Multiplex PCR Assay for Identifi cation and Differentiation of Campylobacter jejuni and Campylobacter coli Isolates.

INTRODUCTION: Campylobacter spp. are important causative agents of gastrointestinal infections in humans. The most frequently isolated strains of this bacterial genus are Campylobacter jejuni and Campylobacter coli. To date, genetic methods for bacterial identification have not been used in Bulgaria. We optimized the multiplex PSR assay to identify Campylobacter spp. and differentiate C. jejuni from C. coli in clinical isolates. We also compared this method with the routinely used biochemical methods. AIM: To identify Campylobacter spp. and discriminate C. coli from C. jejuni in clinical isolates using multiplex PCR assay. MATERIALS AND METHODS: Between February 2014 and January 2015 we studied 93 stool samples taken from patients with diarrheal syndrome and identified 40 species of Campylobacter spp. in them. The clinical material was cultured in microaerophilic atmosphere, the isolated strains being biochemically diff erentiated (hydrolysis of sodium hippurate for C. jejuni, and hydrolysis of indoxyl acetate for C. coli). DNA was isolated from the strains using QiaAmp MiniKit (QIAGEN, Germany). Twenty strains were tested with multiplex PCR for the presence of these genes: cadF, characteristic for Campylobacter spp., hipO for C. jejuni and asp for C. coli. RESULTS AND DISCUSSION: The biochemical tests identified 16 strains of C. jejuni, 3 strains of C. coli, and 1 strain of C. upsaliensis. After the multiplex PCR assay the capillary gel electrophoresis confirmed 16 strains of C. jejuni, 2 strains of C. coli and 2 strains of Campylobacter spp. - because of the presence of the gene cadF. C. jejuni has the gene hipO, and it is possible that this gene may not be expressed in the biochemical differentiation yielding a negative reaction as a result. In comparison, we can conclude that the genetic differentiation is a more accurate method than the biochemical tests. CONCLUSION: The multiplex PCR assay is a fast, accurate method for identifi cation of Campylobacter spp. which makes it quite necessary in the clinical diagnostic practice.

Advances in molecular surveillance of Clostridium difficile in Bulgaria.

The increasing incidence of Clostridium difficile infection (CDI) in Bulgaria has indicated the need to implement better surveillance approaches. The aim of the present work was to improve the current surveillance of CDI in Bulgaria by introducing innovative methods for identification and typing. One hundred and twenty stool samples obtained from 108 patients were studied over 4 years from which 32 C. difficile isolates were obtained. An innovative duplex EvaGreen real-time PCR assay based on simultaneous detection of the gluD and tcdB genes was developed for rapid C. difficile identification. Four toxigenic profiles were distinguished by PCR: A(+)B(+)CDT(-) (53.1 %, 17/32), A(-)B(+)CDT(-) (28.1 %, 9/32), A(+)B(+)CDT(+) (9.4 %, 3/32) and A(-)B(-)CDT(-) (9.4 %, 3/32). PCR ribotyping and multilocus variable number of tandem repeat analysis (MLVA7) were used for molecular characterization of the isolates. In total, nine distinct ribotypes were confirmed and the most prevalent for Bulgarian hospitals was 017 followed by 014/020, together accounting for 44 % of all isolates. Eighteen per cent of the isolates (6/32) did not match any of the 25 reference ribotypes available in this study. Twenty-four MLVA7 genotypes were detected among the clinical C. difficile isolates, distributed as follows: five for 017 ribotype, two for 014/020, 001, 002, 012 and 046 each, and one each for ribotypes 023, 070 and 078. The correlation between the typing methods was significant and allowed the identification of several clonal complexes. These results suggest that most C. difficile cases in the eight Bulgarian hospitals studied were associated with isolates belonging to the outbreak ribotypes 017 and 014/20, which are widely distributed in Europe. The real-time PCR protocol for simultaneous detection of gluD and tcdB proved to be very effective and improved C. difficile identification and confirmation of clinical C. difficile isolates.