a unique P. aeruginosa isolate from Katowice (PFGE type E) produced amplicons of the expected size with primers specific for the blaVIM-1-related genes. For all of the remaining isolates, the amplicons were yielded by PCR specific for blaVIM-2-related genes. DNA sequencing, carried out for isolates representing all PFGE types and subtypes, revealed that the amplicons were consistent with either blaVIM-4 or blaVIM-2 genes, respectively.
Detection of integrons. PCR specific for the integrase gene intI1 revealed the presence of class 1 integrons in all study isolates. Positive PCR results with the forward primer INT1F and reverse primer VIM-1R or VIM-2R indicated that the blaVIM-4 and blaVIM-2 genes resided inside this type of element (results not shown).
PCR-RFLP of the variable regions of the blaVIM-containing integrons. The PCR-RFLP analysis of the variable regions of the blaVIM-containing class 1 integrons was performed for all study isolates. It revealed the presence of eight different MboI restriction patterns of the amplified DNA, which corresponded to eight variants of the integronic gene cassette arrays (variants a to h). In general, the distribution of the integron polymorphs correlated well with the P. aeruginosa PFGE types and/or the geographic origins of all isolates (Table 1 and Fig. 1).
Structure of the cassette arrays with blaVIM genes. Eight isolates representing all of the integron polymorphs identified were selected for sequencing the entire variable regions (Table 1). The array structures comprised from one to four gene cassettes, and the genes with known functions besides blaVIM-2 or blaVIM-4 were exclusively those coding for aminoglycoside-modifying enzymes. Only polymorph h from P. aeruginosa from Cracow (isolate 1885) consisted of a sole blaVIM gene cassette, blaVIM-2, and over the entire sequenced fragment, it was identical to the integron In56 from P. aeruginosa COL-1 from France (22). The same blaVIM-2 cassette was also found in polymorphs b and c from the isolates from the East Pomerania region and in variant f from Mazovia (Fig. 1). Gene cassette arrays b and c (isolates 1262 and 1266, respectively) differed from each other only by the presence of the aacC1 cassette between blaVIM-2 and aphA15 in polymorph c. The aacC1 gene demonstrated five mutations (one amino acid substitution, Glu142Asp) when compared to aacC1 in the blaVIM-2-containing integron In58 from P. aeruginosa RON-1 from France (21). On the other hand, the aphA15 gene cassette, present both in variants b and c, was identical to that often observed in blaVIM-1- containing integrons in Italy (13, 26, 30). In the integron polymorph f (isolate 804), the blaVIM-2 cassette was accompanied by aadB, identical to that described in In34 (19) and a fused gene, aadA6/aadA10. Its 806-bp-long aadA6 part was almost identical to that from a blaVIM-4-containing integron from Greece (GenBank accession no. AY460181), whereas the 19-bp-long 3' end fully matched the aadA10 sequence from the integron in plasmid R388-R151 (18).
Sequencing of the a-type polymorph from P. aeruginosa 1956 from Zabrze (the Upper Silesia region) confirmed the structure obtained earlier by Walsh et al. for isolate 2751. blaVIM-2 was followed by the aacA4 gene cassette in this integron, and the 59-base element (59-be) of blaVIM-2 was unusually short (19 bp) (33). The aacA4 cassette was identical to that identified in In58 (21), and the two integrons might have been related to each other (33). Polymorph e from the isolates from Lower Silesia/Great Poland (isolate 188) contained the same genes but in the opposite configuration. The blaVIM-2 cassette was unique by the fact that its 59-be was almost completely deleted and the qacE1 gene directly followed the inverse core site. aacA4 was identical to those in In58 (21) and polymorph a. The same composition of gene cassettes was identified in In72 from a P. aeruginosa isolate from Italy (17); however, four point mutations in aacA4 and the 59-be deletion in blaVIM-2 rather excluded the close relatedness between polymorph e and In72.
Similarly to all but one of the blaVIM-2-containing integrons, the two variants with blaVIM-4 were found to be of unique cassette compositions. The blaVIM-4 cassettes were identical to each other and to that originally described by Pournaras et al. (24). The aacA4 gene cassette which followed blaVIM-4 in polymorph d from the isolates from East Pomerania (isolate 1264) was specific and contained a single mutation (amino acid substitution Asp164Val) compared to those in variants a and e and in In58 (21). Polymorph g from a single P. aeruginosa isolate (isolate 2611) from Katowice (Upper Silesia) possessed aacA7, aadA6, and orfD cassettes behind blaVIM-4. It differed from the integron identified in Greece only by the presence of aacA7 (GenBank accession no. AY460181).
Location of the blaVIM gene-containing integrons. The undigested total DNA of the isolates was separated by PFGE and hybridized separately with the blaVIM and rRNA gene probes. Each isolate was characterized by a DNA band of the same migration and the highest molecular weight (MW) observed which hybridized with the rRNA gene probe and most probably represented the chromosomal DNA. Some isolates produced additional DNA bands of faster migration, and these could correspond to plasmid molecules of highly diverse MWs. In the case of the Acinetobacter isolate from Upper Silesia (integron variant a) and all of the P. aeruginosa and P. putida isolates from Mazovia (integron variant f), the blaVIM probe hybridized only to the bands of faster electrophoretic migration. For all of the remaining isolates, the signal was detected at the putative chromosomal band (results not shown).
The representative isolates of Acinetobacter (isolate 446), P. aeruginosa (isolates 900, 1381, and 2248), and P. putida (isolate 2596) with the probable plasmidic location of the blaVIM-2-containing integrons were subjected to the imipenem resistance transfer experiment. Transconjugants with the MBL-positive phenotype were produced by the Mazovian isolates P. aeruginosa 900 and 1381 and P. putida 2596, all of which carried integron variant f.
Antimicrobial susceptibility. The MIC patterns that characterized the study isolates were similar to those usually observed for MBL producers (16, 34, 37) (Table 3). In general, the MICs of penicillins, cephalosporins, and carbapenems were significantly increased. Almost all of the isolates were resistant to gentamicin and nonsusceptible to tobramycin and ciprofloxacin.
anges of the -lactam MICs were very wide among the isolates, which was well exemplified by the MICs of piperacillin, ceftazidime, and imipenem (MICs, 8 to 512 μg/ml) or meropenem (MICs, 1 to 512 μg/ml). This diversity could even be observed within particular clones of P. aeruginosa with the same blaVIM integron variant, as it was the case of VIM-2-producing isolates of PFGE types B and C.
DISCUSSION
In 1996, a 6-month survey of antimicrobial susceptibility of P. aeruginosa clinical isolates was performed in 30 hospitals in 26 Polish cities, and it demonstrated that 11.6% and 8.6% of 674 isolates collected were nonsusceptible to imipenem and meropenem, respectively (9). The retrospective analysis revealed that none of these organisms produced MBL (Gniadkowski et al., unpublished). A similar study was conducted in 2003 in 26 centers in 23 cities, the majority of which (23 hospitals) were participants in the 1996 survey. The prevalences of nonsusceptibility to imipenem and meropenem among 621 study isolates were determined to be 15.5% and 12.9%, respectively (Gniadkowski et al., unpublished). Three isolates, included in this report, were identified as MBL producers (3.1% of the imipenem-nonsusceptible organisms). All of these observations suggested that resistance to carbapenems in nosocomial populations of P. aeruginosa in Poland has been increasing in recent years at a moderate rate and that MBLs have played a rather marginal role in it compared to the situation in other countries (34). Nevertheless, the occurrence of MBL producers in a growing number of hospitals as documented by Walsh et al. (33) and this work, and by Patzer et al. [20; J. A. Patzer, M. A. Toleman, A. Grzesik, D. Dzieranowska, and T. R. Walsh, Abstr. 15th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P412, 2005; Clin. Microbiol. Infect. 11(Suppl. 2):100, 2005], even if sporadic in the majority of cases, indicated clearly that MBLs have started to constitute a countrywide problem. (Only three of the hospitals from which the study isolates were derived participated in the P. aeruginosa surveys mentioned above).
Both the PFGE typing of the study P. aeruginosa isolates and the analysis of the variable regions of blaVIM-containing integrons revealed their specific geographic distribution, which indicated that MBL-mediated resistance determinants had emerged several times in separate areas of Poland. Of the eight different polymorphs of the integronic gene cassette arrays, two variants were most likely related to each other (b and c in P. aeruginosa in East Pomerania); therefore, the emergence of the whole set should be assigned to at least seven independent selection events in five regions. If the data collected by others (20; Patzer et al., 15th Eur. Congr. Clin. Microbiol. Infect. Dis.) are also considered, the number of such selection events in Poland may have exceeded the number of 10. The comparison of the gene cassette sequences and configurations with those observed in other countries suggested that most of these integrons were assembled in Poland from existing bacterial populations as opposed to being imported from foreign environments. The probable exception was polymorph h with the sole blaVIM-2 cassette, which was identical to the original blaVIM-2-containing integron In56 from France (22). It is also possible that polymorphs a and g had either common ancestors with or were directly derived from integrons identified in France (In58) (21, 33) and Greece (AY460181), respectively.
Some of the blaVIM integron variants have started to disseminate in local populations of gram-negative nonfermenters, mostly the blaVIM-2-containing variants a, b/c, and e and f, as well as polymorph d with blaVIM-4. The dissemination of variants a and f in the regions of Upper Silesia and Mazovia, respectively, may be attributed to both clonal spread of producer strains and the DNA horizontal transfer. The clonal spread was illustrated well by "fresh" outbreaks of P. aeruginosa in the hospitals in Pock (integron variant f) and Zabrze (variant a). On the other hand, the presence of variant f in P. aeruginosa isolates of three distinct PFGE types and in P. putida from four Mazovian hospitals (in Pock and Warsaw) most probably resulted from the plasmid transfer, as demonstrated by the results of the mating experiment and the fact that at least two of these isolates shared plasmids of the same size (results not shown). Similarly, the integron variant a could have been exchanged between the P. aeruginosa clone from Zabrze and Acinetobacter from Bielsko-Biaa, though only in the Acinetobacter isolate it was located on a plasmid. The combination of clonal spread and DNA horizontal transfer in dissemination of MBL producers has been documented several times until now, although usually at the level of a single center (14, 23). An essential role of the horizontal gene transfer in wider-scale epidemiology of MBLs has been either postulated or demonstrated: e.g., in Japan (27), Taiwan (36), and Italy (30).
The multicenter clonal dissemination occurred in the case of P. aeruginosa of PFGE types B (integron variants b/c or d) and C (variant e) in East Pomerania and Lower Silesia/Great Poland, respectively. In both cases, some level of diversity of PFGE patterns of the isolates indicated the microevolution commenced in the epidemic P. aeruginosa clones. The situation of the PFGE type B clone was complicated by the alternative presence of integrons of two different origins in its isolates, either with blaVIM-2 (b/c) or blaVIM-4 (d). All of the data suggested that the clone had been spread and diversified in hospitals of East Pomerania before the emergence of blaVIM-carrying integrons and that later it continued the dissemination. The clonal spread of MBL producers, though usually at the single-center level, has been reported in a number of papers so far and has been postulated to be the major mechanism of diffusion of these organisms (4, 6, 31). Recently, Riccio et al. described epidemiology of VIM-1-producing P. aeruginosa in Northern Italy, documenting independent introductions of blaVIM-1-containing integrons into the epidemic clonal complex of P. aeruginosa spread a priori in hospitals of the region (26).
Although the number of countries in which clinical isolates with acquired MBLs have been reported is high (16, 34), this work is one of the relatively few studies that show the molecular epidemiology of MBL-producing bacteria on a regional or countrywide scale (7, 26, 27, 30). The fact that it has consisted of five separate regional situations so far indicates the early stage of the dissemination of these organisms in Poland. Already striking are the number and diversity of the phenomena observed, such as the independent selection of several resistance determinants; their horizontal transmission, including the interspecies transfer, clonal spread of producer strains on a single center, and regional scale; parallel penetration of a bacterial clone by two different integron types with MBL genes; and evolution of an integron. This work may be a good starting point for the future precise monitoring of MBL producers in the country.
ACKNOWLEDGMENTS
We thank A. Bliniuk, K. Filczak, B. Findysz-Dylg, E. Gospodarek, B. Jaworska-Bach, J. Kdzierska, E. Kozera, D. Krawiecka, A. Mol, K. Pawlik, W. Piechota, W. wistun, and A. Wrobel who kindly provided the clinical isolates and B. Bukowska for her technical support. We are also thankful to G. Jagura-Burdzy for the P. aeruginosa PAO1161 strain and J. Empel for critical reading of the manuscript.
This work was partially financed by grant no. 6 PCRD LSHM-CT-2003-503-335 from the European Commission.
EFERENCES
Arakawa, Y., N. Shibata, K. Shibayama, H. Kurokawa, T. Yagi, H. Fujiwara, and M. Goto. 2000. Convenient test for screening metallo--lactamase-producing gram-negative bacteria by using thiol compounds. J. Clin. Microbiol. 38:40-43.
Bartosik, A. A., K. Lasocki, J. Mierzejewska, C. M. Thomas, and G. Jagura-Burdzy. 2004. ParB of Pseudomonas aeruginosa: interactions with its partner ParA and its target parS and specific effects on bacterial growth. J. Bacteriol. 186:6983-6998.
Bush, K., G. A. Jacoby, and A. A. Medeiros. 1995. A functional classification scheme for -lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-1233.
Cardoso, O., R. Leito, A. Figueiredo, J. C. Sousa, A. Duarte, and L. Peixe. 2002. Metallo--lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal. Microb. Drug Resist. 8:93-97.
Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing: 15th informational supplement, M100-S15. CLSI, Wayne, Pa.
Cornaglia, G., A. Mazzariol, L. Lauretti, G. M. Rossolini, and R. Fontana. 2000. Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-1, a novel transferable metallo--lactamase. Clin. Infect. Dis. 31:1119-1125.
Giakkoupi, P., G. Petrikkos, L. S. Tzouvelekis, S. Tsonas, The WHONET GREECE Study Group, N. J. Legakis, and A. C. Vatopoulos. 2003. Spread of integron-associated VIM-type metallo--lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals. J. Clin. Microbiol. 41:822-825.
Gniadkowski, M., I. Schneider, A. Paucha, R. Jungwirth, B. Mikiewicz, and A. Bauernfeind. 1998. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing -lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrob. Agents Chemother. 42:827-832.
Gniadkowski, M., A. Skoczyska, J. Fiett, K. Trzciski, and W. Hryniewicz. 1998. Susceptibility of Pseudomonas aeruginosa isolated from hospital infections to antibiotics. Pol. Merk. Lek. 30:346-350. (In Polish.)
Grundmann, H., C. Schneider, D. Hartung, F. D. Daschner, and T. L. Pitt. 1995. Discriminatory power of three DNA-based typing techniques for Pseudomonas aeruginosa. J. Clin. Microbiol. 33:528-534.
Hryniewicz, W., A. Sulikowska, K. Szczypa, J. Krzyszto-Russjan, and M. Gniadkowski. 2002. Recommendations for susceptibility testing to antimicrobial agents of selected bacterial species. Mikrobiologia Medycyna 32:12-39. (In Polish.)
Koeleman, J. G. M., J. Stoof, M. W. Van Der Bijl, C. M. J. E. Vandenbroucke- Grauls, and P. H. M. Savelkoul. 2001. Identification of epidemic strains of Acinetobacter baumannii by integrase gene PCR. J. Clin. Microbiol. 39:8-13.
Lauretti, L., M. L. Riccio, A. Mazzariol, G. Cornaglia, G. Amicosante, R. Fontana, and G. M. Rossolini. 1999. Cloning and characterization of blaVIM, a new integron-borne metallo--lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 43:1584-1590.
Lee, K., J. B. Lim, J. H. Yum, D. Yong, Y. Chong, J. M. Kim, and D. M. Livermore. 2002. blaVIM-2 cassette-containing novel integrons in metallo--lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital. Antimicrob. Agents Chemother. 46:1053-1058.
Levesque, C., L. Piche, C. Larose, and P. H. Roy. 1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39:185-191.
Nordmann, P., and L. Poirel. 2002. Emerging carbapenemases in Gram-negative aerobes. Clin. Microbiol. Infect. 8:321-331.
Pallecchi, L., M. L. Riccio, J. D. Docquier, R. Fontana, and G. M. Rossolini. 2001. Molecular heterogeneity of bla(VIM-2)-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-beta-lactamase. FEMS Microbiol. Lett. 195:145-150.
Partridge, S. R., C. M. Collis, and R. M. Hall. 2002. Class 1 integron containing a new gene cassette, aadA10, associated with Tn1404 from R151. Antimicrob. Agents Chemother. 46:2400-2408.
Partridge, S. R., and R. M. Hall. 2003. In34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob. Agents Chemother. 47:342-349.
Patzer, J., M. A. Toleman, L. M. Deshpande, W. Kamiska, D. Dzieranowska, P. M. Bennett, R. N. Jones, and T. R. Walsh. 2004. Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998-2001). J. Antimicrob. Chemother. 53:451-456.
Poirel, L., T. Lambert, S. Türkoglü, E. Ronco, J.-L. Gaillard, and P. Nordmann. 2001. Characterization of class 1 integrons from Pseudomonas aeruginosa that contain the blaVIM-2 carbapenem-hydrolyzing -lactamase gene and of two novel aminoglycoside resistance gene cassettes. Antimicrob. Agents Chemother. 45:546-552.
Poirel, L., T. Naas, D. Nicolas, L. Collet, S. Bellais, J.-D. Cavallo, and P. Nordmann. 2000. Characterization of VIM-2, a carbapenem-hydrolyzing metallo--lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. 44:891-897.
Pournaras, S., M. Maniati, E. Petinaki, L. S. Tzouvelekis, A. Tsakris, N. J. Legakis, and A. N. Maniatis. 2003. Hospital outbreak of multiple clones of Pseudomonas aeruginosa carrying the unrelated metallo--lactamase gene variants blaVIM-2 and blaVIM-4. J. Antimicrob. Chemother. 51:1409-1411.
Pournaras, S., A. Tsakris, M. Maniati, L. S. Tzouvelekis, and A. N. Maniatis. 2002. Novel variant (blaVIM-4) of the metallo--lactamase gene blaVIM-1 in a clinical strain of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 46:4026-4028
iccio, M. L., N. Franceschini, L. Boschi, B. Caravelli, G. Cornaglia, R. Fontana, G. Amicosante, and G. M. Rossolini. 2000. Characterization of the metallo--lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of blaIMP allelic variants carried by gene cassettes of different phylogeny. Antimicrob. Agents Chemother. 44:1229-1235.
iccio, M. L., L. Pallecchi, J.-D. Docquier, S. Cresti, M. R. Catania, L. Pagani, C. Lagatolla, G. Cornaglia, R. Fontana, and G. M. Rossolini. 2005. Clonal relatedness and conserved integron sequences in epidemiologically unrelated Pseudomonas aeruginosa strains producing the VIM-1 metallo--lactamase from different Italian hospitals. Antimicrob. Agents Chemother. 49:104-110
Senda, K., Y. Arakawa, K. Nakashima, H. Ito, S. Ichiyama, K. Shimokata, N. Kato, and M. Ohta. 1996. Multifocal outbreaks of metallo--lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum -lactams, including carbapenems. Antimicrob. Agents Chemother. 40:349-353.
Stuart-Keil, K. G., A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen. 1998. Plasmids responsible for horizontal transfer of naphthalene catabolism genes between bacteria at a coal tar-contaminated site are homologous to pDTG1 from Pseudomonas putida NCIB 9816-4. Appl. Environ. Microbiol. 64:3633-3640.
Tenover, F. C., R. D. Arbeit, V. R. Goering, P. A. Mickelsen, B. E. Murray, D. H. Pershing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.
Toleman, M. A., D. Biedenbach, D. M. Bennett, R. N. Jones, and T. R. Walsh. 2005. Italian metallo--lactamases: a national problem Report from the SENTRY Antimicrobial Surveillance Programme. J. Antimicrob. Chemother. 55:61-70.
Tsakris, A., S. Pournaras, N. Woodford, M.-F. I. Palepou, G. S. Babini, J. Douboyas, and D. M. Livermore. 2000. Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece. J. Clin. Microbiol. 38:1290-1292.
Vaneechoutte, M., L. Dijkshoorn, I. Tjernberg, A. Elaichouni, P. de Vos, G. Claeys, and G. Verschraegen. 1995. Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 33:11-15.
Walsh, T. R., M. A. Toleman, W. Hryniewicz, P. M. Bennett, and R. N. Jones. 2003. Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from the SENTRY Antimicrobial Surveillance Program. J. Antimicrob. Chemother. 52:116-119.
Walsh, T. R., M. A. Toleman, L. Poirel, and P. Nordmann. 2005. Metallo--lactamases: the quiet before the storm Clin. Microbiol. Rev. 18:306-325.
Watanabe, M., S. Iyobe, M. Inoue, and S. Mitsuhashi. 1991. Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35:147-151.
Yan, J.-J., P.-R. Hsueh, W.-C. Ko, K.-T. Luh, S.-H. Tsai, H.-M. Wu, and J.-J. Wu. 2001. Metallo--lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme. Antimicrob. Agents Chemother. 45:2224-2228.
Zhiyong, Z., L. Xiaoju, and G. Yanyu. 2003. Metallo--lactamases of non-fermenting Gram-negative bacteria. Rev. Med. Microbiol. 14:79-93.
National Institute of Public Health, 00-725 Warsaw, Poland
1 Department of Microbiology, Medical University in Wrocaw, 50-368 Wrocaw, Poland
2 Institute of Microbiology, Wrocaw University, 51-148 Wrocaw, Poland
3 Department of Clinical Microbiology, Public Hospital No. 1, 80-211 Gdask, Poland4(Janusz Fiett, Anna Barani)