L-alanine-induced germination in Bacillus licheniformis -the impact of native gerA sequences.

BACKGROUND: L-alanine, acting through the GerA receptor, was recently found to be an efficient germinant in Bacillus licheniformis ATCC14580/DSM13. RESULTS: In this study, we show that several of 46 examined B. licheniformis strains germinate remarkably slower than the type strain when exposed to L-alanine. These strains are not necessarily closely related, as determined by MLST (multi-locus sequence typing). Three of the slow-germinating strains were further examined in order to see whether nucleotide substitutions in the gerA sequences were responsible for the slow L-alanine germination. This was performed by complementing the transformable type strain derivate MW3ΔgerAA with gerA variants from the three slow-germinating strains; NVH1032, NVH1112 and NVH800. CONCLUSIONS: A wide selection of B. licheniformis strains was evaluated for L-alanine-induced germination efficiency. Our results show that gerA substitutions could only partially explain why spores of some B. licheniformis strains responded slower than others in the presence of L-alanine.

Analysis of the genetic distribution among members of Clostridium botulinum group I using a novel multilocus sequence typing (MLST) assay.

Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partial sequencing of the adk, atpH, gyrB, proC, rpoD and spo0A genes from 51 various C. botulinum/sporogenes isolates was performed, resulting in 37 different sequence types (STs). Analysis of the sequence data revealed a genetic distribution in five larger clusters with a loose correlation to the BoNT serotypes. The developed MLST assay had a slightly lower resolution ability when compared to the MLVA (multilocus variable number of tandem repeat analysis), but the two methods resulted in similar subclusters of the strains possessing the BoNT serotypes A, B and F. The current work presents the development of a novel MLST assay useful for genotyping C. botulinum related to basic phylogenetic research and trace-back analysis in microbial forensic studies.

Genotyping of B. licheniformis based on a novel multi-locus sequence typing (MLST) scheme.

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.

Real-time PCR and NASBA for rapid and sensitive detection of Vibrio cholerae in ballast water.

Transport of ballast water is one major factor in the transmission of aquatic organisms, including pathogenic bacteria. The IMO-guidelines of the Convention for the Control and Management of Ships' Ballast Water and Sediments, states that ships are to discharge <1 CFU per 100 ml ballast water of toxigenic Vibrio cholerae, emphasizing the need to establish test methods. To our knowledge, there are no methods sensitive and rapid enough available for cholera surveillance of ballast water. In this study real-time PCR and NASBA methods have been evaluated to specifically detect 1 CFU/100ml of V. cholerae in ballast water. Ballast water samples spiked with V. cholerae cells were filtered and enriched in alkaline peptone water before PCR or NASBA detection. The entire method, including sample preparation and analysis was performed within 7 h, and has the potential to be used for analysis of ballast water for inspection and enforcement control.

A multiplatform real-time polymerase chain reaction detection assay for Vibrio cholerae.

We report a multiplatform real-time polymerase chain reaction methodology based on genes encoding for the regulatory toxR activator and enterotoxin A protein to determine enterotoxigenic Vibrio cholerae types from other vibrios. This assay, which was successfully validated on a collection of 87 bacterial strains, including 63 representatives of V. cholerae and 8 noncholera vibrios provides a rapid tool for detection and identification of cholera.

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae.

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index=0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.

Genetic distribution of 295 Bacillus cereus group members based on adk-screening in combination with MLST (Multilocus Sequence Typing) used for validating a primer targeting a chromosomal locus in B. anthracis.

The genetic distribution of 295 Bacillus cereus group members has been investigated by using a modified Multilocus Sequence Typing method (MLST). By comparing the nucleic acid sequence of the adk gene fragment, isolates of B. cereus group members most related to B. anthracis may be easily identified. The genetic distribution, with focus on the B. anthracis close neighbours, was used to evaluate a new primer set for specific identification of B. anthracis. This primer set, BA5510-1/2, targeted the putative B. anthracis specific gene BA5510. Real-time PCR using BA5510-1/2 amplified the target fragment from all B. anthracis strains tested and only two (of 289) non-B. anthracis strains analysed. This is one of the most thoroughly validated chromosomal B. anthracis markers for real-time PCR identification, in which the screened collection contained several very closely related B. anthracis strains.

Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification.

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.

Heterologous gene expression in Lactococcus lactis; expression of the Azotobacter vinelandii algE6 gene product displaying mannuronan C-5 epimerase activity.

The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1-7 can be used to improve the properties of the commercially important polysaccharide alginate that is widely used in a variety of products, such as food and pharmaceuticals. Since lactic acid bacteria are generally regarded as safe, they are attractive candidates for production of the epimerases. A. vinelandii genes are GC-rich, in contrast to those of lactic acid bacteria, but we show here that significant expression levels of the epimerase AlgE6 can be obtained in Lactococcus lactis using the nisin-controlled expression system. A 1200-fold induction ratio was obtained resulting in an epimerase activity of 23900 dpm mg(-1) h(-1), using a tritiated alginate substrate. The epimerase was detected by Western blotting and nuclear magnetic resonance spectroscopy analysis of its reaction product showed that the enzyme displayed catalytic properties similar to those produced in Escherichia coli.