1 Department of Pathology & Microbiology, University of Bristol, Bristol, UK; 2 The JONES Group/JMI Laboratories, North Liberty, IA, USA
Received 21 July 2004; returned 23 September 2004; revised 14 October 2004; accepted 24 October 2004
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Abstract |
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Methods: Multidrug-resistant (MDR) strains were identified by MIC analysis followed by genotyping and PCR-based strategies.
Results: Initial MIC analysis identified 31 MDR isolates that displayed an Etest MßL-positive phenotype. Of these, 25 produced either the MßL VIM-1 or IMP-13 as detected by PCR and sequencing. VIM-1-producing isolates were found at all sites, whereas IMP-13-producing isolates were only found in Rome. MßL-producing isolates were found at all Italian SENTRY sites and together amounted to 6.5% of all P. aeruginosa isolates. Genetic analysis indicated that many strains contained multiple integrons and identified two novel MßL integrons, one from the site in Genoa and one from Sicily. Integrons identical in structure and sequence to In70, the first identified and characterized blaVIM-containing integron from Verona, were found in isolates with distinct ribotypes at the Roman and Sicilian sites indicating that this integron has recently disseminated across Italy. All 25 MßL-producing isolates were genetically linked in that all isolates contained Tn5051 sequences and all harboured the insertion sequence IsPa7 which may be involved in the mobilization of these resistance alleles.
Conclusions: Taken together, these results indicate that Italy has a nationwide problem of MDR P. aeruginosa produced by mobile MßL genes.
Keywords: Pseudomonas aeruginosa , IsPa7 , Tn5051 , integrons , Italy
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Introduction |
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MßLs are Ambler class B enzymes and constitute a heterogeneous family. However, they share four main characteristics: (i) activity against carbapenem antibiotics; (ii) no obvious hydrolysis of monobactams; (iii) inhibition by chelating agents, such as EDTA and dipicolinic acid; and (iv) requirement of Zn2+ ions.1 Whilst some environmental bacteria possess MßL genes, recent attention has largely focused on those genes that are transferable.
The genes encoding MßLs can be plasmid or chromosomally mediated and are important resistance determinants considering that most are carried as mobile gene cassettes on class 1 integrons with the potential to spread.3 It has now been established that some of the class 1 integrons carrying MßL genes from Europe are located in a transposition locus (tnp region) termed Tn5051-like.3
Since the early 1990s when IMP-1 was first described in Japan, new MßL genes have been reported all over the world in clinically important pathogens, such as Pseudomonas spp., Acinetobacter spp. and members of the Enterobacteriaceae family. Since the emergence of IMP-1, three other sub-classes of clinically relevant MßLs have been described, the VIM family,46 SPM-17 and GIM-1 present in Germany.8 Whilst SPM-1 and GIM-1 appear to be restricted to Brazil and Germany, respectively, VIM-type MßLs, which currently number seven, appear to be widely disseminated and have been reported from North America, South America, Europe and SE Asia.912 The dominant type of VIM is VIM-2 which has been isolated from strains in most countries within Europe. The earliest isolates reported to carry the blaVIM gene were from Portugal (blaVIM-2) in 1995,13 VIM-1 from Greece in 1996,6 VIM-2 from France in 1996,5 VIM-1 from Italy in 19974 and then blaVIM-4 from Poland in 1998.14 Subsequently, there have been many reports of VIM and IMP-type MßLs across Europe particularly from the Mediterranean area.1517 The MßLs reported from Italy include blaVIM-1,4 blaVIM-2,18 blaVIM-4,19 blaIMP-1,20 blaIMP-2,21 blaIMP-12,22 and blaIMP-13.3
In this study, we have characterized a number of MßL genes embedded in novel integrons from geographically different regions within Italy. We also present data on the nature of the transposition locus carrying the class 1 integrons. The carbapenem-resistant P. aeruginosa clinical isolates from Italy were submitted to the SENTRY Antimicrobial Surveillance Programme between 1999 and 2002.
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Materials and methods |
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A diverse collection of clinical isolates (383 strains) from medical centres at Genoa, Rome and Catania were submitted to the SENTRY Antimicrobial Surveillance Programme. Among other selected pathogens, P. aeruginosa strains resistant to imipenem (MIC 16 mg/L), meropenem (MIC
16 mg/L) and ceftazidime (MIC
32 mg/L) have been routinely screened for MßL genes. Strains fitting these criteria and used in this study are listed in Table 1.
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All isolates collected in the SENTRY Programme were tested for susceptibility using the reference broth microdilution method described by the National Committee for Clinical Laboratory Standards (NCCLS).23 Antimicrobial agents were obtained from: ampicillin, ceftazidime, aztreonam, gentamicin, tobramycin and amikacin (Sigma Chemical Co., Poole, Dorset, UK); ceftazidime (GlaxoSmithKline, Worthing, UK); imipenem (Merck, Sharpe & Dohme, West Point, PA, USA); meropenem (Zeneca Pharmaceuticals, Macclesfield, UK); cefepime (Bristol-Myers Squibb, New York, NY, USA). Quality control was carried out by concurrent testing of Escherichia coli ATCC 25922, P. aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212.
Phenotypic detection of MßL
MßL Etest strips (AB Biodisk, Solna, Sweden) were used to screen class B ß-lactamase production on MuellerHinton Agar (Oxoid, Basingstoke, UK). Tests were carried out and interpreted according to the manufacturer's instructions. In addition, carbapenemase activities on crude-cell extracts from overnight broth cultures were determined using spectrophotometric assays as previously described.7 Replicate changes in absorbance levels were obtained by measuring the hydrolysis of imipenem and meropenem solutions at 299 nm with a total volume of 2.05 mL. Assays were carried out with and without EDTA to confirm the inhibition of MßLs by chelating agents.
PCR experiments
Molecular screening for blaVIM, blaIMP and blaSPM-1 was carried out using primers targeting the conserved regions of the MßL genes (Table 2). Strains known to harbour MßL genes (SENTRY isolates 48-1997, 48-12346 and 81-11963 harbouring blaSPM-1 blaIMP-1 and blaVIM-2 from Brazil, Brazil and Poland) were used as positive controls for the PCR reactions. The presence of class 1 integrons in each strain was assessed using the class 1 specific primers Int1F and QacR designed to anneal to the 5' and 3' conserved sequences, respectively. The genetic structure of each MßL-containing integron was deduced by amplifying the 5' and 3' sections of each integron separately using class 1 5' and 3' specific primers and those designed to each specific MßL gene. Primers used to amplify/sequence VIM-1-containing integrons were combinations of primers VimMF and QacR and VimMR and Int1F. Primers used to amplify/sequence IMP-13-containing integrons were IMP-13MF and QacR and IMP-13MR and Int1F. The two sections of each integron were then sequenced using a combination of primers designed to specific known genes and custom designed primers and aligned together to produce a single contig of each MßL-containing integron.
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DNA sequencing and computer analysis
The PCR fragments obtained with integron primers were sequenced on both strands using the Perkin-Elmer 377 DNA Sequencer. The nucleotide sequences, and deduced amino acid sequences were determined using the Lasergene software package (DNASTAR, Madison, WI, USA) and compared with sequences available over the internet (http://www.ebi.ac.uk/fasta33/).
Plasmid content, conjugation and electroporation
Plasmid extraction was carried out using a Qiagen miniprep kit from P. aeruginosa strains and plasmid preparations subsequently electroporated into E. coli DH5 and a rifampicin-resistant mutant of P. aeruginosa pA01. Experiments were carried out using 2.5 kV, 25 µF and 400
in the Bio-Rad Gene Pulser apparatus (Bio-Rad, Richmond, CA, USA) and selection was carried out on nutrient agar plates containing 10 mg/L ceftazidime. Conjugation experiments were carried out in broth assays with the in vitro obtained rifampicin-resistant plasmid-free E. coli K-12 and P. aeruginosa pA01. Conjugant selection was carried out on nutrient agar with 4 mg/L ceftazidime and 200 mg/L rifampicin.
Ribotyping
Ribotyping was carried out using an automated Riboprinter Microbial Characterisation System (Qualicon, Inc.). The isolates were grown overnight on BHI agar (Difco) and harvested in sample buffer. Genomic DNA was isolated and digested using PvuII. DNA fragments were separated by agarose electrophoresis. Southern hybridization using probes derived from E. coli rRNA operon created characteristic band patterns. These patterns were matched to pre-existing patterns by computer analysis and those showing 93% or more similarity were assigned to the same ribogroup.
Nucleotide sequence accession numbers
The nucleotide sequence data reported in this paper have been assigned the following EMBL/GenBank nucleotide accession numbers: AJ784256, AJ784804 and AJ784805.
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Results |
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Three hundred and eighty three P. aeruginosa were initially tested with Etest strips to look for characteristic metallo-ß-lactamase phenotypes. Thirty-one isolates that were positive by Etest were further investigated using PCR with primer sets designed to amplify all blaVIM and blaIMP variant metallo-ß-lactamase genes. Positive blaVIM-1 PCR products were found in 16/16 Etest-positive isolates from Genoa, 4/4 strains from Catania and 1/11 isolates from Rome, all these PCR products were subsequently identified as blaVIM-1 by sequence analysis. No PCR products were produced from P. aeruginosa isolates isolated in Genoa or Catania using blaIMP-specific primers. However, four of the 11 Etest-positive isolates from Rome were found to harbour the recently identified blaIMP-13 metallo-ß-lactamase.3 All isolates that were positive for MßL genes were also positive in imipenem hydrolysis experiments (data not shown).
PCR analysis of class 1 integron variable regions
The genetic context of the blaVIM-1 and blaIMP-13 gene cassettes was initially assessed with primers designed to amplify the variable region of class 1 integrons. PCR utilizing primers specific to the class 1 5' and 3' conserved sequences (CS) gave multiple products using DNA templates from many of the isolates (Figure 1a) indicating that many of these isolates contained multiple class 1 integrons. In particular, all the isolates isolated from Genoa that harboured blaVIM-1 gave identical profiles of three products in the PCR reaction with the class 1 specific primers, isolates 85-4744 and 85-2966 from Catania gave three identical sized PCR products and strains 85-2394 and 85-14297 were also identical to each other in that they produced two PCR products, whereas all the isolates containing MßL genes from Rome produced a single PCR product. See Figure 1(a) for representatives of each group of strains.
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Isolates were also screened by PCR for the presence of additional resistance gene cassettes using primers designed to amplify gene cassettes often associated in the same integron as MßL gene cassettes. These were primers specific for aacA4, aacA7, aacA29A, aacC1, aacC4, aadA1, aadA2, aphA15, catB4 and catB8 (Table 1). All isolates harboured the gene cassette aacA4 in addition to the MßL gene cassettes. All strains also harboured the aadA1 gene cassette with the exception of all the blaIMP-13-containing isolates from Rome and two isolates from Catania: 85-2966 and 85-4744. Strains 85-2394 and 85-14297 from Catania and isolate 86-10088 from Rome also harboured the aphA15 gene cassette in addition to aacA4 and aadA1 (data not shown).
PCR analysis using combinations of specific MßL gene and class 1 integron CS primers
Since multiple integrons were present in many of the MßL gene cassette-harbouring isolates, the genetic context of the particular MßL gene cassette in each isolate was assessed using PCR. This was achieved with a combination of class 1 integron CS specific primers and primers specific to the particular MßL gene cassette previously identified, so that the 5' and 3' sections of each MßL integron were amplified separately. PCR products produced using primers designed to amplify the 5' section of each integron were all of small size: 600 bp for blaIMP-13-containing integrons and 850 bp for blaVIM-1-containing integrons (Figure 1b). Sequence analysis revealed that all MßL gene cassettes in this study were in the first gene cassette position in their respective integrons (Figure 2). PCR products of the 3' sections of each MßL integron were of various lengths (Figure 1c) and were sequenced individually using combinations of primers specific to resistance gene cassettes known to be in the particular strain together with custom-designed primers to extend sequences of the novel gene cassettes identified (Table 1).
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All the isolates from Genoa in the north of Italy were of an identical ribotype (Table 2). Amplification with class 1 specific primers IntI1F/QacR gave three products, the smallest of which was approximately 1900 bp for all isolates. Two isolates were chosen randomly as template: 76-3634 and 75-3600, and PCR products of 1900 bp from each of these templates were excised from the gel and sequenced. Both consisted of two gene cassettes, an aacA4 gene cassette in the first position followed by an aadA1 gene cassette. An identical integron was further confirmed in all Genoese isolates by restriction mapping.
The blaVIM-1 integron was subsequently amplified in two segments as described above. Identical PCR products of 850 bp and 2600 bp were amplified from all isolates from Genoa. PCR products amplified from isolates 75-3600 and 75-3634 were partially sequenced and consisted of blaVIM-1 in the first position, followed by an aacA4 gene cassette. The cassette preceding the qacE1 3'CS was also an aacA4 gene cassette which leaves approximately 900 bp of unidentified sequence, enough for an as yet unidentified gene cassette which we were unable to sequence due to the presence of the aacA4 gene flanking this sequence on both sides (Figure 2). Therefore, all MßL-containing isolates recovered in Genoa were identical in possessing at least two integrons, one containing the blaVIM-1 MßL gene cassette.
Similar analysis of isolate 86-10088 from Rome revealed an integron containing the blaVIM-1 gene cassette in the first position followed by aacA4, aphA15 and aadA1 gene cassettes and was identical to In70, the integron initially sequenced and described by Lauretti et al.4 from North Italy and subsequently also found in Pavia and Trieste.
All blaIMP-13-containing isolates were found to have an identical integron to that previously found in Rome (Figure 3).3
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The SMR-2 gene cassette included a 59 base element (59be) that was 60 bp long and shared >90% identity with more than 50 similar sequences in the GenBank database including the 59be of numerous antibiotic resistance genes such as aacA4, aadA6, aadA7, qacF and catB8 as well as other gene cassettes encoding hypothetical proteins, biosynthetic genes and a gene from an uncultured bacterium recently identified by Stokes et al.27 This analysis suggests an environmental bacterium or a very closely related group of environmental bacteria are the source of all these gene cassettes.
The third gene cassette contained an ORF encoding a protein of 183 amino acids that displayed highest identities to hypothetical proteins from Gleobacter violaceus (57% identity, GenBank accession no. AP006572) and Bradyrhizobium japonicum (51% identity, GenBank accession no. AP005961) of undetermined function and weak identity to sections of chlorite dismutase proteins (34% identity over 121 amino acids) and ferritin-like proteins (195-24% over 128 amino acids). This gene cassette had a GC content of 47% and included a 59be of 75 bp. The fourth gene cassette of this integron was an aacA4 gene cassette identical to numerous other aacA4 cassettes listed (GenBank).
The final gene cassette in the integron from P. aeruginosa 85-4744 encoded the ß-lactamase PSE-1 that mediates resistance to ampicillin and was 100% identical to the PSE-1 gene cassettes of In28 cloned from a clinical strain of P. aeruginosa,28 a number of Vibrio cholerae O-serotype strains isolated in Thailand29 and the antibiotic resistance gene cluster of the Salmonella genomic island of Salmonella enterica typhimurium DT104.30 The PSE-1 gene cassette included a 59be of 75 bp identical to the other PSE-1 59be and had a GC% of 41%, characteristic of a horizontally transferred/imported gene.
Genetic elements carrying MßL genes
Plasmids. Attempts to transfer the resistance to P. aeruginosa PA01 and E. coli DH5 by conjugation and electroporation following miniprep were unsuccessful for all isolates except the strain 86-16734 harbouring the MßL gene blaIMP-13, which was successfully transferred to recipient Pseudomonas PA01 by conjugation and selection on plates containing ceftazidime 10 mg/L. Therefore, with the exception of the blaIMP-13 integron harboured by strain 86-16734, the vast majority of these MßL genes are probably chromosomally encoded or on non-conjugating/non-mobilizable elements.
Transposons. Since a Tn5051-type transposon was previously identified to be responsible for disseminating blaVIM-2 genes in Poland and blaIMP-13 genes in Rome,3 all isolates were probed by PCR with Tn5051-specific primers to see whether this transposon was present in the isolates in this study. The primers used were tnpAF/R and tnpRF/R that amplify the transposase and resolvase genes of transposon Tn5051. A characteristic 530 bp PCR product was amplified from all 25 clinical isolates when screened with tnpRF/R primers. However, 1300 bp tnpAF/R products were only amplified from blaIMP-13-containing isolates from Rome. This indicates that the Tn5051 transposon is widespread in Italian MßL-gene harbouring isolates but that in many of these isolates, the transposon is defective in self-mobilization sequences. Many of the isolates have multiple integrons, therefore a further PCR reaction was employed to detect if the Tn5051 transposon sequences were found upstream of the MßL gene-containing integrons using primers specific to the tnpR gene of Tn5051 (i.e. primer tnpRF2) and blaVIM-1/blaIMP-13 (primers VimMR, ImpMR), respectively. The Tn5051 tnpR gene was found upstream of the blaVIM-1-containing integrons from Genoese isolates and the blaIMP-13-containing integrons from Roman isolates but no PCR products were produced with the strains 86-10088, 85-2394, 85-14297, 85-2966 and 85-4744. This indicated that the tnpR gene in these isolates was not immediately adjacent to the integron harbouring the MßL gene cassette in these isolates or that the length of the PCR product was such that it could not be amplified under the conditions used (3 min extension at 68°C). These PCR products were also sequenced with the tnpRF2 primer to determine the exact insertion site of the MßL-containing integron in the DNA adjacent to the tnpR gene. Sequence analysis identified an identical insertion site for the blaIMP-13-containing integrons as that determined previously for the blaIMP-13-containing strain 86-14571 (Figure 3).3 However, all the Genoese blaVIM-1-containing integrons inserted at a position 220 bp upstream of the blaIMP-13-containing integron. This was between the resI and resII sites of the tnpM gene of Tn5051. (Figure 3).
Insertion elements. Since the integrons containing blaVIM-1 in isolates 86-10088, 85-2394 and 85-14297 shared 100% identity in primary sequence and structure with In70 first isolated from P. aeruginosa strain VR-143/97, they were investigated to see whether the insertion element IsPa7 found in strain VR-143/97 was also present in these strains. They were probed with primers designed to amplify the insertion sequence IsPa7 found downstream of the integrase yet within the inverted repeat IRi of the In70 integron. These primers were ISPF1/R1 and ISPF2/R2 designed to amplify the 3' and 5' pieces of IsPa7, respectively. PCR products were produced from all three isolates, which were sequenced to reveal 100% identity to IsPa7 of In70. Two further PCR reactions were also undertaken to see if this insertion element was in an identical position. These were PCR amplified with the primers ISPF2 and IntI1R2 designed to anneal to the middle sections of the IsPa7 insertion element and the integrase gene, respectively, and IsPa7F2 and VIMCR designed to anneal to the IsPa7 and the central section of the blaVIM-1 gene. In all cases, PCR products of the correct size were produced indicating that in all respects, the integrons from isolates 86-10088, 85-2394 and 85-14297 were of identical structure and sequence to In70 previously identified in northern Italy.
Since many of the MßL-positive strains possessed multiple integrons, it was hypothesized that this IsPa7 insertion element could possibly be present in other isolates. Therefore, all 25 MßL-positive isolates were then screened with the primer pair IsPa7F2/IntI1R2 to see if this insertion element was present in these isolates. PCR products of identical size were found in all isolates, and individual representative PCR products were also sequenced to reveal an identical sequence. This indicates that the IsPa7 insertion element is present in all MßL-positive strains collected at the three SENTRY sites and in an identical position. PCR reactions designed to investigate whether this insertion element is upstream of the MßL gene in all isolates failed to produce products except for the aforementioned isolates. Since these isolates represent several different ribotypes, these results indicate that the integron carrying IsPa7 has spread through different strains throughout Italy but that it is not always associated in the same integron as an MßL gene cassette.
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Discussion |
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Interestingly, the highest incidence of MßL-producing alleles in this study was from Genoa in the north of Italy, where the first blaVIM-containing isolate was identified and from where most MßL-producing isolates have been reported. All MßL-producing isolates were of an identical ribotype and harboured an identical blaVIM-1-containing integron that was considerably different in structure to the original blaVIM-containing integron In70. These results indicate recent emergence of the blaVIM-1 gene cassette and clonal dissemination of this isolate at this institution.
Many MßL-producing isolates from this study and all the isolates from Genoa harboured multiple integrons, at least three being harboured by the isolates from Genoa and perhaps as many as four being harboured by isolates 85-4744 and 85-2966. This has been reported before e.g. S. enterica typhimurium DT104 has three integrons within the genomic island SGI-1, but has not previously been reported for MßL-producing isolates. The numerous copies of the integrase gene within an individual cell combined with a large number of resistance alleles is likely to produce increased genetic rearrangement and reassortment of gene cassettes and foster greater mobility of individual genes. It is interesting that the non-MßL-encoding integron sequenced from the Genoese isolates consisted of the gene cassettes aacA4 and aadA1 and was identical to the recently published integron In110 from P. putida isolated in northern Italy apart from the absence of the blaVIM-1 gene cassette. This integron may well have been both the progenitor of the non-MßL-containing integron in these strains and the source of the MßL gene cassette for the Genoese MßL-encoding integron.
Isolate 86-10088 from Rome and isolates 85-2394, 85-14297 from Catania (Sicily) harboured an identical integron to In70 initially described from Verona. This demonstrates that this particular integron has spread throughout Italy very recently. The different ribotypes of the isolates from Rome and Catania also demonstrate its mobility. The 5' end of this integron is unusual in that it has a 1600 bp insertion of the insertion element IsPa7 downstream of the integrase gene. PCR detected this insertion element in an identical position in the 5' end of an integron in all MßL-encoding isolates in this study throughout Italy which means that either all the isolates involved in this study are ancestrally related or this particular integron has incredible mobility. Interestingly, this element is not found in the A. xylosoxidans isolate which suggests that the plasmid that it harbours is the progenitor of the integrons found in the P. aeruginosa strains.
The other MßL-encoding isolates from Rome all encoded the MßL IMP-13 in an identical integron, which we have published recently.3 Three isolates from Rome harboured this element on the chromosome whereas one isolate harboured the MßL gene on a plasmid, which adds to the evidence that this transposon is mobile. Very recently, this MßL gene has been detected in P. aeruginosa isolates in a hospital in southern Italy highlighting dissemination.32
PCR analysis of all strains for Tn5051 sequences detected sequences for tnpR of Tn5051 in all strains but only tnpA in strains harbouring blaIMP-13. Interestingly, the sequence immediately upstream of In70 (Y18050) includes a small section of the sequence between tnpM and the resolution site resI of Tn5051 (Figure 3) but the sequence loses identity to Tn5051 sequences at the beginning of the sequence of Y18050 which indicates that In70 was inserted initially into a Tn5051-type transposon but genomic rearrangements have since moved the tnpR sequence away from its initial position adjacent to In70. This may be the reason that a PCR product was not produced with primers tnpF2 and VimMR in strains 86-10088, 85-14297 and 85-2394. tnpR has been implicated in the mobilization of integrons and its presence in all Italian MßL-containing isolates suggests that it was involved in the mobilization of integrons in the past if not now and therefore the Tn5051 transposon has a history of involvement with Italian integrons. It may well be that this transposon is very important in the dissemination of carbapenemases both within Italy and throughout Europe. The different insertion sites between IMP-13 the Genoese VIM-1-encoding integrons and In70 also demonstrates that this transposon has purloined integrons on several occasions (Figure 3).
In addition to the In70 integron harboured by isolates 85-2394 and 85-14297, two other isolates 85-4744 and 85-2966 from Sicily also harboured blaVIM-1-containing integrons. These integrons were identical to each other and were found to have unique variable regions. Included in the variable region was an aacA4 cassette, the small multidrug resistance gene cassette SMR-2, a blaPSE-1 gene cassette and a cassette encoding a hypothetical protein similar to a similar ORF from G. violaceus. The 59be of the SMR-2 cassette was found to share high identities to similar regions in more than 50 gene cassettes including those conferring resistance to various antimicrobial compounds. The source of the gene cassettes found in clinical isolates has been proposed to be superintegrons which appear to be ubiquitous in many environmental organisms27 and are composed of an integrase together with a large number of gene cassettes, e.g. V. cholerae superintegron has 179 gene cassettes.33 Superintegrons differ from the integrons found in clinical isolates not only in the number of resistance genes that they possess but in the sequence of their 59bes. The 59be found among superintegrons are all identical or very similar within a given species or group of highly related organisms but dissimilar between different organisms. The sequences of the 59be are therefore a kind of tag identifying the source organism of any particular gene cassette. In clinical isolates, the integrons generally have gene cassettes with diverse 59bes and diverse GC percentages indicating different origins like the gene cassettes of isolates 85-4744 and 85-2966. The fact that the SMR-2 gene cassette has a 59be that is highly similar to such a large number of gene cassettes suggests that these gene cassettes have all arisen from one species of bacterium or a group of closely related bacteria. Interestingly, one of the 59be that was similar was amplified from soil from an uncultured bacterium.27 This as yet unidentified bacterium may be the source of this gene cassette and numerous other resistance alleles.
In summary, this report has revealed that MßL-encoding alleles are found at all SENTRY sites in Italy in geographically distinct institutions where they correspond to a large proportion of carbapenemase-resistant P. aeruginosa strains. The study has also revealed that the In70 integron has spread throughout the length of Italy whereas other integrons are only found at individual institutions. All MßL-harbouring isolates also appear to be genetically linked to some extent and many harbour multiple integrons, but there is also a large degree of variability and identity in MßL type and integron structure between geographically distinct sites. The mobility of these alleles both genetically and geographically, together with the difficulty of eradicating isolates harbouring them once they have emerged mean that they are a clear and present threat to current antimicrobial chemotherapy.
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Acknowledgements |
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Footnotes |
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References |
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