JAC Advance Access originally published online on December 7, 2005
Journal of Antimicrobial Chemotherapy 2006 57(2):369-371; doi:10.1093/jac/dki438
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Correspondence |
AmpC ß-lactamase-mediated cefpodoxime-resistant Escherichia coli isolated from faecal samples of healthy volunteers
1 Department of Environmental Infectious Diseases, Graduate School of Medical Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa, Japan; 2 Department of Microbiology, School of Medicine, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan
* Corresponding author. Tel: 81-42-778-9349; Fax: 81-42-778-9350; E-mail: matsu{at}kitasato-u.ac.jp
Keywords: E. coli , AmpC promoter , CMY-2 , resistance
Sir,
Escherichia coli is one of the most common pathogens causing nosocomial infections. In recent years, clinical reports of E. coli with extended-spectrum cephalosporin (ESC) resistance, particularly mediated by AmpC ß-lactamase, have been increasing rapidly.1–3 This indicates the spread of either plasmid-borne AmpC or chromosomal AmpC mutants among clinical isolates. In order to analyse the population of drug-resistant microorganisms among normal microflora and detect microorganisms with the potential to cause pandemics in the future, extensive surveillance of drug-resistant microorganisms has been performed in healthy humans and animals. However, little is known about the existence of E. coli with AmpC-mediated ESC resistance in the normal intestinal microflora of healthy humans. Here, we report that two strains of ESC-resistant E. coli were detected in the faeces of healthy volunteers. These two isolates had different resistance mechanisms, which were CMY-2 (a plasmid-borne AmpC) and a mutation in the ampC promoter, respectively.
In 2003, 63 E. coli strains were isolated from faecal samples of healthy third-year medical students. Each bacterial strain was isolated from an individual student, and none of the students had received antibiotic therapy within 1 month before the sampling. MICs of nine ß-lactams and a combination with ß-lactam-inhibitor were determined by the agar dilution method according to CLSI (formerly NCCLS) guidelines (Table 1).
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Two cefpodoxime-resistant E. coli strains (KU6763 and KU6764) were detected (Table 1). KU6763 and KU6764 both exhibited resistance to cefpodoxime (MIC = 128 and 16 mg/L, respectively). Measurement of AmpC activity and detection of plasmid-borne blaAmpC were performed by a colorimetric assay and multiplex PCR, respectively as described previously.4,5 The ß-lactamase activity of both strains was >99-fold higher than that of
1037, a cefpodoxime-susceptible control (1.5 ± 0.7 and 0.99 ± 0.03 U/mg of protein, respectively). Detection of plasmid-borne AmpC by multiplex PCR revealed a 462 bp PCR amplicon in KU6763. Cefpodoxime resistance of KU6763 was transmissible to 1037 at a frequency of 1.1 x 10–7, and this resistance was mediated by a 100 kb plasmid (designated as pKU433). After conjugating pKU433 to 1037, the MICs of cefoxitin and flomoxef were increased by 8- and 128-fold, respectively. In parallel, the ß-lactamase activity of the transconjugant also increased 132-fold compared with that of the recipient strain. Extraction of plasmids from KU6763 was done with a HiSpeed Plasmid Midi kit (Qiagen, Tokyo, Japan). pKU433 thus obtained was digested with BamHI, and then was cloned into the BamHI site of the pHSG398 cloning vector (Takara, Kyoto, Japan). Finally, a cefpodoxime-resistant clone carrying a 4 kb fragment from pKU433 was obtained by selection with cefpodoxime (at a concentration of 25 mg/L). DNA sequence analysis of pHSG398 carrying the BamHI-digested fragment of pKU433 was done with an ABI PRISM 310 DNA sequencer (Applied Biosystems, Tokyo, Japan). Identification of the ß-lactamase gene using the BLASTN database search program showed that the plasmid-borne blaAmpC gene was identical to blaCMY-2 (GenBank accession no. X91840
[GenBank]
). It was previously demonstrated that CMY-2 conferred resistance to cephamycins in Gram-negative bacilli.2 These findings indicated that the ESC resistance of KU6763 was mediated by CMY-2.
KU6764 also exhibited ESC resistance and high ß-lactamase activity compared with 1037, although plasmid-borne AmpC was not detected by PCR amplification. The MICs of cefpodoxime and ceftazidime were increased by 16- and 8-fold, respectively, compared with those for 1037. In parallel, AmpC activity was increased by 99-fold compared with that of 1037. Previous studies have shown that insertion of one or two bases between –35 and –10 in the E. coli ampC promoter causes overexpression of ampC.3 To confirm this, KU6764 chromosomal ampC was amplified by PCR using HotStar Taq (Qiagen) with the following primers: ampC Up#1, 5'-AATGTTTCCTTACTGGTTTTT-3'; and ampC Low#0, 5'-GGTCGCGTATTCCTGTTCCTGATG-3'. DNA sequence analysis of chromosomal ampC revealed insertion of T at a site 73 bp upstream of the ampC coding region, and this mutation corresponded with the ampC promoter mutant reported by Forward et al.3 Therefore, we concluded that AmpC overproduction by KU6764 was due to this mutation of the promoter.
In this study, an E. coli strain harbouring blaCMY-2 and another strain with an ampC promoter region mutation were isolated from the faecal samples of healthy volunteers. To our knowledge, this is the first report concerning blaCMY-2 isolation from healthy volunteers in Japan. CMY-2 is one of the most widely disseminated plasmid-borne AmpCs among patients and animals.6 The isolation of E. coli with ESC resistance due to ampC promoter mutation has also been increasing among both patients and healthy animals.1,3,6 Our findings suggest that chromosomal or plasmid-borne AmpC-mediated ESC resistance is already widely disseminated among E. coli, even in the normal human intestinal flora. We conclude that further surveillance to detect microorganisms with ESC resistance in the human intestinal flora is necessary.
Transparency declarations
We have no conflicts to declare.
Footnotes
These authors contributed equally to this work. 
Acknowledgements
This work was supported by grants (14370096) from the Japanese Ministry of Education, Culture, Sports, Science and Technology; the COE Program of the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor and Welfare (the Research Project of Emerging and Establishment of Rapid Identification Methods, H15-Sinko-9); and the Charitable Trust Clinical Pathology Research Foundation of Japan.
References
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