Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998–2001)

Jan Patzer1, Mark A. Toleman2,*, Lalitagauri M. Deshpande3, Wanda Kaminska1, Danuta Dzierzanowska1, Peter M. Bennett2, Ronald N. Jones3 and Timothy R. Walsh2

1 The Children’s Memorial Health Institute, Warsaw, Poland; 2 Department of Pathology and Microbiology, The Medical Building, University Walk, The University of Bristol, Bristol BS8 1TD, UK; 3 The Jones Group, JMI Laboratories, North Liberty, Iowa, USA

Received 10 October 2003; returned 4 November 2003; revised 1 December 2003; accepted 1 December 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: During 1997–2001, 151 isolates of imipenem-resistant Pseudomonas aeruginosa were obtained from clinical specimens taken from children hospitalized in Warsaw, Poland. These strains were investigated further to determine the mechanism of resistance.

Methods: The strains were analysed by a combination of genotyping and PCR-based strategies.

Results: Eleven of these strains were found to contain the metallo-ß-lactamase (MßL) gene blaVIM-4. The first strain appeared in 1998, and P. aeruginosa strains harbouring this MßL have become endemic in this hospital since then. All P. aeruginosa strains belonged to serotype O:6, and PFGE analysis revealed four different patterns and three sub-types. All 11 MßL-producing strains contained an identical class 1 integron with the usual 5' and 3' conserved sequences. The integron included two resistance cassettes, aacA4 in the first position and the blaVIM-4 cassette in the second position. The blaVIM-4 gene included an unusual direct repeat of 169 bp of the 3' portion of the blaVIM-4 gene.

Conclusions: An unusual blaVIM-4 MßL has become endemic in P. aeruginosa isolates infecting Polish children hospitalized on surgical wards. The formation of this unusual blaVIM-4 gene cassette could be explained by a mechanism involving deletion of a segment of an ancestral tandem repeat of blaVIM-4 via slipped strand replication, mediated by a combination of polymerase and integrase.

Keywords: metallo-ß-lactamases, serotypes, PFGE, ß-lactams, carbapenems


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomial infections, particularly in immunocompromised patients.1 Included in this broad-risk category are neonates and children who have previously received antimicrobial agents and those with primary or secondary immunodeficiencies. P. aeruginosa is characterized by inherent resistances to a wide variety of antimicrobials. Its resistance to anti-pseudomonal ß-lactams, advanced-generation cephalosporins, monobactams and carbapenems is also an increasing clinical problem.2

Carbapenems, mainly imipenem and meropenem, are potent agents for the treatment of infections due to multidrug-resistant (MDR) P. aeruginosa. However, the prevalence of carbapenem-resistant P. aeruginosa has been increasing recently. Mechanisms of low-level resistance to carbapenems (MIC 8–32 mg/L) in P. aeruginosa are associated with reduced uptake as a result of loss of the OprD porin3 combined with de-repression of the chromosomal ampC ß-lactamase gene4 or by over-expression of an efflux pump system.5,6 High-level resistance to carbapenems (MIC > 32 mg/L) is still uncommon in P. aeruginosa, but can be caused by the presence of class B ß-lactamases, the metallo-ß-lactamases (MßL).7 These enzymes possess divalent cations, usually zinc, at the active site and are therefore characterized by inhibition by metal chelators. They are also resistant to inhibition by the commercially available enzyme inhibitors (clavulanic acid, sulbactam and tazobactam), which are active against class A ß-lactamases. MßLs are capable of hydrolysing both imipenem and meropenem, in addition to most ß-lactam agents, and confer resistance to these agents in pathogenic bacteria.8

The first MßL in P. aeruginosa, IMP-1, was detected in Japan;9 more recently, however, IMP-1 and IMP-variant enzymes have been reported across four continents.912 To date, 13 IMP variants, differing in primary sequence by 0.4%–22.2%, have been described.13 Since the emergence of IMP-1, three other types of clinically relevant MßL have been discovered, SPM-1,14 GIM-115 and the VIM family.1618 The first enzyme of the VIM group (VIM-1) was found in a strain of P. aeruginosa isolated in Verona, Italy. At present the VIM family consists of seven MßL members that differ in amino acid sequence identity from 77% to 99%.19 With the exception of SPM-1, all the clinically relevant MßL enzymes are encoded by gene cassettes, as part of integrons, which in turn are often found as part of complex transposons. This genetic arrangement allows for several degrees of mobility for the MßL gene cassette and as such these gene cassettes have disseminated into various Gram-negative pathogens.20 The aim of this study was to screen imipenem-resistant P. aeruginosa isolates obtained from hospitalized children in Warsaw, Poland, during 1997–2001 for the presence of MßLs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and serotyping

The 1313 P. aeruginosa isolates used in this study were recovered from a variety of specimens obtained from children hospitalized in the Children’s Memorial Health Institute, (Warsaw, Poland) during 1997–2001. The isolates were identified by standard laboratory methods. Multiple isolates from individual patients were excluded if they belonged to the same serotype and had the same antimicrobial resistance pattern. P. aeruginosa strains 48–501 and 75–3677 (clinical isolates from Sao Paulo and Genoa, containing blaIMP-1 and blaVIM-1, respectively) were used as positive controls for the Etest, and blaIMP and blaVIM PCR reactions. A streptomycin-resistant mutant of P. aeruginosa strain PA01 was used as the recipient strain for conjugation experiments.

The O serotype of the isolates was determined by slide agglutination using commercially available antisera (BIO-RAD, Marnes la Coquette, France).

Antimicrobial susceptibility testing

MICs of selected antimicrobials were determined by the agar dilution method on Mueller–Hinton II agar (BD Microbiology Systems, Cockeysville, MD, USA), as recommended by the NCCLS.21,22 P. aeruginosa ATCC strain 27853 was used as a negative control for antimicrobial susceptibility testing.

Detection of MßLs

Imipenem-resistant P. aeruginosa isolates were screened for the presence of MßLs using a simple disc diffusion test. Briefly, a colony of each isolate was suspended in saline to 0.5 McFarland standard and spread on Mueller–Hinton II agar. Two discs, one containing imipenem (IPM 10 mg/L) and a filter disc containing 5 µL of 100 mM EDTA were placed on the agar. For strains producing an MßL, the growth inhibitory zone between the two discs expanded. Production of an MßL was confirmed using Etest MßL strips (AB Biodisk, Solna, Sweden).23

PFGE

PFGE was performed as previously stated.24 Ethidium bromide-stained gels were examined visually. Isolates showing fewer than three different bands were considered identical/clonally related.24

PCR screening for integrons and blaVIM/blaIMP MßL genes

Primers used for amplification of blaVIM/blaIMP genes and class 1 integron-specific primers were VimF/R, ImpF/R and QACR and VAF, respectively, (Table 1) and were designed using the computer program Primer—designer version 1.01 (Scientific and Educational Software). PCR was performed as described previously.13 PCR products were visualized by electrophoresis on 0.8% agarose gels in Tris Boric Acid/EDTA buffer (pH 7.0) and staining with 1% ethidium bromide.


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Table 1. Primer sequences used in this study
 
DNA sequencing and sequence analysis

Sequencing was carried out on both DNA strands by the dideoxynucleotide chain termination method with a Perkin Elmer Biosystems 377 DNA sequencer (Advanced Biotechnology Centre, Imperial College London, London, UK). Primers used to sequence the variable region of the class 1 integron are listed in Table 1. Sequence analysis was performed using the Lasergene DNASTAR software package.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial susceptibility testing

The results of antimicrobial susceptibility testing of 1313 P. aeruginosa isolates showed a significant increase of imipenem-resistant strains, from 7.9% in 1997 to 19.3% by 2001 (data not shown). Among 151 isolates of imipenem-resistant P. aeruginosa, 11 produced MßL, as detected by the disc diffusion test and confirmed using Etest MßL strips.23 The MIC values for each isolate for imipenem and imipenem + EDTA were found to be >256 mg/L and 1.0–1.5 mg/L, respectively. Since the emergence of MßL-producing strains among imipenem-resistant P. aeruginosa isolates, the incidence of the enzyme producers has only varied 6.5%–11.1% (average = 7.3%), with no evidence of increasing prevalence. All P. aeruginosa isolates producing MßLs were obtained from children hospitalized on either general or urology surgical wards (Table 2).


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Table 2. Characteristics of P. aeruginosa strains producing MßLs
 
Serotype/genotype and resistance profiles of MßL-containing strains

Isolates producing MßL enzymes were all found to be of the O:6 serotype and showed identical antimicrobial susceptibility profiles. These isolates displayed resistance to: imipenem (MIC >128 mg/L), meropenem (64 mg/L), ceftazidime (128 mg/L), azlocillin (>128 mg/L), carbenicillin (>512 mg/L), piperacillin–tazobactam (>128 mg/L), cefoperazone (>128 mg/L), cefepime (128 mg/L), amikacin (64–128 mg/L), gentamicin (32–64 mg/L), netilmicin (>128 mg/L) and tobramycin (>128 mg/L), and remained susceptible to aztreonam (8 mg/L) and ciprofloxacin (0.25 mg/L). The 11 P. aeruginosa isolates producing MßLs had four distinct PFGE patterns, including three subgroups. PFGE patterns A (six strains) and B (three strains) were the most common (Table 2).

Screening for known MßL genes

To determine if the strains produced MßLs, PCR analysis was undertaken using primers specific for VIM- and IMP-type MßL genes. Products of ~650 bp were amplified using genomic DNA from all 11 strains using VimF and VimR primers (Table 1), which were subsequently sequenced in both directions. The isolates were then further analysed by amplification of the entire integron using the class 1-specific primers VAF and VAR. Products of 2148 bp resulted, which were digested with the restriction endonuclease HincII to reveal an identical restriction fragment pattern for each isolate, demonstrating that all strains harbour an integron of apparently identical structure. Representative PCR products amplified from each distinct PFGE pattern were sequenced completely, confirming an identical integron sequence among these isolates.

Sequence analysis

Sequence analysis revealed the common arrangement of antimicrobial resistance gene cassettes and the 5'/3' conserved sequences of a class 1 integron. Two gene cassettes were found in each integron. In the first position was an aacA4 gene cassette identical to the aacA4 gene found in numerous other integrons. The second gene cassette position was occupied by the blaVIM-1 variant, blaVIM-4. The blaVIM-4 gene is additionally unique in that it contains a 163 bp direct repeat of the 3' end of the gene. The direct repeat starts 15 bp after the blaVIM-4 stop codon and immediately after the natural inverse core site of the 59 base element (59-be) (Figure 1). The extent of the gene duplication is 169 bp of the 3' end of the blaVIM-4 gene. The 59-be at the end of the direct repeat is identical to a normal blaVIM-1 59-be and suggests that a normal blaVIM-4 59-be is identical to a blaVIM-1 59-be (the 59-be of previous blaVIM-4 gene cassettes are not represented in the GenBank database).



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Figure 1. Schematic of the blaVIM-4-containing integron amplified with the primers VAF and QACR (not to scale). (a) The amplicon is 2148 bp long and contains a partial sequence of the class 1 conserved sequences intI1 and qacE{Delta}1 at the 5' and 3' ends of the integron, respectively, together with the two gene cassettes found in this integron. The position of the HincII restriction enzyme sites and the expected sizes of fragments that would be generated by a HincII digest of this amplicon are shown above. Open boxes represent the positions of the various genes and the arrows show the direction of their transcription. The attI1 site of this integron is represented by an open ellipse and the 59-be of the gene cassettes by filled ellipses. The position of the 169 bp direct repeat of the 3' end of blaVIM-4 is shown by an empty box at the 3' end of the blaVIM-4 gene. (b) Represents an enlarged diagram of the blaVIM-4 gene and its putative 59 base elements. Filled rectangles represent the pseudo-core sequence found within the blaVIM-4 structural gene, the pseudo-core sequence of the 3' blaVIM-4 repeat and the conserved core site of the 59-be. Checked rectangles represent inverse core sequences and the circled regions represent possible 59 base elements. The sequences of the pseudo-core sites, the core site and the inverse core sites are given below.

 
The sequence GTTGAGC found within the blaVIM-4 gene coding sequence was located 20 bp downstream of the start of the direct repeat (Figure 1). This pseudo-core sequence matched the core site of a 59-be perfectly, thus effectively forming a shortened 59-be of 34 bp directly after the complete copy of the blaVIM-4 gene. The direct repeat also includes its own 59-be and therefore it appears that the blaVIM-4 gene cassette has two 59-be (Figure 1).

A GenBank/FASTA search using the entire Polish integron sequence revealed an almost identical match with a blaVIM-1-containing class 1 integron isolated from a strain of Escherichia coli in Greece, accession number AY152821. The only differences were a single nucleotide change resulting in the substitution S 205 R of the blaVIM-4 gene and two transitions of C to T at positions 60 and 68 of the 59-be of the blaVIM-1 gene.

Gene transfer experiments

Repeated attempts at isolating plasmids from all the MßL-containing strains were unsuccessful, as were attempts to transfer resistance to P. aeruginosa PA01 by conjugation. This indicates that the blaVIM-4-containing integron is probably chromosomally encoded in all isolates.

Nucleotide sequence accession number

The nucleotide sequence of the novel blaVIM-4-containing integron has been assigned the GenBank accession number AJ585042


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During 1986–1992, the incidence of imipenem-resistant P. aeruginosa clinical isolates was in the range 0–1.8%,25 and in the period 1994–1996 was 0–2.6% at the monitored institution (J. Patzer, unpublished data). However, by 2001 the incidence had risen to 19.3%, which correlates with the increase in carbapenem usage at this hospital—from 1182 defined daily doses (DDD) in 1997 to 2132 DDD in 2002. The first MßL-producing, MDR P. aeruginosa strain was isolated from faeces of a 2-week-old child, hospitalized in the general surgery ward in 1998. Subsequently, during 1997–2001, MDR P. aeruginosa isolates producing MßLs were obtained from 11 children hospitalized in general surgery or urology wards. Five of these 11 patients received imipenem therapy at this hospital, although none received imipenem therapy immediately before the isolation of MßL-containing organisms. However, 10 of the 11 patients received either a third- or fourth-generation cephalosporin. Epidemiological typing (PFGE analysis) identified two distinct PFGE types—A and B. Despite an intensive epidemiological search, the sources of these strains were not identified and the strains appear to have become endemic.

Genetic analysis revealed that all the resistant isolates contained the same class 1 integron. An aacA4 gene cassette and a blaVIM-4 gene cassette were inserted between the two conserved regions of this integron. The latter gene is unusual in that it has a 3' terminal direct repeat of 169 bp comprising the end of blaVIM-4.

The blaVIM-4 gene cassette was first identified in a strain of P. aeruginosa isolated in April 2001 in Greece.26 This strain was responsible for a major epidemic27 and the cassette was subsequently found in strains of P. aeruginosa with different genotypes, reflecting its ability to transfer from one bacterium to another. Very recently the same gene was identified in a P. aeruginosa isolate from Sweden.28 In this case the link between Greece and Sweden was clear in that the patient was a Greek citizen who emigrated to Sweden in 2001.28 In all the strains investigated, the blaVIM-4 gene was the only gene cassette found in the integrons.

A homology search of the GenBank databases, using the Polish blaVIM-4 integron as the search sequence, identified a near perfect match with a blaVIM-1-containing integron from an E. coli strain isolated in Greece (accession number AY152821). This integron has blaVIM in the second gene cassette position preceded by an aacA4 cassette and differs from the Polish integron by only three nucleotides. One substitution creates the difference between blaVIM-4 and blaVIM-1 and there are two T for C transitions in the 59-be. Three lines of evidence suggest that the integrons found in Polish isolates are older than that found in the Greek isolate. These are: (1) the integrons found in Poland have a perfect blaVIM-1 59-be, whereas the Greek integron has two base changes; (2) the MßL genes usually appear in Pseudomonas or Acinetobacter sp. prior to their isolation in members of the Enterobacteriaceae; and (3) the earlier isolation date of the Polish strain.

The 169 bp duplication that is part of the blaVIM-4 gene cassette described here is, we believe, an insertion that was generated by deletion of part of a blaVIM-4 tandem cassette. The repeat is positioned precisely at the end of an inverse core site, which we may presume is derived from the original copy of the 59-be that formed part of the first blaVIM-4 cassette. The deletion removed most of the 59-be of the first blaVIM-4 cassette together with 643 bp of the second copy of the blaVIM-4 gene, fusing the first cassette to the end of the second. Integrase-mediated generation of tandem repeats of gene cassettes is not an unusual event and has been observed both naturally29 and experimentally.30 The more important question is how did the deletion arise? Looking at the probable sequences that gave rise to the deletion junctions, there are no obvious stretches of sequence homology and therefore no case can reasonably be made for intramolecular recA-mediated recombination. However, an inverse core sequence is found on one side of the junction and a pseudo-core sequence that is embedded in blaVIM-4 is found fairly close to the other side of the junction of the deletion, i.e. 20 bp downstream. One explanation for the deletion would be the participation of integrase in a replication slippage event leading to deletion. This would involve integrase being responsible for bringing the two regions of DNA that define the ends of the deletion close together, looping out the intervening DNA. Replication across the synapse would generate the deletion seen. This would produce a short pseudo 59-be (34 bp) immediately after the first blaVIM-4 gene, followed by a direct repeat of the 3' end of the second blaVIM-4 gene cassette, including the associated 59-be. The resulting hybrid cassette would then have a blaVIM-4 gene followed apparently by two 59 base elements, one of which is unlikely to be functional, not only because it is short (34 bp) but also because the sequence between the inverse core and pseudo-core sequences is not of 59-be origin. The second 59-be has three single bp changes relative to the previously reported blaVIM-1 cassette, which may affect its function. Further experiments are planned to assess the functionality of this unusual cassette.

The discovery of clinical bacterial isolates carrying genes that confer broad spectrum antibiotic resistance is a major cause for concern. This document reports the second recorded independent emergence of this type of resistance mechanism in Poland, relative to the recent discovery of blaVIM-2, also in P. aeruginosa.31 Similarly, this report highlights the persistence of these strains once they have emerged in the clinical setting, the mobility of the resistance determinants and the paucity of current anti-infective therapy to eradicate infection caused by bacteria harbouring these resistance determinants.


    Acknowledgements
 
The excellent technical assistance of Teresa Guzik and Maßgorzata Walus is gratefully appreciated.

The financial support of Astra Zeneca is gratefully acknowledged.


    Footnotes
 
* Corresponding author. Tel:+44-117-928-7522; E-mail: mark.toleman{at}bris.ac.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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