a Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK; b Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand; c Division of Infection and Immunity, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
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Abstract |
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Introduction |
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Materials and methods |
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From the surveys undertaken in November 1994 and between February and April 1996 by Lulitanond et al.,17 52 of 200 Enterobacteriaceae isolates and 28 of 100 Pseudomonadaceae isolates were found to produce ESBLs. These bacteria were isolated from clinical specimens submitted to the clinical microbiology laboratory, Srinagarind Hospital (700 beds), Khon Kaen University, Thailand. The ESBL-producing isolates were used in this study. The laboratory records of the isolates were reviewed. Isolates collected from the same patients were included only when identified as different species or different strains by pulsed-field gel electrophoresis (PFGE). All isolates were shown to have ESBLs by a double disc diffusion test.18 Isolates that appeared to have lost their ESBL activities during storage were excluded. The isolates were identified by standard biochemical tests.19,20 Species of Enterobacter, Serratia and Pseudomonas putida were identified by API 20E and API 20NE strip tests as appropriate (bioMérieux SA, Marcy-l'Étoile, France). Sixty-one ESBL-producing isolates were finally included in the study as follows: 27 K. pneumoniae, 17 P. aeruginosa, five E. coli, five Serratia marcescens, four Enterobacter cloacae, one Enterobacter amnigenus, one Citrobacter freundii and one P. putida (Table 1).
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Antimicrobial agents used in this study were supplied as follows: aztreonam from Bristol-Myers Squibb (Hounslow, UK); cefotaxime from Roussel Laboratories (Uxbridge, UK); ceftazidime from Glaxo Wellcome (Greenford, UK) and clavulanic acid from SmithKline Beecham Pharmaceuticals (Brentford, UK). The MICs of aztreonam, cefotaxime, ceftazidime and ceftazidime plus clavulanic acid (at a fixed concentration of 4 mg/L) were determined using an agar dilution method.21 E. coli strain NCTC 10418 and P. aeruginosa strain NCTC 10662 were used as antibiotic-susceptible controls.
Transfer of ceftazidime resistance
Plasmids encoding ceftazidime resistance were transferred by conjugation in broth culture, as described by Shannon et al.22 using E. coli strain UB1637 (recA, his, lys, trp, StrepR), E. coli strain UB5201 (recA, pro, met, NalR) or P. aeruginosa strain NCTC 50814 (RifR) as recipient, as appropriate. The procedure was followed as described previously,22 except that the mating time was 4 h. Transconjugants were selected on nutrient agar containing ceftazidime (2 mg/L) (Sigma, Poole, UK) and either streptomycin (25400 mg/L) (Sigma) to select for UB1637 transconjugants or nalidixic acid (25 mg/L) (Sigma) if UB5201 was used as the recipient. NCTC 50814 transconjugants were selected on nutrient agar containing ceftazidime (4 mg/L) and rifampicin (100800 mg/L) (Sigma).
PCR amplification
The DNA primers described by M'Zali et al.23 were used to amplify a 475 bp fragment of the blaSHV gene. A fragment of 861 bp within the blaTEM gene was amplified using primers B and H.24,25 Bacterial DNA was prepared by suspending one or two fresh colonies in 50 µL of sterile distilled water and heating at 95°C for 5 min. PCR amplifications of the blaSHV and blaTEM sequences were performed using 30 amplification cycles of 15 s at 94°C (denaturation), 30 s at 52°C (annealing) and 90 s at 72°C (chain elongation), with final elongation at 72°C for 5 min. Primer sets specific for blaPER-1,26 blaCTX-M-127 and blaVEB-115 genes were used to screen for the presence of the genes in isolates producing non-TEM and non-SHV ESBLs. PCR amplification was carried out under the following conditions: 95°C for 3 min followed by 30 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min, and finally 72°C for 5 min. Primers OXA-10F and OXA-10B were used to amplify 759 bp of blaOXA-10 in the blaVEB-like-carrying isolates.28 The variable regions of the blaVEB-like genes associated with integrons were amplified using two pairs of primers, 5'Conserved Segment (5'CS) and VEBINV1, and VEBINV2 and 3'Conserved Segment (3'CS) as described previously.28 In addition, regions downstream from the blaVEB-like genes were detected using primers VEBINV2 and AADB-B.28
PCRSSCP and PCRRFLP
The SHV-producing isolates were characterized by PCRsingle strand conformational polymorphism (SSCP) analysis23 and PCRrestriction fragment length polymorphism (RFLP) analysis with DdeI and NheI restriction endonucleases.29 PCRRFLP analysis was carried out as described previously,29 except that a substitute primer (5'-AGTCATATCGCCCGGCAC-3'), was used as the reverse primer to yield the desired amplimers for NheI digestion. Restriction fragments were analysed by gel electrophoresis in a 2% (w/v) agarose gel (Gibco-BRL, Paisley, UK). E. coli C600 (R1010) encoding SHV-1 and E. coli HB101 (pAFF611) encoding SHV-5 were used as reference strains. Molecular characterization of the blaTEM- carrying isolates by PCRRFLP analysis was as described previously.30 Restriction endonucleases used to characterize TEM ß-lactamase genes included AluI, BclI, BpmI, BsmaI, HhaI, HpaII, HphI, MseI, NlaIII and Sau3AI. Restriction endonucleases AluI and BclI were obtained from Boehringer Mannheim (Lewes, UK); the remaining restriction endonucleases were purchased from New England Biolabs (Beverley, MA, USA). Four reference strains, E. coli C600 (pCFF04) encoding TEM-3, E. coli C600 (pUD16) encoding TEM-4, E. coli C600 (pCFF14) encoding TEM-5 and E. coli C600 (pIF100) encoding TEM-7, were used.
Nucleotide sequence determination
PCR products were used as templates for nucleotide sequence determination. A fragment of 826 bp within blaSHV was amplified,23 as were the entire blaVEB-1 and blaTEM genes.15,24 The amplification products were purified with the QIAquick PCR purification kit (Qiagen, Crawley, UK) and their nucleotide sequences were determined using an ABI PRISM (ABI Biosystems, Pasadena, CA, USA) automated sequencing machine (Model 373, version 3.3), according to the manufacturer's instructions. Nucleotide sequence determinations were performed on both DNA strands and on two independently generated amplimers.
Plasmid DNA analysis, Southern blotting and hybridization
In general, plasmid DNA was extracted by the method of Bennett et al.31 However, plasmid DNA extraction from S. marcescens strain 2 and P. aeruginosa strain 17 was according to Sambrook et al.32 for large-scale preparation. Plasmid DNA was detected by agarose gel electrophoresis and plasmid sizes were determined by comparison with plasmids of known sizes isolated from E. coli strains NCTC 50192 and NCTC 50193. Plasmid DNA from transconjugants was digested with either BamHI or EcoRI endonuclease (Gibco-BRL) and the fragments were analysed by agarose gel electrophoresis, using DNA digested with BglI as a DNA size marker. After electrophoresis, uncut and digested plasmid DNA were transferred to nylon membranes (Boehringer Mannheim) by the method of Southern33 and hybridized with a digoxigenin-labelled blaSHV, blaTEM or blaVEB-1 gene fragment (PCR DIG labelling mixture, Boehringer Mannheim).
PFGE
PFGE34 was used to type all isolates in the study. Bacteria were grown in 5 mL of tryptone soya broth at 37°C, overnight for Pseudomonadaceae and for 4 h for Enterobacteriaceae. Chromosomal DNA was digested with XbaI (Gibco-BRL) at 37°C for 4 h, according to the supplier's instructions. The resultant DNA fragments were separated by electrophoresis in 1.2% (w/v) agarose gel (Agarose NA; Amersham Pharmacia Biotech AB, Uppsala, Sweden) using a Pharmacia LKB (Sweden) system at 175 V and 12°C. The pulse time was increased from 5 to 20 s for Pseudomonas DNA and from 5 to 35 s for Enterobacteriaceae DNA. The running time was 18 h for Pseudomonas DNA and 20 h for Enterobacteriaceae DNA. A 48.5 kb ladder (Bio-Rad) was used as the DNA size marker. PFGE DNA patterns were classified after visual inspection. Only fragments >48.5 kb, which is the smallest rung of the
DNA ladder, were considered when comparing PFGE patterns. Patterns were considered to be of the same type if there were no more than three band differences.35
Analytical isoelectric focusing
Isoelectric focusing was performed on ß-lactamases expressed by the Pseudomonas isolates. Crude ß-lactamase preparations were obtained using the sonication method of Danel et al.26 Analytical isoelectric focusing was performed as described previously,36 except that agarose gels containing pharmalyte (pH 310, Pharmacia Biotech) were used.
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Results |
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Among the Enterobacteriaceae, all isolates except one strain of C. freundii carried blaSHV and 23 harboured both blaTEM and blaSHV (Table 1). The C. freundii strain has blaTEM and blaVEB. The sole P. putida isolate and all P. aeruginosa isolates, except strain 17, which produces SHV, have blaVEB. The blaOXA-10-like gene was detected in all isolates carrying blaVEB, except P. aeruginosa strains 1 and 2 (Table 2
). All isolates of Enterobacteriaceae examined in this study were screened by PCR for the blaVEB-1 gene. No evidence for this gene in these isolates was found, except for the C. freundii strain, as noted.
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Genes coding for SHV and TEM ß-lactamases were investigated by PCRSSCP and PCRRFLP analyses. Among the SHV-producing Enterobacteriaceae, three isolates gave the ß-lactamase gene PCRSSCP pattern designated A (Figure 1, lane B), indicating carriage of blaSHV-1, blaSHV-2a, blaSHV-3 or blaSHV-11;29 28 showed SSCP patterns designated B (Figure 1
, lanes CF), indicating that they have either blaSHV-5 or blaSHV-12;29 11 K. pneumoniae gave SSCP patterns of type A+B (Figure 1
, lanes HK), consistent with the presence of two different SHV genes in the same strain, whereas representatives of their transconjugants yielded only SSCP pattern B (data not shown). PCRSSCP analysis of the ß-lactamase gene of P. aeruginosa strain 17 showed a type B SSCP pattern.
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Despite using a battery of 10 restriction endonucleases, all TEM-producing isolates gave amplimers with identical RFLP profiles (data not shown). Only one silent mutation, at nucleotide position 436 (numbering according to Sutcliffe38) was detected, identifying a variant of blaTEM-1. From 24 isolates carrying blaTEM, a representative C. freundii strain that carries only blaTEM was selected and blaTEM was sequenced. The gene identified was blaTEM-1B, as described by Goussard & Courvalin.39
Identification of VEB-1-like ß-lactamase genes and their integron contexts
Of the 18 isolates harbouring blaVEB-like, two representative strains, the sole C. freundii strain and P. aeruginosa strain 9, were selected for nucleotide sequence analysis of blaVEB-like. Both contained blaVEB-1.15 The flanking regions, containing aadB, downstream of blaVEB-like genes were amplified from all 18 isolates. Using the 5'CS and VEBINV1 primers, fragments ranging in size from 0.85 to 2.8 kb were detected; no amplification product was obtained from the C. freundii transconjugant tested (Table 2). Using the 3'CS and VEBINV2 primers, amplimers of sizes 0.93.5 kb were obtained. No PCR products were recovered using DNA from the C. freundii strain or its transconjugant.
Molecular epidemiology of the ß-lactamase genes
Of the 61 isolates used in this study, 19 were isolated in November 1994 and 42 between February and April 199617 (Table 1). They were obtained from 57 patients, three of whom were found to carry more than one ESBL-producing strain: one had three different strains, K. pneumoniae strain 17 and E. coli strains 4 and 5, a second carried K. pneumoniae strain 19 and E. cloacae strain 1, while a third had K. pneumoniae strain 5 and E. cloacae strain 2. Fifty-three isolates were from patients in 16 of 26 wards. Eight isolates were from patients admitted to outpatient departments (OPD). The most common specimens yielding bacteria resistant to extended-spectrum cephalosporins were sputum (45.9%) and urine (21.3%). Unfortunately, a history of diagnosis and antibiotic treatment was not available for any of the patients in the study.
Genetic contexts of blaSHV-5
Of the isolates investigated, 13 (11 K. pneumoniae and two E. coli) were found to produce SHV-5. They were isolated in 1994 and 1996 from patients admitted to different wards except K. pneumoniae strains 4 and 7, which were obtained from different patients admitted to the same ward in 1996. Transfer of ceftazidime resistance from each of these isolates to E. coli UB1637 was successful in all cases. Analysis of the plasmids in the transconjugants revealed that in each case blaSHV-5 is encoded on a plasmid of c. 130 kb. Restriction digestion of these plasmids with BamHI or EcoRI showed that nine from K. pneumoniae and two from E. coli have the same restriction pattern, designated pattern I (Figure 3a). In the cases of transconjugants of strains 2, 5, 7 and 8, two extra bands were found in what is otherwise restriction pattern I. These were uncut small plasmids, which had been transferred from their donors together with the 130 kb plasmid. Subsequent hybridization of Southern blots of these and similar gels with the blaSHV probe revealed that the blaSHV-5 genes of these isolates are present in all cases on an EcoRI restriction fragment of c. 4.5 kb (Figure 3b
). The plasmid from the transconjugant of K. pneumoniae strain 10 gave EcoRI restriction pattern II, which differed from pattern I by two fragments. The blaSHV probe hybridized to two fragments from this plasmid. The plasmid from the representative transconjugant of K. pneumoniae strain 11 yielded a very different restriction pattern from patterns I and II, designated III. In this case, the blaSHV-5 gene was found on a 3.5 kb EcoRI fragment. PFGE analysis of the 11 K. pneumoniae isolates carrying blaSHV-5 revealed that the nine isolates with plasmids with identical EcoRI restriction patterns also have identical PFGE patterns. K. pneumoniae strain 10, which has the plasmid with the modified EcoRI restriction pattern II, has the same PFGE pattern as that of the nine isolates. K. pneumoniae strain 11, which has a different plasmid, as judged by its EcoRI restriction pattern, also has a different PFGE DNA profile from the other 10 isolates. In addition, E. coli strains 1 and 2, which also have the blaSHV-5 gene on a plasmid indistinguishable from that found in nine of the K. pneumoniae isolates, have different PFGE patterns.
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Of the 61 isolates, 27 produce SHV-12. These include 14 K. pneumoniae, five S. marcescens, three E. coli, three E. cloacae, one E. amnigenus and P. aeruginosa strain 17. They were obtained from patients admitted to 11 wards and OPD, in 1994 and 1996. All isolates, except the five S. marcescens and P. aeruginosa strain 17, have large plasmids that hybridized with the blaSHV probe. Ceftazidime resistance was successfully transferred to E. coli UB1637 from 15 isolates (55.6%). The blaSHV-12 gene was found on several plasmids of sizes c. 100130 kb. Restriction endonuclease digestion of the 15 conjugative plasmids with EcoRI or BamHI showed most to be unique, except those of transconjugants of K. pneumoniae strains 22 and 23, and those of E. coli strains 4 and 5, which have identical restriction patterns (data not shown). Consistent with these findings, the blaSHV-12 gene was found on EcoRI fragments of different sizes when hybridized with the blaSHV probe. Among the 14 K. pneumoniae isolates producing SHV-12, eight PFGE patterns were identified. Strains 12 and 13 have identical PFGE patterns but have different plasmid profiles and plasmid restriction patterns; strains 18, 19 and 20 also have the same PFGE pattern but have different plasmids. Strain 21, collected in 1994, showed the same PFGE pattern as those of strains 24 and 25 isolated in 1996, but each has a different plasmid profile. Strains 22 and 23, obtained in 1996 from different patients admitted to the same ward, have identical PFGE patterns, plasmid profiles and plasmid restriction patterns. The four remaining isolates have different PFGE and plasmid profiles. The three E. coli isolates producing SHV-12 have different PFGE and plasmid profiles, even though strains 4 and 5 were isolated from the same patient and both carry blaSHV-12 on the same plasmid. Among the three E. cloacae isolates producing SHV-12, strains 2 and 3 have the same PFGE pattern but different plasmid profiles. Irrespective of the isolation method used, all S. marcescens isolates gave an identical plasmid profile consisting of a single plasmid of <1.9 kb. PFGE analysis of these isolates revealed that they are very closely related.
Genetic contexts of blaSHV-2a and blaVEB-1-like
Two isolates, K. pneumoniae strain 26 and E. cloacae strain 4, were found to produce SHV-2a. The former was isolated in 1994, the latter in 1996. Each isolate has a unique PFGE pattern and plasmid profile compared with other E. cloacae isolates in the set (Table 1).
Among the Pseudomonas spp. isolates, two have no plasmids. Six plasmid profiles were obtained from the remaining isolates (Table 1). The blaTEM and blaSHV probes did not hybridize to any of the plasmids or to chromosomal DNA, except from strain 17. In this case chromosomal DNA hybridized weakly to the blaSHV probe. Despite repeated attempts, no evidence for plasmids in strain 17 was found. Sixteen isolates of P. aeruginosa and one each of P. putida and C. freundii were found to produce VEB-1-like ß-lactamases. The isolates were collected in 1994 and 1996 from patients admitted to seven wards and the OPD. Attempts to transfer the resistance determinant to another strain were successful in two cases, namely from the C. freundii strain and P. aeruginosa strain 1. Attempts to hybridize the blaVEB-1 probe to plasmid and chromosomal DNA from the 16 P. aeruginosa harbouring blaVEB-like all met with failure. However, a single plasmid, of c. 100 kb, from the P. putida isolate hybridized with the blaVEB-1 probe, as did chromosomal DNA from the VEB-1-producing C. freundii. No hybridization with either the 70 or 150 kb plasmid of the C. freundii strain was detected. No plasmids were found in the transconjugant of P. aeruginosa strain 1. PFGE analysis of the 16 P. aeruginosa isolates carrying blaVEB-like revealed that nine (56.3%) have the same profile; strains 1 and 2 are also indistinguishable, as are strains 12 and 13 (Table 1
). The three remaining isolates each has a unique PFGE DNA pattern, as does strain 17, which produces SHV-12. Among the nine isolates with identical PFGE profiles (Table 1
), all except isolate 6 carry a 100 kb plasmid. In addition, isolates 4 and 9 have an additional 8.3 kb plasmid, while isolate 8 has two extra plasmids of 50 kb and one >150 kb.
Resistance phenotypes
The MICs of three ß-lactams for all isolates are shown in Table 1. Among the Enterobacteriaceae, MIC50 and MIC90 values of both aztreonam and ceftazidime were
128 mg/L. The MIC50 and MIC90 values of cefotaxime were 8 and 32 mg/L, respectively. For the Pseudomonas isolates, the MIC50 and MIC90 values of all the ß-lactam antibiotics tested were
128 mg/L. Clavulanic acid, at 4 mg/L, greatly reduced the MIC of ceftazidime for all isolates, except for E. cloacae strain 4. The MICs of various ß-lactam antibiotics for the transconjugants are also summarized in Table 1
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Isoelectric focusing
Isoelectric focusing studies revealed that the P. putida strain and P. aeruginosa strains, except isolates 1, 2 and 17, produced two ß-lactamases, with pIs of approximately 6.2 and 7.3 (data not shown). Although it proved difficult to ascribe very accurate values to the bands, the findings are consistent with production of VEB-1-like and OXA-10-like ß-lactamases, as indicated by the PCR results. Strains 12 and 13 and the P. putida strain also produced ß-lactamases with pIs of c. 5.7, indicating production of another ß-lactamase(s). Strains 1 and 2 produce ß-lactamases with pIs of approximately 5.7 and 7.3. Strain 17 produces a ß-lactamase with a pI of 8.2.
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Discussion |
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ESBLs SHV-2a, SHV-5, SHV-12 and VEB-1 were found in the isolates investigated. TEM-1B and SHV-11, narrow-spectrum ß-lactamases, were also found, and one isolate produces an ESBL that belongs to neither the TEM nor the SHV family. These findings augment others concerning the occurrence of SHV ß-lactamases in the Far East. SHV-2 and SHV-12 have been reported in China,13 SHV-12 and SHV-2a in Korea8 and SHV-12 and SHV-2 have recently been reported in Japan.7 SHV-12, SHV-5 and SHV-2 have been found in Taiwan1012 and SHV-5 has been reported in Singapore1 and Thailand.13 This paper is the first report of the presence of SHV-12 and SHV-2a in Thailand, although these ß-lactamases have been found in other parts of Asia. SHV-12 ß-lactamase is the most common ESBL, emerging apparently as the best-adapted ESBL for the prevailing antibiotic selection pressure in the Far East. In this study, SHV-12 was found in several species, including K. pneumoniae, E. coli, E. cloacae, S. marcescens and P. aeruginosa, indicative of horizontal gene transfer.
The molecular epidemiology of SHV ESBLs among the isolates studied was investigated. Plasmid fingerprinting of the 15 transconjugants carrying blaSHV-12 indicated that this resistance gene is carried on 13 different plasmids, as defined by restriction endonuclease profiles. PFGE analysis of the clinical isolates possessing blaSHV-12 in general gave no evidence for dissemination of this gene by clonal spread. Relationships between isolates were established in only a few instances and did not involve more than three strains, except for the five S. marcescens isolates. In this particular instance, the isolates are very closely related, indicating clonal spread of this strain in the hospital. In contrast, the restriction profiles of plasmids from the nine transconjugants that acquired blaSHV-5, and the PFGE patterns of the respective donors, indicate clonal spread through several wards during 19941996 (Table 1). In addition, two isolates of E. coli were found to carry blaSHV-5 on a plasmid indistinguishable from that found in the K. pneumoniae isolates, suggesting horizontal gene transfer on a transmissible plasmid. In general, the plasmid carries a single copy of blaSHV-5, but in one case (K. pneumoniae strain 2-7) evidence for two copies of blaSHV was found.
We characterized blaSHV genes by PCRSSCP23 and PCRRFLP analysis.29 These techniques identified the SHV variants, including SHV-2a, SHV-5 and SHV-12, confirmed by nucleotide sequence analysis. To our knowledge, this is the first time that these techniques have been applied extensively in an epidemiological study, validating their utility. Isolates carrying both blaSHV-5 and blaSHV-11 were identified with these techniques. It is possible to identify two different SHV types in a single strain using a combination of both techniques, thus preventing the need for cloning studies.
Of the TEM ß-lactamases, only TEM-1B, a narrow- spectrum ß-lactamase, was found in this study. Twenty-four of 61 isolates carried the gene for this enzyme. Recently, TEM-1 ß-lactamase was also reported from Enterobacteriaceae isolated from China3 and Taiwan.11,12 In the Far East, TEM ESBLs that have been reported include TEM-26 from Japan7 and TEM-52 from Korea.9 It appears that TEM ESBLs are not as common as SHV ESBLs in bacteria isolated in Asian countries. This may be because of different selective pressures in these countries compared with other geographical locations.
Clavulanic acid-inhibited ESBLs detected in P. aeruginosa include PER-1,41 TEM-42,42 OXA-18,43 SHV-2a,44 SHV-5 (GenBank accession no. AF096930), VEB-114,15 and TEM-24.45 In this study, almost all P. aeruginosa isolates and an isolate of P. putida produced VEB-1-like ß-lactamase. The blaVEB-1 gene described previously is plasmid located in E. coli and P. mirabilis.16,28 In this study, it was found on a plasmid in P. putida; however, the blaVEB-1 probe failed to hybridize to the plasmids from the P. aeruginosa isolates. This indicates that the gene is on the chromosome in these isolates, as reported previously.14,15 PFGE analysis of the P. aeruginosa isolates showed them to be indistinguishable, indicating the dissemination of the strain by clonal spread.
The blaVEB-1 probe hybridized only to chromosomal DNA from the sole C. freundii isolate and that of its transconjugant, although plasmids were found in both. In addition, no plasmids were found in the transconjugant of P. aeruginosa strain 1 that was tested. One explanation for these results is that blaVEB-1 is on a self-transmissible plasmid that is integrated into the chromosomal DNA of the donor and has integrated into the chromosome of the transconjugant, in a similar manner to Hfr E. coli strains. Amplimers of the blaVEB-like genes were sequenced from two representative strains only; there may be other variants of blaVEB-1 among these isolates.
Integron carriage of blaVEB-1 was investigated by PCR (Table 2). At least six different structures of blaVEB-like- containing integrons were detected. Interestingly, the sizes of amplification products from P. aeruginosa strains 14 and 15 with the 5'CS and VEBINV1, and VEBINV2 and 3'CS primers were c. 2800 and 900 bp, respectively, compared with the predicted sizes of the PCR products from P. aeruginosa JES from Bangkok15 of 2735 and 881 bp, respectively. This suggests that strains 14 and 15 may have the same integron as strain JES from Bangkok.15 Using primers VEBINV2 and 3'CS, no PCR product was obtained from the C. freundii strain or its transconjugant (Table 2
). The fragment may be as large as those reported in E. coli MG-116 and P. mirabilis Lil-128 in which case no product would be expected. It was also found that PCR fragments amplified from the transconjugant of the C. freundii strain and that of P. aeruginosa strain 1 were of different sizes from those of the donors, indicating that rearrangement and gene cassette acquisition has occurred.
VEB-1 ESBL has only recently been described in a few strains of P. aeruginosa from Bangkok, the central part of Thailand.14,15 The isolates examined in this study were from the Srinagarind Hospital, Khon Kaen, in the north-east of Thailand, 450 km from Bangkok, indicating widespread dissemination of the ß-lactamase gene among P. aeruginosa in Thailand. VEB-1 ß-lactamase has also been reported in E. coli, K. pneumoniae and P. mirabilis isolated from Vietnamese patients in France.16,28 These findings suggest that the VEB-1 ESBL originated in and is common in Southeast Asia. However, the origin of the blaVEB-1 gene is still uncertain. In the present study, all isolates of Enterobacteriaceae were screened for the presence of blaVEB-1 but only the C. freundii strain has it. From this, it appears that blaVEB-like genes are more common in P. aeruginosa than in members of the Enterobacteriaceae. However, the isolates from Vietnamese patients that produced this enzyme were members of the Enterobacteriaceae.16,28 Further study of the sequences flanking blaVEB-like genes among Pseudomonadaceae and Enterobacteriaceae may help to elucidate the evolutionary history of this resistance determinant.
Only one isolate of P. aeruginosa was found to produce SHV-12. No plasmids were found in the isolate, suggesting that the gene is on the chromosome. There have only been two previous reports of SHV ESBLs in P. aeruginosa: SHV-2a in a strain isolated in France44 and SHV-5 in one from Greece (GenBank accession no. AF096930). The blaSHV-2a gene was presumed to be chromosomal, since no plasmids were detected in the strain.36 This paper presents the first report of blaSHV-12 in P. aeruginosa, as well as of blaVEB-1 in C. freundii and blaVEB-like in P. putida. It appears that SHV-12 ß-lactamase is the most common ESBL produced by Enterobacteriaceae from the Srinagarind Hospital, whereas VEB-1-like ß-lactamase is more common among Pseudomonadaceae from the same source. This report demonstrates that there has been substantial transfer of ESBL genes between Enterobacteriaceae and Pseudomonadaceae, enlarging the known gene pools among which ESBL genes can move.
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Acknowledgements |
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Notes |
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References |
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Received 4 October 2000; returned 1 February 2001; revised 27 March 2001; accepted 14 August 2001