OBJECTIVES: This study was conducted to examine the genetic variation occurring in the cmeB gene encoding the transporter component of the CmeABC efflux pump. METHODS: Expression of the CmeABC pump in 21 strains of Campylobacter jejuni and Campylobacter coli was studied by western-blot analysis. MIC determination was conducted in the presence or absence of an efflux pump inhibitor (EPI). Inactivation of the cmeB gene and sequencing of the cmeABC operon were performed for a single strain. The remaining strains were compared by RFLP analysis of the cmeB-specific PCR amplicon. The cmeB genes of two C. coli strains with different RFLP patterns were sequenced completely. RESULTS: Conflicting results were obtained in the western-blot analysis with anti-CmeB and anti-CmeC antibodies for one strain, whereas MIC determinations with EPI and cmeB gene inactivation confirmed the efflux pump's activity. The cmeB gene of this isolate showed only 78% nucleotide sequence identity with the sequence of reference strains. PCR-RFLP analysis identified 4 different patterns among the 5 C. jejuni and 14 different patterns among the 16 C. coli strains investigated. At the amino acid sequence level, variation was higher in the periplasmic loops of the transporter. CONCLUSIONS: A total of 18 different cmeB-specific PCR-RFLP patterns were detected among the 21 C. jejuni and C. coli strains. These sequence variations might have an impact on the function and substrate recognition of this transporter. The sequence data obtained in this study will help to design suitable tools to study the presence or the expression of the gene cmeB.
OBJECTIVES: This study was conducted to examine the genetic variation occurring in the cmeB gene encoding the transporter component of the CmeABC efflux pump. METHODS: Expression of the CmeABC pump in 21 strains of Campylobacter jejuni and Campylobacter coli was studied by western-blot analysis. MIC determination was conducted in the presence or absence of an efflux pump inhibitor (EPI). Inactivation of the cmeB gene and sequencing of the cmeABC operon were performed for a single strain. The remaining strains were compared by RFLP analysis of the cmeB-specific PCR amplicon. The cmeB genes of two C. coli strains with different RFLP patterns were sequenced completely. RESULTS: Conflicting results were obtained in the western-blot analysis with anti-CmeB and anti-CmeC antibodies for one strain, whereas MIC determinations with EPI and cmeB gene inactivation confirmed the efflux pump's activity. The cmeB gene of this isolate showed only 78% nucleotide sequence identity with the sequence of reference strains. PCR-RFLP analysis identified 4 different patterns among the 5 C. jejuni and 14 different patterns among the 16 C. coli strains investigated. At the amino acid sequence level, variation was higher in the periplasmic loops of the transporter. CONCLUSIONS: A total of 18 different cmeB-specific PCR-RFLP patterns were detected among the 21 C. jejuni and C. coli strains. These sequence variations might have an impact on the function and substrate recognition of this transporter. The sequence data obtained in this study will help to design suitable tools to study the presence or the expression of the gene cmeB.
Authors: Isaac P Kashoma; Issmat I Kassem; Julius John; Beda M Kessy; Wondwossen Gebreyes; Rudovick R Kazwala; Gireesh Rajashekara Journal: Microb Drug Resist Date: 2015-07-08 Impact factor: 3.431