a Sera and Vaccines Central Research Laboratory, ul. Chelmska 30/34, 00-725 Warsaw b Health Care Center Praga-Pólnoc, ul. Jagielloñska 34, 03-719 Warsaw, Poland
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Abstract |
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Introduction |
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An ESBL variant may be selected de novo in a given hospital,10,11,12 or it may be introduced from another centre.13,14,15 Its further spread within the hospital can be a consequence of plasmid transmission among non-related Enterobacteriaceae strains,8,16,17 or clonal dissemination of the enzyme-producing microorganism.18,19 Parallel outbreaks caused by ESBL-expressing strains, resulting from both plasmid transfer and clonal spread have also been reported.20,21 Often, more than one ESBL variant is present in the microflora of a given hospital environment.22,23,24 Persistence and outbreaks of ESBL producers have been convincingly correlated with intensive use of third-generation cephalosporins.14,19,25
In 1996, routine surveillance for ESBL-producing Enterobacteriaceae was introduced at the Praski hospital. Initial analysis of the first four identified isolates (three Citrobacter freundii and one Escherichia coli) resulted in the characterization of a novel ESBL variant, CTX-M-3, and gave some indication of the complex epidemiology of strains producing the enzyme.26 In this study, results of detailed analysis performed on all ESBL-producing strains identified in the hospital over a period of 4 months, are presented.
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Materials and methods |
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Thirty-five ESBL-producing isolates belonging to the family Enterobacteriaceae: K. pneumoniae (n = 12), Serratia marcescens (n = 8), E. coli (n = 7), C. freundii (n = 4), Enterobacter cloacae (n = 2), Klebsiella oxytoca (n = 1) and Morganella morganii (n = 1) were collected between November 1996 and February 1997. The Praski hospital has approximately 600 beds located in 11 wards. The isolates were recovered from patients on seven different wards (mostly from the department of urology and one of three surgical wards), and from patients seen as outpatients after discharge (Tables I and II). All these patients were receiving antibiotics, 30 of them being treated with ceftriaxone or cefuroxime. The majority of isolates were identified as aetiological agents of urinary tract infections; one isolate, E. coli 6, was cultured from a blood sample. Species were identified by the hospital laboratory using the ID32E ATB test (bioMérieux, Charbonnieres-les-bains, France). Putative production of ESBL was detected by the double-disc test.27 One of the isolates, E. coli 173, was included in another analysis,28 the results of which partially overlap with the data presented here.
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MICs of various antibiotics were determined by the agar dilution method as described in NCCLS guidelines.29 The following antibiotics were used: ampicillin, cefotaxime and gentamicin (Polfa, Tarchomin, Poland), aztreonam, cefepime and amikacin (Bristol-Myers Squibb, New Brunswick, NJ, USA), cefoxitin (Sigma Chemical Company, St Louis, MO, USA), ceftazidime (Glaxo Wellcome, Stevenage, UK), lithium clavulanate (SmithKline Beecham Pharmaceuticals, Letchworth, UK), imipenem (Merck, Sharp & Dohme Research, Rahway, NJ, USA), piperacillin (Lederle Piperacillin Inc., Carolina, Puerto Rico), and tazobactam (Lederle Laboratories, Pearl River, NY, USA). In all ß-lactaminhibitor combinations, concentrations of clavulanate and tazobactam were 2 and 4 mg/L, respectively. E. coli ATCC 25922 was used as the reference strain for antimicrobial susceptibility testing.
Resistance transfer
Ceftazidime- or cefotaxime-resistance transfer experiments were carried out on all the isolates. E. coli A15 R-, resistant to nalidixic acid or rifampicin, was used as the recipient strain. Equal volumes (1 mL) of cultures of the donor and the recipient strain (109 cfu/mL), grown with agitation in tryptic soy broth (Oxoid, Basingstoke, UK), were mixed and incubated statically for 18 h at 35°C. Transconjugants were selected on MacConkey agar (Oxoid) supplemented with nalidixic acid (64 mg/L; Sigma Chemical Company) or rifampicin (128 mg/L; Polfa) to inhibit the growth of donor strains, and ceftazidime or cefotaxime (2 mg/L) to inhibit the growth of recipient strains.
Isoelectrofocusing (IEF) of ß-lactamases
Crude preparations of ß-lactamases30 were subjected to IEF, using a method described by Matthew et al.,31 utilizing a Model 111 Mini IEF Cell (Bio-Rad, Hercules, CA, USA). Following IEF, ß-lactamase bands were visualized by staining with nitrocefin (Oxoid). Gels were run over a pH range of 310.
Bioassays for the detection of ESBL activity
After isoelectric focusing, ESBL activity was assigned to visualized ß-lactamase bands, by bioassay, as described by Bauernfeind et al.30 The concentration of ceftazidime or cefotaxime used in the experiment was 2 mg/L.
Typing by randomly amplified polymorphic DNA analysis (RAPD)
Genomic DNA for typing was extracted from bacterial cells using the Genomic DNA Prep Plus kit (A & A Biotechnology, Gdañsk, Poland). RAPD analyses were performed using the RAPD-732 oligonucleotide as a primer. Reactions were run under conditions described previously.24 The following strains were used in typing as epidemiologically non-related controls: K. pneumoniae L-95,24 S. marcescens 1020/96, E. coli ATCC 25922, C. freundii L-60126 and E. cloacae 1019, all (apart from the ATCC strain) isolated previously in other hospitals.
Plasmid DNA preparation and plasmid fingerprinting
Plasmid DNA was purified with the Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany), following the manufacturer's procedure, as described by Bauernfeind et al.33 For fingerprinting analysis, approximately 5 µg of plasmid DNA was digested with 10 U of PstI restriction enzyme (MBI Fermentas, Vilnus, Lithuania) for 2 h at 37°C. DNA was electrophoresed in 1% agarose gels (FMC Bioproducts, Rockland, ME, USA).
PCR detection of ESBL-encoding genes
Total DNA preparations from clinical isolates were used as templates in specific PCR reactions for the detection of genes coding for ESBLs. SHV-A24 and SHV-C: 5'-CGCACCCCGCTTGCT-3' primers were used for amplification of blaSHV genes. The SHV-C primer anneals to the part of a blaSHV gene that codes for amino acid residues from positions 238 to 243,34 and was designed to amplify genes coding for SHV ß-lactamases containing the G238S and E240K substitutions specifically.35 The SHV-C and SHV-A primers did not amplify any products from DNA preparations of strains of K. pneumoniae, which produced ß-lactamases with pI values of 7.47.6 but not 8.2 (results not shown). P1C and P2D primers26 designed for recognition of the blaCTX-M-1 gene sequence36 were used for amplification of blaCTX-M genes. PCR reactions were run under conditions described previously.24,26
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Results |
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Typing using the RAPD-7 primer32 was carried out on all the isolates analysed, except for the single strains of K. oxytoca and M. morganii. Results of the analysis are shown in Tables I and II. All four C. freundii isolates (including three isolates from the urological ward) produced the same RAPD pattern. Four different RAPD types were revealed in the group of eight S. marcescens isolates, the predominant pattern (pattern a) characterizing five isolates from one of the surgical wards. Of 12 K. pneumoniae isolates, four isolates from three different wards were characterized by the same RAPD pattern (pattern a), and in the collection of seven E. coli strains, three isolates were shown to be indistinguishable by RAPD (pattern c). Typing differentiated the two E. cloacaeisolates. Typing of the E. coli 173 isolate, performed in the context of another collection of strains, has been published elsewhere.28
IEF of ß-lactamases
Crude sonicates of all the clinical isolates were subjected to IEF. The pI values of the ß-lactamases visualized are listed in Tables I and II. ß-lactamases with a pI of 8.4 were found in extracts of 27 isolates, including eight of 12 K. pneumoniae, three of seven E. coli, and all S. marcescens, C. freundii, E. cloacae, K. oxytoca and M. morganii (Table I). In all but one of the cases, the 8.4 ß-lactamases were co-identified with enzymes with a pI of 5.4. Four K. pneumoniae and four E. coli isolates produced ß-lactamases with a pI of 8.2, which often were also accompanied by the 5.4 ß-lactamases (Table II). In some species (K. pneumoniae, S. marcescens, E. cloacae, K. oxytoca and M. morganii), additional major ß-lactamase bands with characteristic pI values were detected. These most probably represented species-specific enzymes. In almost all cases, isolates of a given species representing the same RAPD type were also characterized by identical ß-lactamase content (the only exception was the E. coli RAPD type c; Table II). The IEF data for the E. coli 173 isolate has already been published.28
Detection of ESBL activity
Protein extracts of all the isolates were used for the detection of ceftazidime- and cefotaxime-hydrolysing activities. Results are shown in Tables I and II. For each isolate, the ESBL activity was assigned to either pI 8.2 or 8.4 ß-lactamases. In each case, the 8.2 ß-lactamase was able to hydrolyse both ceftazidime and cefotaxime. The pI 8.4 enzyme hydrolysed only cefotaxime under the conditions used in this experiment.
Mating and ß-lactamase content of transconjugants
All clinical isolates were subjected to conjugation experiments, in which cefotaxime or ceftazidime were used as selective agents for recombinant strains. ß-Lactamases produced by transconjugants were studied by IEF. Results of the analyses are presented in Tables I and II.
Twenty of the 27 isolates expressing the ESBLs with a pI of 8.4 produced recombinants with high frequency (up to 10-2 transconjugants per donor cell). These transconjugants expressed ß-lactamases with pIs of 8.4 and 5.4 (Table I). Five of eight isolates expressing the pI 8.2 ESBLs produced transconjugants, in which ß-lactamases of 8.2 alone or accompanied by enzymes of pI 5.4 (if only these were produced by the clinical isolates) were identified (Table II). In almost all cases, isolates of a given species belonging to a single RAPD type behaved uniformly in mating experiments (the only exception being the S. marcescens RAPD type a, grouping both conjugating and non-conjugating isolates; Table I).
Identification of ESBLs by PCR
Total DNA preparations of all isolates were used as templates in specific PCR reactions, in order to identify the ESBLs produced. For all the isolates expressing the 8.4 cefotaxime-hydrolysing enzymes (Table I), PCR products were of the expected size of approximately 1 Kb, using P1c and P2d primers (specific for the blaCTX-M-1 gene) revealing the presence of genes coding for the CTX-M family of ESBLs. These are related to the CTX-M-1/MEN-1 enzyme. For all isolates expressing pI 8.2 ESBLs (Table II), PCR products of the expected size of approximately 220 bp were found using SHV-A and SHV-C primers. These reactions indicated that these enzymes belonged to the SHV family of ß-lactamases, and contained the G238S and E240K substitutions (a slightly different PCR with DNA of the E. coli 173 isolate has been reported elsewhere28).
Antimicrobial susceptibility testing of clinical isolates and transconjugants
MICs were determined for clinical isolates representing each distinguished RAPD type or different ß-lactamase pattern, and those exhibiting different behaviour in mating experiments. These results are shown in Tables III and IV, with data obtained for the transconjugants. Clinical isolates expressing the CTX-M ESBLs (Table III) were found to have MICs of cefotaxime (32512 mg/L) substantially higher than those of ceftazidime (14 mg/L). The MICs of cefepime and aztreonam for these isolates were in between those of cefotaxime and ceftazidime (cefepime, 464 mg/L; aztreonam, 432 mg/L). ß-lactamase inhibitors reduced the MICs of ß-lactams in all the combinations tested; this effect being particularly remarkable in the case of cefotaxime. Only S. marcescens, E. cloacae, C. freundii and M. morganii isolates were resistant to cefoxitin (MICs 32128 mg/L). All isolates were susceptible to imipenem (MICs 0.060.5 mg/L). The vast majority of isolates were resistant to gentamicin and amikacin (MICs >256 mg/L).
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Higher MICs of ceftazidime (4256 mg/L) and aztreonam (8256 mg/L) were characteristic of the group of K. pneumoniae and E. coli isolates expressing SHV ESBLs (Table IV), when compared with those of cefotaxime (MICs 132 mg/L) and cefepime (MICs 0.258 mg/L). ß-Lactamase inhibitors decreased MICs of ß-lactams in all the combinations tested. K. pneumoniae isolates were more resistant to the majority of ß-lactams tested than were the E. coli strains. No isolates was found to be resistant to gentamicin, with the exception of the K. pneumoniae 440 strain (MIC 128 mg/L). MICs for the E. coli 173 isolate have been presented elsewhere.28
The MIC patterns of transconjugants expressing the SHV ESBLs (Table IV) reflected data obtained for clinical isolates. The MICs of ceftazidime (232 mg/L) and aztreonam (464 mg/L) were higher than those of cefotaxime (0.58 mg/L), and transconjugants of K. pneumoniae strains were characterized by higher MICs than recombinants produced by E. coli isolates.
Plasmid fingerprinting
Plasmid DNA was purified from representative isolates for each distinguishable RAPD type, different ß-lactamase pattern or distinctive product of mating experiments. If a plasmid profile contained more than one plasmid and the transconjugant was available, plasmid DNA was also isolated from the recombinant strain. Results of the analysis are shown in Tables I and II, and Figures 1 and 2.
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The SHV ESBLs-producing isolates contained high molecular weight plasmids (Table II; Figure 2), in some cases in association with plasmids of lower molecular weight (K. pneumoniae 174, 280, 440; E. coli 22). All the PstI restriction patterns were found to be different from each other; the pattern of the E. coli 173 isolate plasmid has been presented elsewhere.28
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Discussion |
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Analysis revealed ESBLs of two different pI values. The most prevalent were enzymes with a pI of 8.4, produced by 27 isolates that exhibited cefotaxime but not ceftazidime-hydrolysing activity. This activity was reflected in the antimicrobial susceptibility patterns of the strains. MICs of cefotaxime were significantly higher than those of ceftazidime for all isolates producing the 8.4 ß-lactamases. PCR with specific primers has revealed that these enzymes belong to the CTX-M family of ESBLs, and are closely related to the CTX-M-1/MEN-1 ß-lactamase.36 ,37 ,38 It is likely that the isolates produced the same enzyme (CTX-M-3 ß-lactamase pI 8.4), originally identified in three isolates of C. freundii from the same hospital collected in July 1996.26 These three CTX-M-3-producing C. freundii isolates were found to represent the same RAPD type, the same EcoRI1HindIII ribotype and to carry plasmids of the identical PstI restriction pattern (A1) as the C. freundii isolates analysed here (data not shown). The expression of other sequence variants of CTX-M enzymes by some isolates cannot be excluded. In particular, E. coli 6, which with transconjugant was characterized by remarkably elevated MICs of ß-lactams. These data, together with recent reports on outbreaks caused by Salmonella typhimurium strains producing CTX-M-2-related enzymes from Latvia39 and Russia,40,41 suggest that the CTX-M family of ESBLs is becoming more common in European health care facilities.
The second group of ESBLs observed were the SHV enzymes, with a pI of 8.2, produced by four E. coli and four K. pneumoniae isolates. These isolates were characterized by different antimicrobial susceptibility patterns when compared with CTX-M producers, and MICs of ceftazidime that were higher than those of cefotaxime. The 8.2 ESBLs were most probably SHV-5,35,42 SHV-9,43 SHV-1244 or other related ß-lactamases, as indicated by the pI value and results of amplification of the genes. SHV-5 and related enzymes seem to be the most prevalent ESBLs all over the world15,44,45,46,47,48,49,50 as well as in Poland, where they have been identified in several small studies.24,51
The epidemiology of CTX-M-producing microorganisms in the Praski Hospital is multi-faceted. The ESBL-encoding genes have spread efficiently, by plasmid transmission. Two distinct plasmid types carrying the blaCTX-M genes, PstI restriction types A and B, have been identified. Type A plasmids have been purified from multiple isolates of K. pneumoniae, S. marcescens, C. freundii, E. coli, E. cloacae and K. oxytoca. The majority produced transconjugants with ease, indicating the transmission potential of the A-type plasmids. The ß-lactamase content and susceptibility patterns of transconjugant strains indicated that these plasmids also contained genes encoding the 5.4 ß-lactamases (probably TEM-1), as well as a gene(s) determining resistance to aminoglycosides. Some variability in the fingerprints of the A family plasmids could be correlated either with the loss of transfer functions by particular variants (the A4 and A5 plasmids of K. pneumoniae 179 and S. marcescens12, respectively) or with some differences in antimicrobial susceptibilities among isolates and their transconjugants (higher MICs of ß-lactams for E. coli6, plasmid A2; lower MICs of aminoglycosides for S. marcescens275 and 278, plasmids A6 and A7, respectively). The heterogeneity of the A-type plasmids may have resulted from various DNA recombination and mutation events, and may be a reflection of the advanced process of plasmid microevolution within the population of CTX-M producers. The non-conjugative plasmid B, characteristic of the single E. coli279 isolate, was specified by a markedly different PstI fingerprint from that of the A-type plasmids, and from the plasmid present in the E. coli2527/96 strain, isolated in July 199626 (not identified in the material analysed here). This revealed that the blaCTX-M genes were present in at least three different types of plasmid in 1996/1997. It could be that all these types of plasmid are related, evolution resulting in molecules with dissimilar fingerprints, but it seems more likely that the blaCTX-M genes have been transferred to plasmids of different origins.
It is likely that clonal spread has accounted for other aspects of the epidemiology of CTX-M-producing organisms. The four C. freundiiisolates were characterized by the same RAPD patterns and the same ribotypes (results not shown) which, as mentioned above, were also identical to those produced by the original three C. freundiiCTX-M-3-expressing isolates from July 1996.26 The majority of isolates of this species were collected from patients in the urological ward. This ward was probably the source of spread for other wards in the hospital. RAPD analysis also suggested close similarities between five isolates of S. marcescens (type a, all of which were recovered from patients in the single surgical ward) and four isolates of K. pneumoniae (also type a), two of which were collected from patients in the one internal medicine ward). All these results suggest that the hospital-wide outbreak of CTX-M-producing strains was due to plasmid dissemination, with clonal spread of several strains and potentially also to ESBL gene transfer between different plasmids.
The epidemiology of SHV-type ESBL-producing strains was less clear than that of the CTX-M producers. All four K. pneumoniae isolates expressing these enzymes represented different RAPD types. They were also characterized by different ß-lactamase profiles or behaved in a different way in mating experiments. Plasmids purified from these strains produced various PstI fingerprints. Among the four E. coliisolates, three had the same RAPD pattern, but only two of these (E. coli20 and 437) contained identical ß-lactamases. The high degree of heterogeneity of SHV producers may suggest the multiple selection of the SHV-type ESBL variants and/or multiple introduction events of these strains from other hospitals by means of patient transfer.
Data presented in this paper illustrate the complexity and extent of spread of ESBL-producing microorganisms in Polish hospitals. If the rate of increase in consumption of extended ß-lactamases is not halved and the spread of resistance not monitored, organisms producing different ESBLs will continue to be selected and parallel outbreaks, such as the one described here, will continue.
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Acknowledgments |
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Notes |
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References |
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Received 10 December 1998; returned 20 April 1999; revised 20 May 1999; accepted 14 June 1999