Two Different Extended-Spectrum β-Lactamases (ESBLs) in One of the First ESBL-Producing Salmonella Isolates in Poland

Various types of extended-spectrum β-lactamases (ESBLs) constitute one of the major mechanisms of resistance of gram-negative bacteria to oxyimino-β-lactam antibiotics (10, 13, 17). In general they are most frequently identified in Klebsiella pneumoniae and Escherichia coli; however, more and more papers in recent years have documented their growing incidence in other organisms of the family Enterobacteriaceae, including Salmonella spp. (10, 17). Sporadic infections or nosocomial outbreaks caused by ESBL-producing salmonellae have been reported in numerous countries of Latin America, Africa, Asia, and Europe (4, 8, 18, 19). Salmonellae have been found to express a wide variety of ESBL types, including TEM, SHV, PER, and CTX-M enzymes (10, 13, 18). The situation is of particular concern because it demonstrates the danger of ESBL spread among pathogens circulating in livestock and the community (17).

Although frequently found in many enterobacterial species, ESBL had not been reported in Salmonella spp. in Poland until 1999. The two cefotaxime-resistant isolates analyzed in this work belong to the first ESBL-producing salmonellae identified in the country. They were recovered in 1999 and 2000 in two regional hospitals located in geographically distant cities (Table 1). Both strains were cultured from the stools of patients with diarrhea. Biochemical identification of the isolates was performed with the ATB ID32E test (bioMérieux sa, Marcy l'Etoile, France). Serogroups and serotypes with respect to somatic (O) and flagellar (H) antigens were determined in the Public Health Sanitary-Epidemiological Station in Warsaw. Isolate 9197 from Grajewo was classified as Salmonella enterica serovar Typhimurium, and isolate 33/01 from Koszalin was identified as Salmonella enterica serovar Enteritidis. ESBL production was detected by the double disk synergy test (12).

The two salmonella isolates were subjected to the cefotaxime resistance transfer experiment as described previously (3), with E. coli A15, resistant to nalidixic acid, as the recipient strain. Transconjugants were selected on MacConkey agar (Oxoid, Basingstoke, United Kingdom) supplemented with cefotaxime (2 μg/ml; Polfa Tarchomin, Warsaw, Poland) and nalidixic acid (64 μg/ml; Sigma Chemical Company, St. Louis, Mo.). Both isolates produced transconjugants with an efficiency of around 10⁻⁴ per donor cell. MICs of various antibiotics were evaluated for the isolates and the transconjugants by the agar dilution method according to the NCCLS guidelines (14). Antimicrobial standards were supplied by the corresponding manufacturers. Both clinical isolates and their transconjugants revealed MIC patterns that are typical for ESBL-producing strains (13) (Table 2). They were characterized by raised MICs of a variety of β-lactams, except for cefoxitin and imipenem, and β-lactamase inhibitors reduced effectively MICs of β-lactams in all combinations tested. Cefotaxime MICs were significantly higher than those of ceftazidime. Apart from β-lactams the clinical isolates were also resistant to aminoglycosides and cotrimoxazole, and resistance to these drugs was cotransferred with β-lactam resistance to the transconjugants.

β-Lactamases of the clinical isolates and their transconjugants were visualized by isoelectric focusing (IEF), and their cefotaxime-hydrolyzing activity was detected by the bioassay approach. IEF and the bioassay were performed as described by Bauernfeind et al. (3), with the use of a Model 111 Mini Cell (Bio-Rad, Hercules, Calif.) for IEF. The concentration of cefotaxime in the bioassay was 2 μg/ml. Results of the analyses are shown in Table 1. Both isolates produced β-lactamases with isoelectric points (pIs) of 8.4 and 5.4, and these two enzymes were also found in extracts of the transconjugants. The serovar Typhimurium isolate, 9197, but not its transconjugant, expressed an additional β-lactamase with a pI of 7.6. Only enzymes with pIs of 8.4 and 7.6 demonstrated the cefotaxime-hydrolyzing activity under the conditions used in the bioassay (the β-lactamase with a pI of 5.4 was probably TEM-1).

Genes coding for the cefotaxime-hydrolyzing β-lactamases were detected by PCR in DNA of the clinical isolates. Both isolates were tested for the presence of bla_CTX-M genes (supposed to encode the pI 8.4 enzymes), and DNA of serovar Typhimurium 9197 was also checked by PCR specific for a bla_SHV gene (likely coding for the β-lactamase with a pI of 7.6). PCRs were run as reported previously (6, 11). Amplicons of the expected size of around 1 kb were obtained in all three cases and subjected to sequencing using an ABI PRISM 310 sequencer (PE Biosystems, Foster City, Calif.). Sequencing was performed as previously described (1, 6, 11). The results are shown in Table 1. Coding regions of the two bla_CTX-M genes were identical to that of the bla_CTX-M-3 gene (11), which indicated that the β-lactamase with a pI of 8.4 was CTX-M-3. The bla_SHV gene coding region was of the same nucleotide sequence as in the originally reported bla_SHV-2a gene (16), and this revealed that the ESBL with a pI of 7.6 was SHV-2a.

Plasmid DNA was purified from the transconjugants with the use of the QIAGEN Plasmid Midi Kit (QIAGEN, Hilden, Germany) and subjected to the fingerprinting analysis along with all the variants of bla_CTX-M-3 gene-carrying plasmids that had been identified before in Enterobacteriaceae isolates in Poland (1). The PstI restrictase (MBI Fermentas, Vilnius, Lithuania) fingerprinting was performed as reported previously (11). Both transconjugants contained large plasmids (of around 90 to 110 kb) that produced different fingerprints (Table 1 and Fig. 1). The plasmid specific for serovar Enteritidis 33/01 had a fingerprint identical to that of the widely spread bla_CTX-M-3 gene-carrying plasmid in Poland (variant A1) (1), whereas the molecule present in serovar Typhimurium 9197 demonstrated a unique PstI restriction pattern.

With the two multiresistant serovar Typhimurium and serovar Enteritidis isolates analyzed here and an S. enterica serovar Mbandaka isolate reported elsewhere (21), Poland has joined the group of countries in which ESBL-producing salmonellae have been described. Both strains produced the CTX-M-3 enzyme, identified originally in 1996 in Citrobacter freundii isolates from a hospital in Warsaw (11). CTX-M β-lactamases have been relatively frequently found in ESBL-producing salmonellae, including CTX-M-2 in Argentina (2), CTX-M-4 in Russia (9), CTX-M-5 in Latvia (4), CTX-M-6 and -7 in Greece (8), and probably CTX-M-9 in Spain (19). The CTX-M-4 and -5-producing serovar Typhimurium strains caused clonal outbreaks in hospitals in St. Petersburg and Riga, respectively (4, 7, 22), and serovar Typhimurium strains related to those from St. Petersburg have been also observed in Hungary and Greece (22).

The CTX-M-3 β-lactamase is widely spread in Poland, having been identified in numerous strains of seven Enterobacteriaceae species in 16 medical centers throughout the country (1, 11, 15). The isolation of CTX-M-3-producing salmonella strains in hospitals in Grajewo and Koszalin has extended the list by two more institutions. The bla_CTX-M-3 gene-carrying plasmids of both isolates demonstrated a high transmission potential; moreover, one of these had been observed before in nine hospitals, which suggests its nosocomial origin (1, 11, 15). These data support the hypothesis that plasmid dissemination has been the major mechanism of the CTX-M-3 spread in Poland (1, 15). What is noteworthy is that these plasmids also contained a gene coding for another β-lactamase (likely TEM-1) and genes responsible for resistance to aminoglycosides and cotrimoxazole. One of the salmonella isolates, apart from CTX-M-3, also produced another ESBL variant, SHV-2a, which had previously never been identified in Poland. Genes coding for the two ESBLs have probably been acquired independently by the strain, as they were located on separate DNA replicons. Such double-ESBL-producer strains, although occasionally reported (1, 5, 20), are rare, and to our knowledge they have not been observed in salmonella to date.

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FIG. 1.

Plasmid fingerprinting analysis performed with the use of the PstI restriction enzyme (MBI Fermentas). Lanes: 1, GeneRuler 100-bp DNA Ladder Plus (MBI Fermentas); 2, Serovar Enteritidis 33/01, pattern A1; 3, Serovar Typhimurium 9197, pattern E; 4, C. freundii 2526/96, pattern A1 (1, 11, 15); 5, E. coli 279, pattern B (15); 6, E. coli SU 8350, pattern C (1); 7, E. coli BB 1775, pattern D (1).

• American Society for Microbiology