Detection of TEM-52 in Salmonella enterica serovar Enteritidis isolated in Scotland

Abstract

Sir, Non-typhoidal salmonellae are one of the principal pathogens implicated in cases of food poisoning worldwide. In the UK, Europe and USA, Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most commonly isolated serotypes,1 and is thought to be spread to humans through the food chain from reservoirs in food-producing animals.1 Antibiotic resistance is relatively uncommon in S. Enteritidis.2 During 1996–2000, the overall incidence of multidrug resistance (resistance to four or more antibiotics) in this serovar was less than 1% in England and Wales.1 Although antibiotics are rarely required in cases of salmonella enterocolitis, they are crucial if the infection spreads from the intestine. In the treatment of extra-intestinal salmonella infections, the antibiotics of choice are extended-spectrum cephalosporins and fluoroquinolones.3 Recently, Salmonella isolates harbouring extended-spectrum β-lactamases (ESBLs) capable of hydrolysing third-generation cephalosporins have been reported.2,3 This is of particular concern for the treatment of salmonellosis in children, because fluoroquinolones cannot be used in this age group. Here we report the presence of the ESBL TEM-52 in an S. Enteritidis strain isolated during a hospital outbreak in Scotland. Previously, TEM-52 has only been reported in salmonellae isolated in Hungary2 and Korea.3 An outbreak of salmonellosis was identified in a general hospital in Glasgow, Scotland, in the period 20 December 2001–21 January 2002. During this outbreak, nine isolates were obtained from five patients with salmonella gastroenteritis, and two asymptomatic members of staff. Isolates were identified as S. enterica serovar Enteritidis PT21 by agglutination to polyvalent antisera and phage typing. Isolates from two patients and one member of staff were susceptible to 14 common antibiotics. Isolates from the remaining patients and one member of staff were resistant to ampicillin and cefotaxime, and carried a plasmid of 95 kb. Plasmid profiling and PFGE revealed these isolates to be clonal, one of which (020003) was used to determine the mechanism of resistance. Susceptibility testing to ampicillin, cefaclor, cefotaxime and ceftazidime was performed using Etest strips (AB Biodisk, Solna, Sweden) following the manufacturer’s instructions. The presence of an ESBL was identified by double disc synergy tests, and Etest strips specific for the detection of ESBLs, containing cefotaxime or ceftazidime, with and without clavulanic acid. Plasmid DNA extraction was performed using standard methods, and plasmid transfers carried out in broth-mating experiments with Escherichia coli K12 J53-2 (lactose positive, rifampicin resistant). The β-lactamase genes blaTEM-1 and blaSHV-1 were sought by PCR using a Taq polymerase kit (Promega) and the following oligonucleotide primer pairs: blaTEM, 5′-ATGAGTATTCAACATTTCCG-3′, 5′-CCAATGCTTAATCAGTGAGG-3′; and blaSHV, 5′-GCCCGGGTTATTCTTATTTGTCGC-3′, 5′-CTTTCCGATGCCGCCGCCAGTCA-3′. Sequencing was performed in forward and reverse directions, and the sequences were compared with sequences in public databases. The salmonella isolates were resistant to ampicillin, cefaclor (both >128 mg/L), cefotaxime (8 mg/L) and ceftazidime (>32 mg/L). All these resistances were carried on the 95 kb conjugative plasmid. PCR with blaSHV-1 primers failed to detect a gene encoding an SHV-1 derivative. The blaTEM-1 PCR yielded an 858 bp product. DNA sequencing showed that this gene encoded TEM-52,3,4 which differs from TEM-15 by amino acid substitutions at positions Glu-104→Lys (GAG→AAG), Met-182→Thr (ATG→ACG) and Gly-238→Ser (GGT→AGT) (Table 1). A change in residue 104 from either glutamic or aspartic acid to lysine is the second most common substitution of the TEM-type ESBLs.6 Here, a negatively charged residue is replaced by a positively charged residue with a long basic side chain, which may interact with the carboxylic acid group in ceftazidime, ceftibuten or aztreonam, increasing initial binding.6 In TEM-type ESBLs, the substitution from methionine to threonine at position 182 adds an extra hydrogen bond between the hydroxyl group of threonine and the carbonyl group of the glutamic acid in position 64. This may increase the stability of the protein where multiple mutations are present.4 The substitution Gly-238→Ser occurs in 27 TEM variants. With the exception of TEM-50, all these enzymes also contain methionine, which has a relatively large side-chain, at position 69 (as does the enzyme from isolate 020003). As the side chain of amino acid 238 lies very close to that of residue 69,6 the presence of large side chains may slightly expand the lower portion of the β-lactam binding site, allowing cephalosporins with rigid branched acylamido substituents (such as ceftazidime) to form hydrogen bonds with residue 237 in the binding site.6 A silent mutation was present in the blaTEM-52 allele of isolate 020003 at position Ala-134 (GCT→GCG) that is not present in other published salmonella TEM-52 sequences (GenBank no. AF126444, AY220520), but which has been reported in a Klebsiella pneumoniae TEM-52 sequence (GenBank no. Y13612) (Table 1). In conclusion, antibiotic resistance is not common in S. Enteritidis, hence the detection of TEM-52, previously unreported in salmonellae in the UK, is significant. The TEM-52 gene described here differed from all published salmonella TEM-52 sequences by one silent mutation.