a Microbiology Department, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma 08025 Barcelona; b Microbiology Department, Complejo Hospitalario Donostia, Universidad del País Vasco, Avenida Dr. Beguiristain s/n, 20014 San Sebastián, Spain
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Abstract |
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Introduction |
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Materials and methods |
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The isolates included in the study were selected from species of Enterobacteriaceae obtained in two clinical microbiology laboratories in two distant regions of Spain (Barcelona in Catalonia, and Gipuzkoa in Basque Country). The screening included clinical isolates of E. coli, Shigella spp., K. pneumoniae, K. oxytoca, P. mirabilis and Salmonella spp. Identification was performed by standardized methods4 using home-made galleries and the API System 20E (bioMérieux, Marcy lÉtoile, France). All isolates included in the study were clinically relevant.
Antibiotic susceptibility testing
The susceptibility studies were performed by the disc diffusion method on MuellerHinton agar according to NCCLS5 guidelines and by broth microdilution method based on NCCLS guidelines,6 using cation-adjusted Mueller Hinton broth in custom-dried 96-well trays (Sensititre; Trek Diagnostic Systems, West Sussex, UK). Cultures were incubated at 35°C for 1820 h in ambient air. E. coli ATCC 25922 was used as a control strain.
The inhibitory effect of cefotaxime and ceftazidime in combination with clavulanic acid was determined by Etest (AB Biodisk, Solna, Sweden) and/or by disc diffusion (using co-amoxiclav discs).
Selection criteria used with the clinical strains for probable production of AmpC type ß-lactamase
The isolates selected for the study were those which, according to the antibiogram obtained by disc diffusion and/or microdilution methods, showed resistance to co-amoxiclav and/or cefoxitin, as well as reduced susceptibility to cefotaxime (MIC 2 mg/L), ceftriaxone (MIC
2 mg/L) or ceftazidime (MIC
2 mg/L). In a second step, the loss of synergy between cefotaxime and/or ceftazidime and clavulanic acid was assessed. Later, a PCR assay using the ampC gene specific primers was performed on all isolates that yielded the aforementioned phenotype of resistance. ß-Lactamase analysis by isoelectric focusing (IEF) and transfer of resistance determinants were performed with five of the isolates.
Transfer of resistance determinants
The conjugation assay was performed on solid media using E. coli BM694 C1a (NalR) as recipient strain. Transconjugants were selected on MuellerHinton agar supplemented with ampicillin (100 mg/L), cefotaxime (1 mg/L) and nalidixic acid (50 mg/L).
Extraction of ß-lactamases and analysis by IEF
Crude extracts of ß-lactamase were obtained by ultrasonication. Analytical IEF of these extracts was performed on polyacrylamide gel with pH ranging from 3.5 to 11 (Amersham Pharmacia Biotech, Uppsala, Sweden). Enzymatic activity was assayed by the iodometric method7 with 250 mg/L of ceftriaxone and 250 mg/L of penicillin G. Crude extracts of the TEM-1 (pI 5.4), TEM-2 (pI 5.6), TEM-3 (pI 6.3), SHV-3 (pI 7.0), SHV-2 (pI 7.6), CTX-M-9 (pI 8.0), SHV-5 (pI 8.2) and CTX-M-4 (pI 8.4) plasmid ß-lactamases were used as controls.
Detection and characterization of ß-lactamase by PCR amplification of DNA and sequencing
The gene that encoded CMY-2 (blaCMY-2) was amplified using the ampC1 and ampC2 consensus primers.8 Amplification conditions were as follows: denaturation at 94°C for 5 min, 30 cycles (denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 1 min), and final extension at 72°C for 10 min. The primer sequences are shown in Table 1.
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The presence of the TEM-1 gene in the clinical isolates and transconjugants was detected by IEF and by PCR assay with specific TEM primers (Table 1). The amplification conditions were 24 cycles with denaturation at 96°C for 15 s, annealing at 50°C for 15 s and extension at 72°C for 2 min.
Detection of ampR regulator gene
The presence or absence of the ampR regulator gene was determined by PCR with the CFAmpRd primer for the ampCampR intercistronic region, specifically at nucleotides 52 to 33, based on the initial codon of several sequences of the ampR gene, which is also common in the initial region of some class C ß-lactamases (GenBank accession numbers: M37839, Y17716, M27222, X78117, X91840, AB016612, AB016611, D13207, D44479, AJ007826, X74512, X51632, X76636, X04730, D85910, AF211348) and the CFAmpRFr primer for the 947965 region of the ampR9 gene (Table 1). In this case the amplification conditions were as follows: denaturation at 94°C for 5 min, 30 cycles (denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min), and final extension at 72°C for 10 min. The expected amplified product of c. 928 bp was obtained in a clinical strain of Citrobacter freundii used as positive control.
Pulsed-field gel electrophoresis
Genomic DNA patterns of the E. coli clinical isolates were analysed by pulsed-field gel electrophoresis (PFGE) as described previously10 with the following modifications: cells were adjusted to an optical density of 1.00 at a wavelength of 560 nm; before lysis buffer incubation, plugs were incubated for 3 h at 37°C in a lysozyme buffer (10 mM Tris, 50 mM EDTA pH 8.0, 0.2% sodium desoxycholate, 0.5% sodium lauryl sarcosine and 0.25 mg/L lysozyme, all from Sigma Chemical Co., St Louis, MO, USA). After washes, plug slices were incubated for 16 h at 37°C in 1x restriction buffer containing 30 U of XbaI (Amersham Pharmacia, Amersham, UK). The electrophoretic conditions were as follows: linear pulse time ramp from 5 to 50 s, run time 22 h, angle 120°, gradient 6 V/cm.
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Results |
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These transconjugants harbouring the two ß-lactamases (pI of 9 and pI of 5.4) demonstrated a multidrug resistance pattern similar to that of the clinical isolates (Table 4
). The PCR with the specific primers of the ampR gene was negative for the five clinical isolates and their transconjugants.
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Discussion |
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Since 1989, plasmid-mediated AmpC-type ß-lactamases have been identified throughout the world, and although the prevalence of the different types may vary with geographical region, most are widespread. Most of these plasmid-mediated AmpC-type ß-lactamases detected up until 1998 in the Mediterranean area belonged to a homogeneous group (CMY-2 to CMY-5, and LAT-1 to LAT-4) related to the chromosomally encoded AmpC-type ß-lactamase of C. freundii.8,11,1420 In Italy, and recently in Spain, the cephalosporinases FOX-3 and FOX-4, which belong to another group of AmpC type ß-lactamases, have been reported in K. oxytoca21 and E. coli.22
In the cases described previously, CMY-2 has been transferred primarily by conjugation with TEM-1 ß-lactamase, and has shown a pattern of multidrug resistance that nearly always included chloramphenicol, tetracycline, streptomycin, gentamicin and tobramycin.8,9,13,14Research is being conducted on the identity of the plasmid encoding CMY-2 as well as the other non-ß-lactam resistance determinants. The initial findings seem to indicate that TEM-1 ß-lactamase and CMY-2 are found in different plasmids, since transconjugants were obtained with only TEM-1 ß-lactamase.
In strains with plasmid-mediated AmpC ß-lactamases the presence of a regulator gene associated with the structural gene of ß-lactamase, such as those found in the strains with chromosomally encoded ß-lactamases, has not been generally observed. The only exception has been a strain of S. enterica serovar Enteritidis with cephamycinase DHA-1, which expressed an inducible resistance phenotype and contained the ampR regulator gene.23 In the isolates considered in this study, no induction was observed, nor was an amplicon obtained, when specific primers of the ampR gene were used.
To our knowledge, with the exception of isolation of an E. coli strain with FOX-422 (isolated in Spain, but far from the Mediterranean area, in the Canary Islands), this study documents the first time that plasmid-mediated AmpC-type ß-lactamase strains have been isolated in Spain. It is also the first time that CMY-2 has been detected in P. mirabilis, K. oxytoca and S. enterica serovars Mikawasima and Montevideo.
CMY-2 ß-lactamase was described for the first time in 1990.24 It is one of the most frequent plasmid-mediated AmpC ß-lactamases which, in less than 10 years, has spread widely. In our area, in just 15 months, strains with CMY-2 and associated multidrug resistance have been detected in 21 strains from five different species of Enterobacteriaceae, with two serovars among the Salmonella. The overall prevalence of clinical isolates harbouring the CMY-2 among the enterobacteria studied was relatively frequent (0.17%), with no statistical differences between the species studied. The two hospitals where the study was performed are approximately 530 km apart. This fact, as well as the variety of species involved, argued against the occurrence of an epidemic focus in a particular area. Moreover, the nine E. coli isolated in the same region were analysed by PFGE and the different patterns obtained ruled out the dissemination of a single clone of this species.
The systematic study began at the end of 1999. Based on a retrospective review, it was not possible to hypothesize on the actual prevalence of most of the bacterial species included in this study during previous years. None the less, the present prevalence of CMY-2 among enterobacteria is high enough to be constitute a source of further spread, especially among subjects receiving antibiotic therapy. It would not be surprising if the incidence of these strains increased in the future. Moreover, this is a cause for concern, as the microorganisms that carry these plasmid-mediated AmpC ß-lactamases frequently harbour additional resistance to several non-ß-lactam drugs. The only ß-lactam antibiotics available to treat infections produced by these microorganisms are the carbapenems. However, carbapenem resistance has been reported in these strains.25,26
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Acknowledgements |
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Notes |
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References |
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Received 18 December 2000; returned 6 April 2001; revised 29 May 2001; accepted 3 July 2001