CMY-2-producing Salmonella enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Escherichia coli strains isolated in Spain (October 1999–December 2000)

Ferran Navarroa, Emilio Perez-Trallerob,*, José M. Marimonb, Roxana Aliagaa, Maria Gomarizb and Beatriz Mirelisa

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
CMY-2 plasmid-mediated AmpC ß-lactamase (CMY-2) was detected in 21 isolates from two hospitals located in different geographical regions of Spain between October 1999 and December 2000. The isolates comprised two Salmonella enterica serovars (Mikawasima and Montevideo), 16 Escherichia coli, one Klebsiella pneumoniae, one Klebsiella oxytoca and one Proteus mirabilis. In addition to the expected resistance to ß-lactams, including extended-spectrum cephalosporins and cefoxitin, all isolates showed a broad spectrum of associated resistance. All were resistant to sulfamethoxazole, chloramphenicol, tetracycline and streptomycin, and all but two were also resistant to gentamicin. Five isolates were studied in detail and all transferred CMY-2 and other resistance determinants by conjugation. Genomic DNA restriction pattern analysis of the E. coli isolates excluded the dissemination of a single clone. To the best of our knowledge this is the first time that CMY-2 has been detected in P. mirabilis, K. oxytoca and S. enterica serovars Mikawasima and Montevideo. It is also the first time that CMY-2 has been described in Spain.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Until recently, in enterobacteria such as Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enterica and probably Proteus mirabilis, which naturally lack chromosomal class C ß-lactamases, or in Escherichia coli and Shigella, which express them at a very low level, resistance to third- generation cephalosporins was associated with the presence of plasmid-mediated class A ß-lactamases, referred to generically as extended-spectrum ß-lactamases (ESBLs).1,2 In the past 10 years this panorama has become even more complex owing to the appearance of strains harbouring plasmid-mediated ß-lactamases derived from chromosomal class C ß-lactamases, referred to as plasmid-mediated AmpC-type ß-lactamases.1 These strains are characterized by a pattern of resistance to ß-lactams similar to the ESBLs, but which also includes combinations of ß-lactams with ß-lactamase inhibitors as well as cefoxitin,1 with the rare exception of strains harbouring the ß-lactamase ACC-1, which are cefoxitin susceptible.3 The objective of this study is to report on the presence of enterobacteria strains in Spain that carry CMY-2 plasmid-mediated AmpC ß-lactamase (CMY-2).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Selection of strains

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 l’Étoile, France). All isolates included in the study were clinically relevant.

Antibiotic susceptibility testing

The susceptibility studies were performed by the disc diffusion method on Mueller–Hinton 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 18–20 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 Mueller–Hinton 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 {approx}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 1Go.


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Table 1. Oligonucleotide sequences used for PCR and sequencing
 
The products obtained by PCR of the blaCMY-2 gene were sequenced using a ABI PRISM 310 Genetic Analyzer (Perkin Elmer Applied Biosystem, Foster City, CA, USA). The primers used were the ampC1 and ampC2 mentioned previously, and the CMY-2MIG and ampC A22 intermedi-ate primers. The nucleotide sequence obtained and the amino acid sequence deduced were analysed with the software available at the Web site of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih. gov).

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 1Go). 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 ampC–ampR 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 947–965 region of the ampR9 gene (Table 1Go). 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Among 12 090 clinical isolates of enterobacteria studied, 153 had a resistance phenotype, suggesting that they harboured a cefoxitin-resistant AmpC ß-lactamase (Table 2Go). With the ampC-specific primers the expected amplicon was obtained in 21 isolates. All amplicons showed a high level of homology in terms of nucleotide sequence (>97%) and 100% identity with the amino acid sequence in comparison with the previously described CMY-2.11 (GenBank accession number X91840).


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Table 2. AmpC and CMY-2 producing clinical isolates from two Spanish hospitals (October 1999–December 2000)
 
The first clinical isolate with CMY-2, detected in October 1999, was an E. coli isolated from the urine of an 83-year-old woman suffering from uncomplicated cystitis who did not require hospitalization. The other strains were isolated between November 1999 and December 2000 from hospitalized patients and outpatients (Table 3Go). Two outpatients had acute Salmonella gastroenteritis, with fever, abdominal pain, vomiting and diarrhoea. No imported cases were observed.


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Table 3. Characteristics of the clinical isolates studied
 
Broth microdilution was used to determine the MIC50, MIC90 and MIC range for the 21 clinical isolates, which were as follows (in mg/L): 16, 32 (2–32) for cefotaxime; 16, >16 (4–>16) for ceftazidime; and 8, 16 (<8–>16) for aztreonam, respectively. All isolates were susceptible to imipenem. The pattern of cross-resistance to non-ß-lactam drugs was nearly identical for all strains although two clinical isolates were susceptible to kanamycin, gentamicin and tobramycin and several strains were resistant to co-trimoxazole, nalidixic acid and ciprofloxacin (Table 3Go). The different PFGE patterns of nine E. coli clinical isolates ruled out the dissemination of a single clone (FigureGo).



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Figure. PFGE patterns of 12 E. coli strains. Lanes 1–9: E. coli clinical isolates (273235, 287811, 289164, 215568, 266582, 226927, 272340, 226689, 233405); lane 10: lambda ladder (Bio-Rad); line 11: E. coli BM694 Cla (recipient strain); lanes 12 and 13: E. coli harbouring the pI {approx}9 and pI 5.4 ß-lactamases transconjugants from S. enterica serovar Montevideo and E. coli 278811, respectively.

 
The five clinical isolates studied in detail (E. coli 273235, S. enterica serovar Mikawasima, S. enterica serovar Montevideo, K. pneumoniae and P. mirabilis) harboured two ß-lactamases, one with a pI of {approx}9 and another with a pI of 5.4. The five strains were conjugated with E. coli BM694 Cla (NalR), and one or several transconjugants of each strain were selected. Both ß-lactamases (pI of {approx}9 and pI of 5.4) were transferred by conjugation in the same step into E. coli BM694 C1a, with ampicillin (100 mg/L), cefotaxime (1 mg/L) and nalidixic acid (50 mg/L) as selector antibiotics. No transconjugants harbouring only the ß-lactamase with a pI of {approx}9 were obtained. Transconjugants of E. coli harbouring only the ß-lactamase with a pI of 5.4 were obtained using ampicillin and nalidixic acid without cefotaxime as selector antibiotics. In these strains, the PCR assay with the specific TEM primers produced the expected band. Therefore, this enzyme was identified as a possible TEM-1 ß-lactamase. The K. pneumoniae clinical isolate also produced a band of pI 7.6, which was not transferred to the transconjugant and was compatible with chromosomal SHV-1. Following iodometric analysis with ceftriaxone, only the ß-lactamase with a pI {approx}9 showed activity. Table 4Go shows the MICs of a variety of antibiotics for the five clinical isolates, one transconjugant of each and the E. coli BM694 Cla recipient strain.


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Table 4. MICs (mg/L) obtained by broth microdilution of 17 antibiotics
 
The amplification reaction performed on the transconjugants using ampC-specific primers was also positive, and a 1143 bp fragment was detected in all of them. The sequence obtained for the transconjugants and for the corresponding clinical isolates showed a 100% amino acid identity to CMY-2 (GenBank accession number X91840).

These transconjugants harbouring the two ß-lactamases (pI of {approx}9 and pI of 5.4) demonstrated a multidrug resistance pattern similar to that of the clinical isolates (Table 4Go). The PCR with the specific primers of the ampR gene was negative for the five clinical isolates and their transconjugants.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study describes the presence of CMY-2 in five species of enterobacteria: S. enterica (serovars Mikawasima and Montevideo), K. pneumoniae, K. oxytoca, P. mirabilis and E. coli. The resistance pattern observed in both these clinical isolates and their transconjugants was similar to that reported earlier in other enterobacteria which harboured CMY-2 or CMY-2b.8,1114

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


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Guillem Prats for his helpful advice and comments and the foundations ‘Mª Francisca Roviralta’ and ETIP for financial support. This work was partially supported by grants FIS 98/1293 and FIS 98/1522.


    Notes
 
* Corresponding author. Tel: +34-943-00-70-46; Fax: +34-943-00-70-63; E-mail: labmikro{at}terra.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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15 . Bret, L., Chanal-Claris, C., Sirot, D., Chaibi, E. B., Labia, R. & Sirot, J. (1998). Chromosomally encoded AmpC-type ß-lactamase in a clinical isolate of Proteus mirabilis. Antimicrobial Agents and Chemotherapy 42, 1110–4.[Abstract/Free Full Text]

16 . Verdet, C., Arlet G. A., Redjeb, S. B., Hassen, A. B., Lagrange, P. H. & Philippon, A. (1998). Characterisation of CMY-4, an AmpC-type plasmid-mediated ß-lactamase in a Tunisian clinical isolate of Proteus mirabilis. FEMS Microbiology Letters 169, 235–40.[ISI][Medline]

17 . Tzouvelekis, L. S., Tzelepi, E. & Mentis, A. F. (1994). Nucleotide sequence of a plasmid-mediated cephalosporinase gene (blaLAT-1) found in Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 38, 2207–9.[Abstract]

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19 . Gazouli, M., Tzouvelekis, L. S., Prinarakis, E., Miriagou, V. & Tzelepi, E. (1996). Transferable cefoxitin resistance in enterobacteria from Greek hospitals and characterization of a plasmid-mediated group 1 ß-lactamase (LAT-2). Antimicrobial Agents and Chemotherapy 40, 1736–40.[Abstract]

20 . Gazouli, M., Tzouvelekis, L. S., Vatopoulos, A. C. & Tzelepi, E. (1998). Transferable class C ß-lactamases in Escherichia coli strains isolated in Greek hospitals and characterization of two enzyme variants (LAT-3 and LAT-4) closely related to Citrobacter freundii AmpC ß-lactamase. Journal of Antimicrobial Chemotherapy 42, 419–25.[Abstract]

21 . Marchese, A., Arlet, G., Schito, G. C., Lagrange, P. H. & Philippon, A. (1998). Characterization of FOX-3, an AmpC-type plasmid-mediated beta-lactamase from an Italian isolate of Klebsiella oxytoca. Antimicrobial Agents and Chemotherapy 42, 464–7.[Abstract/Free Full Text]

22 . Bou, G., Oliver, A., Ojeda, M., Monzón, M. & Martínez-Beltrán, J. (2000). Molecular characterization of FOX-4, a new AmpC-type plasmid-mediated ß-lactamase from an Escherichia coli strain isolated in Spain. Antimicrobial Agents and Chemotherapy 44, 2549–53.[Abstract/Free Full Text]

23 . Barnaud, G., Arlet, G., Verdet, C., Gaillot, O., Lagrange, P. H. & Philippon, A. (1998). Salmonella enteritidis: AmpC plasmidmediated inducible ß-lactamase (DHA-1) with an ampR gene from Morganella morganii. Antimicrobial Agents and Chemotherapy 42, 2352–8.[Abstract/Free Full Text]

24 . Bauernfeind, A., Schweighart, S., Dornbusch, K. & Giamarellou, H. (1990). A transferable cephamycinase in Klebsiella pneumoniae. In Program and Abstracts of the Thirtieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, 1990. Abstract A190, p. 118. American Society for Microbiology, Washington, DC.

25 . Stapleton, P. D., Shannon, K. P. & French, G. L. (1999). Carbapenem resistance in Escherichia coli associated with plasmiddetermined CMY-4 ß-lactamase production and loss of an outer membrane protein. Antimicrobial Agents and Chemotherapy 43, 1206–10.[Abstract/Free Full Text]

26 . Bradford, P. A., Urban, C., Mariano, N., Projan, S. J., Rahal, J. J. & Bush, K. (1997). Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrobial Agents and Chemotherapy 41, 563–9.[Abstract]

Received 18 December 2000; returned 6 April 2001; revised 29 May 2001; accepted 3 July 2001