Background

Bacillus licheniformis has for many years been used in the industrial production of enzymes, antibiotics and detergents. However, as a producer of dormant heat-resistant endospores B. licheniformis might contaminate semi-preserved foods. The aim of this study was to establish a robust and novel genotyping scheme for B. licheniformis in order to reveal the evolutionary history of 53 strains of this species. Furthermore, the genotyping scheme was also investigated for its use to detect food-contaminating strains.

Results

A multi-locus sequence typing (MLST) scheme, based on the sequence of six house-keeping genes (adk, ccpA, recF, rpoB, spo0A and sucC) of 53 B. licheniformis strains from different sources was established. The result of the MLST analysis supported previous findings of two different subgroups (lineages) within this species, named “A” and “B” Statistical analysis of the MLST data indicated a higher rate of recombination within group “A”. Food isolates were widely dispersed in the MLST tree and could not be distinguished from the other strains. However, the food contaminating strain B. licheniformis NVH1032, represented by a unique sequence type (ST8), was distantly related to all other strains.

Conclusions

In this study, a novel and robust genotyping scheme for B. licheniformis was established, separating the species into two subgroups. This scheme could be used for further studies of evolution and population genetics in B. licheniformis.

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.