The first characterization of extended-spectrum ß-lactamase-producing Salmonella in Japan

Ashraf M. Ahmed, Hiroyuki Nakano and Tadashi Shimamoto*

Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan

Keywords: ESBLs , Salmonella enterica serovar Senftenberg , CTX-M-3

Sir,

In recent years, an expanding group of extended-spectrum ß-lactamases (ESBLs), the CTX-M enzymes, has emerged from many countries. They were initially reported in Japan by Matsumoto et al.,1 who discovered FEC-1, a non-TEM, non-SHV ESBL, in a cefotaxime-resistant isolate of Escherichia coli. CTX-M enzymes have spread worldwide and have been detected in many Gram-negative bacteria, including various Salmonella enterica serovars.2 They are usually plasmid mediated, and confer high-level resistance to cefotaxime and ceftriaxone, but have only marginal effects on MICs of ceftazidime.2

In Japan, CTX-M enzymes have been detected in E. coli,1 Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens.3 We now report the first cefotaximase-producing salmonella isolated in Japan, a non-clinical isolate of S. enterica serovar Senftenberg that harboured CTX-M-3.

Fifty-eight clinical and non-clinical isolates of various Salmonella serovars were screened for ESBL production. Only one isolate, S. enterica serovar Senftenberg strain 9-4, which was isolated from river water in Hiroshima prefecture in 1999, displayed an ESBL phenotype; it was more resistant to cefotaxime, ceftriaxone and cefpodoxime than to ceftazidime by disc testing, and gave a positive cephalosporin–co-amoxiclav synergy test. For the preliminary detection of CTX-M genes, PCR was performed with primers CTX-M-F, 5'-CGCTTTGCGATGTGCAG-3' and CTX-M-R, 5'-ACCGCGATATCGTTGGT-3', which are specific to conserved regions of blaCTX-M alleles and generate a 552 bp amplicon. Then, primers CTX-M-F2, 5'-CCAGAATAAGGAATCCCATG-3' and CTX-M-R2, 5'-GCCGTCTAAGGCGATAAAC-3', were used to amplify the entire allele (876 bp) for sequencing. This revealed that S. enterica serovar Senftenberg strain 9-4 produced CTX-M-3, which was first identified in Poland.4 Southern hybridization using the whole blaCTX-M gene as a probe showed that the gene was located on a plasmid of <38 kb in size. Attempts to transfer this plasmid to E. coli HB101 were unsuccessful. The blaCTX-M gene was cloned into pBluescript II SK(–) and expressed in E. coli XL1-Blue cells. The MICs for both S. enterica serovar Senftenberg 9-4 and E. coli XL1-Blue transformants are shown in Table 1.


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Table 1. MICs of extended-spectrum ß-lactams for S. enterica serovar Senftenberg 9-4 and E. coli XL1-Blue transformants

 
Detection of CTX-M-3 in S. enterica serovar Senftenberg may have public health implications. Previously, the ESBL SHV-5 was detected in clinical isolates of this serovar causing an outbreak in burn wounds in a hospital in Delhi, India.5 Our results show that non-clinical isolates can constitute a reservoir for blaCTX-M alleles, and can facilitate the spread of cefotaximases to clinical isolates. In support of this hypothesis, the metallo-ß-lactamase Sfh-I was recently detected in bacteria not of clinical origin.6 Therefore, researchers working on ESBLs especially in endemic areas should also direct their attention to non-clinical isolates found in these regions.

Nucleotide sequence accession number

The nucleotide sequence of the CTX-M-3 gene of S. enterica serovar Senftenberg has been assigned the DDBJ/GenBank/EMBL accession no. AB168117.

Acknowledgements

This work was supported by a Grant-in-Aid for scientific research to T.S. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Footnotes

* Corresponding author. Tel./Fax: +81-82-424-7897; Email: tadashis{at}hiroshima-u.ac.jp

References

1 . Matsumoto, Y., Ikeda, F., Kamimura, T. et al. (1988). Novel plasmid-mediated ß-lactamase from Escherichia coli that inactivates oxyimino-cephalosporins. Antimicrobial Agents and Chemotherapy 32, 1243–6.[ISI][Medline]

2 . Bonnet, R. (2004). Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrobial Agents and Chemotherapy 48, 1–14.[Free Full Text]

3 . Yamasaki, K., Komatsu, M., Yamashita, T. et al. (2003). Production of CTX-M-3 extended-spectrum ß-lactamase and IMP-1 metallo ß-lactamase by five Gram-negative bacilli: survey of clinical isolates from seven laboratories collected in 1998 and 2000, in the Kinki region of Japan. Journal of Antimicrobial Chemotherapy 51, 631–8.[Abstract/Free Full Text]

4 . Gniadkowski, M., Schneider, I., Palucha, A. et al. (1998). Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing ß-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrobial Agents and Chemotherapy 42, 827–32.[Abstract/Free Full Text]

5 . Revathi, G., Shannon, K. P., Stapleton, P. D. et al. (1998). An outbreak of extended-spectrum, ß-lactamase-producing Salmonella senftenberg in a burns ward. Journal of Hospital Infection 40, 295–302.[CrossRef][ISI][Medline]

6 . Saavedra, M. J., Peixe, L., Sousa, J. C. et al. (2003). Sfh-I, a subclass B2 metallo-ß-lactamase from a Serratia fonticola environmental isolate. Antimicrobial Agents and Chemotherapy 47, 2330–3.[Abstract/Free Full Text]





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