SGM Journals
Research Article

Characterization of two plasmid-encoded cefotaximases found in clinical Escherichia coli isolates: CTX-M-65 and a novel enzyme, CTX-M-87

Jun Yin, Jun Cheng, Zhen Sun, Ying Ye, Yu-Feng Gao, Jia-Bin Li, Xue-Jun Zhang

  • 1Department of Infectious Diseases, First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, PR China
  • 2Clinical Medicine Post-Doctorate Position of Anhui Medical University, Hefei, Anhui 230032, PR China
  • 3Institute of Dermatology, Anhui Medical University, Hefei, Anhui 230032, PR China
  • Correspondence
    Jia-Bin Li
    lijiabin948{at}vip.sohu.com
    Xue-Jun Zhang
    ayzxj{at}vip.sina.com

    • Journal of Medical Microbiology 2009; 58(6):811–815 · https://doi.org/10.1099/jmm.0.006007-0

    View at publisher PubMed

    Abstract

    Three clinical strains of Escherichia coli (p168, p517 and p667) were collected in 2006 from three hospitals in Anhui Province (China). PCR and DNA sequencing revealed that E. coli p168 carried a novel extended-spectrum β-lactamase (ESBL), which was designated CTX-M-87. The extended-spectrum β-lactamase which was carried by E. coli p517 and E. coli p667 was previously named CTX-M-65. The deduced amino acid sequence of CTX-M-87, with pI 9.1, differed from that of CTX-M-14 by the substitutions Ala77→Val and Pro167→Leu. Like CTX-M-14, CTX-M-87 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and had high affinity for cefuroxime and cefotaxime. These data show that mutations at position 167 in CTX-M do not always affect catalytic activity and substrate preference.

    • ↵†These authors contributed equally to this work.

    • The GenBank/EMBL/DDBJ accession number for the CTX-M-87 gene sequence is EU545409.

    INTRODUCTION

    The first plasmid-borne extended-spectrum β-lactamase (ESBL) of the CTX-M type (MEN-1, CTX-M-1) was reported in 1990 (Bauernfeind et al., 1990; Bush, 2001). In contrast to many ESBLs found previously, the early CTX-M enzymes determined high-level resistance to cefotaxime but did not significantly affect ceftazidime (Barthélémy et al., 1992). CTX-M enzymes have caused outbreaks of cefotaxime-resistant enterobacteria mainly in South America, Eastern Europe and Japan (Bradford et al., 1998; Gniadkowski et al., 1998; Ishii et al., 1995; Ma et al., 1998). From the early 1990s to the present, members of the Enterobacteriaceae (mostly Escherichia coli) producing different ESBLs, such as the CTX-M enzymes, have emerged within the hospital and community setting as an important cause of urinary tract infections (Babic et al., 2006). Like Eastern Europe and Japan (Gazouli & Legakis, 1998; Gniadkowski et al., 1998; Bonnet, 2004; Gazouli et al., 1998a), China is an important source of CTX-M-producing bacteria. Our studies that have investigated community-onset infections caused by CTX-M-producing E. coli isolates showed that these isolates are often not clonally related (Cheng et al., 2008).

    The CTX-M-type enzymes are much more active against cefotaxime than against ceftazidime and aztreonam. The amino acid residues proposed to be critical for their extended-spectrum activity, Ser237 and Arg276, seem to be involved in the cefotaxime-hydrolysing activity, but mutagenesis experiment of these residues led to only slight changes in their catalytic activities (Gazouli et al., 1998a, b). Pro167 is located in the omega loop. Substitutions at position 167 were observed in more than 10 CTX-M mutants which exhibited an improved enzymic activity against ceftazidime (Delmas et al., 2006). In the current study, we report a novel CTX-M-type cefotaxime-hydrolysing β-lactamase, which we have designated CTX-M-87. The other enzyme which we studied was CTX-M-65. The deduced amino acid sequence of CTX-M-87 differed from that of CTX-M-14 by the substitutions Ala77→Val and Pro167→Leu; CTX-M-65 differed from CTX-M-14 by the substitutions Ala77→Val and Ser272→Arg.

    METHODS

    Clinical strains.

    Three clinical strains were isolated from three different patients hospitalized in 2006 at upper first class hospitals with more than 500 beds in Anhui Province (China). E. coli p168 was obtained from an incisional secretion, and E. coli p517 and E. coli p667 were obtained from blood. E. coli ATCC 25922, Klebsiella pneumoniae ATCC 700603 and E. coli strain CTX-M-14 (a transformant carrying CTX-M-14 in a vector) were used as control strains. All strains were stored in the Anhui Center of Surveillance Bacterial Resistance.

    Conjugation experiment.

    To determine whether resistance was transferable, conjugations were performed with a streptomycin-resistant recipient, E. coli C600 (Lac−). Conjugation mixtures were plated on MacConkey agar (Tianhe) containing streptomycin (500 μg ml−1) and cefotaxime (2 μg ml−1) and then incubated for approximately 20 h at 37 °C.

    Antimicrobial susceptibility testing.

    MICs were determined with the Mueller–Hinton agar (Oxoid) dilution method. Throughout this study, the results were interpreted by using the Clinical and Laboratory Standards Institute guidelines (CLSI, 2007). The final inoculating concentration of bacteria was 5×105 c.f.u. ml−1. ESBL phenotype was detected by using the double-disc synergy method (CLSI, 2007).

    Preparation of crude extracts of β-lactamase and IEF.

    Crude cell homogenates for the detection of β-lactamases were prepared from fresh overnight culture by sonication. β-Lactamase activity was revealed by staining the gel with 0.5 mg of the chromogenic β-lactam nitrocefin (Oxoid) ml−1. IEF was performed with a PhastSystem apparatus (Amersham Pharmacia Biotech) as described previously (Weill et al., 2004).

    PCR amplification of β-lactamase genes and sequence analysis.

    The full-length amplification products of blaCTX-M-9T (F: 5′-TTGAATTCGCGCATGGTGACAAAGAGAGTGCAA-3′; R: 5′-TTGGATCCGTTACAGCCCTTCGGCGATGATTC-3′) were amplified using 30 cycles of denaturation for 40 s at 94 °C, annealing for 40 s at 57 °C, and extension for 1 min at 70 °C. All nucleotide sequences were determined by bidirectional sequencing with the 3730 automatic DNA sequencer (Sangon).

    Cloning of the CTX-M gene.

    Open reading frames encoding CTX-M-type enzymes were amplified by PCR with primers CTX-M-9T-F and CTX-M-9T-R, which included restriction sites for the enzymes EcoRI (TaKaRa) and BamHI (TaKaRa). The PCR product was purified using purification kits (Axygen). The PCR products were digested with these two enzymes and ligated into the corresponding restriction sites of modified plasmid pHSG398 (Cheng et al., 2008). The resulting plasmids, pHSG398-CTX-M-87 and pHSG398-CTX-M-65, which encoded CTX-M-87 and CTX-M-65, respectively, were transformed into E. coli JM109. The transformant was selected on Mueller–Hinton agar supplemented with cefotaxime (2 μg ml−1) and chloramphenicol (50 μg ml−1). All nucleotide sequences were determined by bidirectional sequencing with the 3730 automatic DNA sequencer.

    Determination of steady-state kinetic parameters.

    The β-lactamase from each recombinant strain was purified by fast protein liquid chromatography as described previously (Ma et al., 1998). Seven different substrate concentrations were used to determine the kinetic parameters for each substrate. Each reported parameter is a mean of three independent measurements.

    PFGE.

    PFGE of chromosomal DNA for strain typing was carried out as described by Gautom (1997). Chromosomal DNA was digested with 10 U XbaI (TaKaRa) at 37 °C for 4 h. The DNA fragments were separated by electrophoresis in a 1.2 % (w/v) agarose gel (Agarose NA; Amersham Pharmacia Biotech) with a CHEF Mapper XA System (Bio-Rad) at 175 V and 12 °C for 20 h. The pulse time was increased from 5 to 35s.

    RESULTS

    Susceptibility testing, conjugation and IEF of β-lactamases

    The clinical isolates E. coli p168, E. coli p517 and E. coli p667 were not clonally related (six or more bands at different sizes according to PFGE; data not shown). All three exhibited resistance to broad-spectrum cephalosporins including cefotaxime. The transferability of cefotaxime resistance from the three E. coli strains was assessed using conjugation experiments with an E. coli recipient; transconjugants were selected at similar frequencies (10−4) from each donor. The MICs of piperacillin, cefotaxime and ceftriaxone against the clinical isolate and the corresponding transconjugant were ≥256 μg ml−1, ≥64 μg ml−1 and ≥64 μg ml−1, respectively. The MICs of various antimicrobial agents for clinical isolates and transconjugants are listed in Table 1⇓. All clinical isolates produced two or three bands on IEF gel. All transconjugants produced one band. Transconjugants where p168 was the donor produced a β-lactamase with a pI value of 9.1; transconjugants where p517 or p667 were the donors both produced a β-lactamase with a pI value of 9.3. All clinical strains produced a second β-lactamase with a pI value of 5.4, which was shown to be TEM-1.

    Table 1.

    MICs (μg ml−1) of antibiotics against wild-type isolates, transconjugants and transformants

    PIP, piperacillin; TZP, piperacillin–tazobactam; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; ATM, aztreonam; IPM, imipenem; MEM, meropenem; LVX, levofloxacin; CIP, ciprofloxacin; GAT, gatifloxacin; AMK, amikacin; GEN, gentamicin.

    Cloning of the β-lactamase genes and DNA sequencing

    Given the likelihood of the reason for cephalosporin resistance being production of a CTX-M-type β-lactamase, PCR with primers CTX-M-9T-F and CTX-M-9T-R was performed. This was positive for all three clinical isolates, and the PCR products were ligated into plasmid pHSG398. The obtained recombinant plasmids were designated pSp168, pSp517 and pSp667 depending upon the template DNA used. They contained an approximately 0.9 kb insert encoding a novel CTX-M enzyme designated CTX-M-87 (from E. coli 168); the other is CTX-M-65 (from E. coli 517 and E. coli 667) which was previously reported, but not characterized (Doi et al., 2008). The deduced amino acid sequence of CTX-M-87 differed from that of CTX-M-14 by the substitutions Ala (GCC)77→Val (GTC) and Pro (CCT)167→Leu (CTT); CTX-M-65 differed from CTX-M-14 by the substitutions Ala (GCC)77→Val (GTC) and Ser (AGC)272→Arg (CGC). When the signal peptide of 28 amino acids is removed (Ambler et al., 1991), the putative mature enzymes CTX-M-87 and CTX-M-65 consist of 263 amino acid residues with calculated molecular masses of 28 018 Da and 28 071 Da, respectively. MICs conferred by the cloned blaCTX-M genes are shown in Table 1⇑.

    Kinetic parameters of CTX-M-87 and CTX-M-65

    We measured kinetic parameters of both purified enzymes against selected β-lactams. The purity of enzyme was >95 %. The Kcat and Km values were determined for a representative set of β-lactam antimicrobial agents (Table 2⇓). Penicillin G, cefaloridine, cefuroxime and cefotaxime were good substrates for CTX-M-87 and CTX-M-65, with Kcat values ranging from 950 to 4700 s−1. The catalytic efficiency of CTX-M-87 and CTX-M-65 against these drugs was greater than 7 μM−1 s−1. However, aztreonam, ceftazidime and cefepime were poorly hydrolysed (Kcat/Km ≤0.02 μM−1 s−1). The results were coincident with the MICs.

    Table 2.

    Kinetic parameters (kcat and Km) and catalytic efficiency (kcat/Km) of enzymes CTX-M-87, CTX-M-65 and CTX-M-14 against various β-lactams

    DISCUSSION

    The molecular epidemiology of CTX-M-producing E. coli strains on a countrywide scale has been previously described in the UK (Woodford et al., 2004), Spain (Oteo et al., 2006), Italy (Mugnaioli et al., 2006) and Canada (Mulvey et al., 2004). There are over 70 different types of CTX-M β-lactamases, which can be divided into five groups based on their amino acid identities (Bonnet, 2004): the CTX-M-1 group, the CTX-M-2 group, the CTX-M-8 group, the CTX-M-9 group and the CTX-M-25 group. In China, CTX-M-1 and CTX-M-9 are most common. In China, extended-spectrum β-lactams such as oxyimino-cephalosporins are widely prescribed, and CTX-M-type β-lactamases have been increasingly reported in recent years. CTX-M-13 and CTX-M-14 were found in 2002 in China (Chanawong et al., 2002), and we found CTX-M-46 in 2004 and reported it in 2008 (Cheng et al., 2008).

    The aim of the present study was to characterize a novel CTX-M enzyme designated CTX-M-87; the other enzyme that we characterized was CTX-M-65 β-lactamase. By studying wild-type clinical isolates, transconjugants and transformants, together with purified enzymes, we have shown that CTX-M-87 has very similar properties to those of CTX-M-14.

    Amino acid residues Asn104, Asn132, Phe160, Pro167, Gly232, Ser237 and Arg276 play an important role in catalysis by CTX-M β-lactamases (Bonnet et al., 2001; Ma et al., 2002). Residue 167 located in the omega loop plays an important role in influencing substrate specificity. The cefotaxime-bound crystal structure of Toho-1 indicates that Pro167 of CTX-M type β-lactamases has good contact with the AT moiety. Thus the substitution of Pro167 appears to play an important role in substrate binding, and substitutions at this position are known to lead to the change of resistance pattern conferred by the enzyme, particularly causing an increase in ceftazidime hydrolysis. Poirel et al. (2001) showed that the change of the single amino acid Pro to Ser at position 167 in CTX-M-19 enlarges the catalytic site of the enzyme for ceftazidime; therefore CTX-M-19 confers ceftazidime resistance. Stepanova et al. (2008) reported a Pro167Thr variant of CTX-M-3 conferring higher-level resistance to ceftazidime. However, the results of Kimura et al. (2004) from modelling studies suggest that CTX-M-18 (Pro167) has a larger cavity than CTX-M-19 (Ser167) for binding the AT moiety. As a result of this small but significant difference in the binding cavity within these two enzymes, the recognition mode of the enzymes against β-lactams may differ for different β-lactams. Thus the mutation at the omega loop site may cause a change (Kimura et al., 2004). The Pro167Gln substitution indicates the further evolutionary potential of CTX-M enzymes. The observed changes increased the ability to confer resistance against ceftazidime, but tended to reduce resistance to cefepime (Karisik et al., 2006). CTX-M-45 (Pro167Gln) has an expanded activity towards ceftazidime (Bae et al., 2006). In 2007, Kimura et al. (2007) reported that the kcat/Km values for Pro167Ser mutants and their respective wild-type enzymes were identical for cefalothin, penicillin and nitrocefin. For cefotaxime, the catalytic efficiency (kcat/Km) for wild-type enzymes was 3.13–7.12 times higher than that of their respective Pro167Ser mutants. Pro167Ser mutants had kcat/Km values 1.73–2.21 times higher than those of their respective wild-type enzymes. These results indicate that the CTX-M-type β-lactamase family can hydrolyse ceftazidime more efficiently because of the point mutation at position 167. However, in our study, CTX-M-87 was shown to differ from CTX-M-14 by two amino acids, at positions 77 (Ala→Val) and 167 (Pro→Leu), but there was no obvious effect of this mutation relative to CTX-M-14 (Tables 1⇑ and 2⇑). Accordingly, we can conclude that not all mutations at position 167 result in alterations in the kinetic parameters or substrate specificity of CTX-M type β-lactamases.

    Acknowledgments

    This project was supported by the National Natural Science Foundation of China (No. 30571654).

    References

    1. Ambler, R. P., Coulson, A. F., Frère, J. M., Ghuysen, J. M., Joris, B., Forsman, M., Levesque, R. C., Tiraby, G. & Waley, S. G. (1991). A standard numbering scheme for the class A beta-lactamases. Biochem J 276, 269–270.
    2. Babic, M., Hujer, A. M. & Bonomo, R. A. (2006). What's new in antibiotic resistance? Focus on beta-lactamases. Drug Resist Updat 9, 142–156.
    3. Bae, I. K., Lee, B. H., Hwang, H. Y., Jeong, S. H., Hong, S. G., Chang, C. L., Kwak, H. S., Kim, H. J. & Youn, H. (2006). A novel ceftazidime-hydrolysing extended-spectrum beta-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop. J Antimicrob Chemother 58, 315–319.
    4. Barthélémy, M., Péduzzi, J., Bernard, H., Tancrède, C. & Labia, R. (1992). Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum beta-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca. Biochim Biophys Acta 1122, 15–22.
    5. Bauernfeind, A., Grimm, H. & Schweighart, S. (1990). A new plasmidic cefotaximase in a clinical isolate of Escherichia coli. Infection 18, 294–298.
    6. Bonnet, R. (2004). Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 48, 1–14.
    7. Bonnet, R., Dutour, C., Sampaio, J. L., Chanal, C., Sirot, D., Labia, R., De Champs, C. & Sirot, J. (2001). Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240→Gly. Antimicrob Agents Chemother 45, 2269–2275.
    8. Bradford, P. A., Yang, Y., Sahm, D., Grope, I., Gardovska, D. & Storch, G. (1998). CTX-M-5, a novel cefotaxime-hydrolyzing beta-lactamase from an outbreak of Salmonella typhimurium in Latvia. Antimicrob Agents Chemother 42, 1980–1984.
    9. Bush, K. (2001). New beta-lactamases in gram-negative bacteria: diversity and impact on the selection of antimicrobial therapy. Clin Infect Dis 32, 1085–1089.
    10. Chanawong, A., M'Zali, F. H., Heritage, J., Xiong, J. H. & Hawkey, P. M. (2002). Three cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's Republic of China. Antimicrob Agents Chemother 46, 630–637.
    11. Cheng, J., Ye, Y., Wang, Y. Y., Li, H., Li, X. & Li, J. B. (2008). Phenotypic and molecular characterization of 5 novel CTX-M enzymes carried by Klebsiella pneumoniae and Escherichia coli. Acta Pharmacol Sin 29, 217–225.
    12. CLSI (2007). Performance Standards for Antimicrobial Susceptibility Testing; 16th Informational Supplement. M100-S15. Wayne, PA: Clinical and Laboratory Standards Institute.
    13. Delmas, J., Robin, F., Carvalho, F., Mongaret, C. & Bonnet, R. (2006). Prediction of the evolution of ceftazidime resistance in extended-spectrum beta-lactamase CTX-M-9. Antimicrob Agents Chemother 50, 731–738.
    14. Doi, Y., Adams-Haduch, J. M. & Paterson, D. L. (2008). Escherichia coli isolate coproducing 16S rRNA methylase and CTX-M-type extended-spectrum beta-lactamase isolated from an outpatient in the United States. Antimicrob Agents Chemother 52, 1204–1205.
    15. Gautom, R. K. (1997). Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in 1 day. J Clin Microbiol 35, 2977–2980.
    16. Gazouli, M. & Legakis, N. J. (1998). Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A beta-lactamase CTX-M-4. FEMS Microbiol Lett 169, 289–293.
    17. Gazouli, M., Tzelepi, E., Markogiannakis, A., Legakis, N. J. & Tzouvelekis, L. S. (1998a). Two novel plasmid-mediated cefotaxime-hydrolyzing beta-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium. FEMS Microbiol Lett 165, 289–293.
    18. Gazouli, M., Tzelepi, E., Sidorenko, S. V. & Tzouvelekis, L. S. (1998b). Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A beta-lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis. Antimicrob Agents Chemother 42, 1259–1262.
    19. Gniadkowski, M., Schneider, I., Pałucha, A., Jungwirth, R., Mikiewicz, B. & Bauernfeind, A. (1998). Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing beta-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrob Agents Chemother 42, 827–832.
    20. Ishii, Y., Ohno, A., Taguchi, H., Imajo, S., Ishiguro, M. & Matsuzawa, H. (1995). Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A beta-lactamase isolated from Escherichia coli. Antimicrob Agents Chemother 39, 2269–2275.
    21. Karisik, E., Ellington, M. J., Pike, R., Livermore, D. M. & Woodford, N. (2006). Development of high-level ceftazidime resistance via single-base substitutions of blaCTX-M-3 in hyper-mutable Escherichia coli. Clin Microbiol Infect 12, 803–806.
    22. Kimura, S., Ishiguro, M., Ishii, Y., Alba, J. & Yamaguchi, K. (2004). Role of a mutation at position 167 of CTX-M-19 in ceftazidime hydrolysis. Antimicrob Agents Chemother 48, 1454–1460.
    23. Kimura, S., Ishii, Y., Tateda, K. & Yamaguchi, K. (2007). Predictive analysis of ceftazidime hydrolysis in CTX-M-type beta-lactamase family members with a mutational substitution at position 167. Int J Antimicrob Agents 29, 326–331.
    24. Ma, L., Ishii, Y., Ishiguro, M., Matsuzawa, H. & Yamaguchi, K. (1998). Cloning and sequencing of the gene encoding Toho-2, a class A beta-lactamase preferentially inhibited by tazobactam. Antimicrob Agents Chemother 42, 1181–1186.
    25. Ma, L., Ishii, Y., Chang, F. Y., Yamaguchi, K., Ho, M. & Siu, L. K. (2002). CTX-M-14, a plasmid-mediated CTX-M type extended-spectrum beta-lactamase isolated from Escherichia coli. Antimicrob Agents Chemother 46, 1985–1988.
    26. Mugnaioli, C., Luzzaro, F., De Luca, F., Brigante, G., Perilli, M., Amicosante, G., Stefani, S., Toniolo, A. & Rossolini, G. M. (2006). CTX-M-type extended-spectrum beta-lactamases in Italy: molecular epidemiology of an emerging countrywide problem. Antimicrob Agents Chemother 50, 2700–2706.
    27. Mulvey, M. R., Bryce, E., Boyd, D., Ofner-Agostini, M., Christianson, S., Simor, A. E. & Paton, S., The Canadian Hospital Epidemiology Committee of the Canadian Nosocomial Infection Surveillance Program, Health Canada (2004). Ambler class A extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian hospitals. Antimicrob Agents Chemother 48, 1204–1214.
    28. Oteo, J., Navarro, C., Cercenado, E., Delgado-Iribarren, A., Wilhelmi, I., Orden, B., García, C., Miguelañez, S., Pérez-Vázquez, M. & other authors (2006). Spread of Escherichia coli strains with high-level cefotaxime and ceftazidime resistance between the community, long-term care facilities, and hospital institutions. J Clin Microbiol 44, 2359–2366.
    29. Poirel, L., Naas, T., Le Thomas, I., Karim, A., Bingen, E. & Nordmann, P. (2001). CTX-M-type extended-spectrum beta-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrob Agents Chemother 45, 3355–3361.
    30. Stepanova, M. N., Pimkin, M., Nikulin, A. A., Kozyreva, V. K., Agapova, E. D. & Edelstein, M. V. (2008). Convergent in vivo and in vitro selection of ceftazidime resistance mutations at position 167 of CTX-M-3 beta-lactamase in hypermutable Escherichia coli strains. Antimicrob Agents Chemother 52, 1297–1301.
    31. Weill, F. X., Demartin, M., Tandé, D., Espié, E., Rakotoarivony, I. & Grimont, P. A. (2004). SHV-12-like extended-spectrum-beta-lactamase-producing strains of Salmonella enterica serotypes Babelsberg and Enteritidis isolated in France among infants adopted from Mali. J Clin Microbiol 42, 2432–2437.
    32. Woodford, N., Ward, M. E., Kaufmann, M. E., Turton, J., Fagan, E. J., James, D., Johnson, A. P., Pike, R., Warner, M. & other authors (2004). Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum beta-lactamases in the UK. J Antimicrob Chemother 54, 735–743.