Characterization of genetic diversity of Bacillus anthracis in France by using high-resolution melting assays and multilocus variable-number tandem-repeat analysis.

Using high-resolution melting (HRM) analysis, we developed a cost-effective method to genotype a set of 13 phylogenetically informative single-nucleotide polymorphisms (SNPs) within the genome of Bacillus anthracis. SNP discrimination assays were performed in monoplex or duplex and applied to 100 B. anthracis isolates collected in France from 1953 to 2009 and a few reference strains. HRM provided a reliable and cheap alternative to subtype B. anthracis into one of the 12 major sublineages or subgroups. All strains could be correctly positioned on the canonical SNP (canSNP) phylogenetic tree, except the divergent Pasteur vaccine strain ATCC 4229. We detected the cooccurrence of three canSNP subgroups in France. The dominant B.Br.CNEVA sublineage was found to be prevalent in the Alps, the Pyrenees, the Auvergne region, and the Saône-et-Loire department. Strains affiliated with the A.Br.008/009 subgroup were observed throughout most of the country. The minor A.Br.001/002 subgroup was restricted to northeastern France. Multiple-locus variable-number tandem-repeat analysis using 24 markers further resolved French strains into 60 unique profiles and identified some regional patterns. Diversity found within the A.Br.008/009 and B.Br.CNEVA subgroups suggests that these represent old, ecologically established clades in France. Phylogenetic relationships with strains from other parts of the world are discussed.

A quantitative PCR assay for the detection and quantification of Shiga toxin-producing Escherichia coli (STEC) in minced beef and dairy products.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are amongst major causes of food-borne infectious diseases and outbreaks. A new quantitative PCR (qPCR) assay was designed to detect all known stx gene subtypes in a single reaction, including the most distant variant stx2f. Performance of this assay was evaluated in combination with two different internal amplification controls (IAC), a competitive one specific for the assay and a universal IAC based on plasmid pUC19. The qPCR assay was 100% specific and showed analytical sensitivity of two STEC genome copies per reaction. The diagnostic approach proposed, combining enrichment, automated DNA extraction and qPCR detection, could reliably detect the presence of STEC in minced beef and cheese inoculated before enrichment at <4 CFU per 25 g. A comparative study performed on 240 minced beef and 113 raw milk cheese samples demonstrated that the method developed was as effective as two PCR screening assays used routinely in our laboratory to detect STEC. The new assay also proved to be appropriate for the direct quantification of STEC in milk and minced meat. It was found to be quantitative over a five log dynamic range, from 4 × 106 to 40 CFU/mL for milk and from 107 to 10² CFU/g for minced beef. In conclusion, the qPCR assay developed here represents a valuable tool for rapid detection and quantification of STEC in foods such as minced beef and dairy products as it ensures a high sensitivity and an optimal STEC diagnostic spectrum, taking into account the genetic stx variability observed in STEC population.

Development of a real-time PCR assay with an internal amplification control for the screening of Shiga toxin-producing Escherichia coli in foods.

AIMS: To develop and evaluate a real-time PCR assay incorporating an internal amplification control (IAC) suitable for the screening of Shiga toxin (Stx)-producing Escherichia coli (STEC) in foods. METHODS AND RESULTS: A competitive IAC was constructed and included in an stx-specific real-time PCR assay. Coupled to 18-h enrichment and automated DNA extraction, the assay could reliably detect the presence of STEC in minced meats inoculated at 10 CFU per 25 g. Its performance was evaluated on 415 minced beef and 112 raw milk cheese samples and compared with that of a PCR-ELISA method. Fifty-three minced meats and 31 cheeses were found stx-positive, giving 98.3% and 93.75% concordance, respectively, with the PCR-ELISA reference method. CONCLUSIONS: A highly sensitive stx-specific real-time PCR method including an IAC was developed, facilitating monitoring of false-negative results due to PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined with automated DNA extraction, the stx-IAC real-time PCR assay represents a suitable method for rapid screening of STEC in foods.

Screening of food raw materials for the presence of Shiga toxin-producing Escherichia coli O91:H21.

AIMS: To provide information on the prevalence and detection, in foods, of Shiga toxin-producing Escherichia coli (STEC) O91:H21. METHODS AND RESULTS: Seven hundred fifteen minced beef meats and 205 raw milk samples were analysed by stx-specific PCR-ELISA. Samples positive for stx were subsequently tested for the presence of wzy-O91, fliC-H21 and the adhesin-encoding gene saa. For minced meat, 16 (2.2%) and 11 (1.5%) samples were found positive for (stx, wzy-O91, fliC-H21) and (stx, wzy-O91, fliC-H21, saa) combinations, respectively. For raw milk, seven (3.4%) samples were found positive for the (stx, wzy-O91, fliC-H21) combination but none of these contained saa. Two STEC O91:H21 saa-positive strains and three STEC O91 H21- and saa-negative strains were isolated by colony hybridization. CONCLUSIONS: A low prevalence of potentially pathogenic STEC O91:H21 in food products was found using a combination of PCR assays targeting stx, wzy-O91, fliC-H21 and saa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-based approach described here represents a valuable method for rapid screening of food samples contaminated by STEC O91:H21.

H-NS and H-NS-like proteins in Gram-negative bacteria and their multiple role in the regulation of bacterial metabolism.

In Escherichia coli, the H-NS protein plays an important role in the structure and the functioning of bacterial chromosome. A homologous protein has also been identified in several enteric bacteria and in closely related organisms such as Haemophilus influenzae. To get information on their structure and their function, we identified H-NS-like proteins in various microorganisms by different procedures. In silico analysis of their amino acid sequence and/or in vivo experiments provide evidence that more than 20 proteins belong to the same class of regulatory proteins. Moreover, large scale technologies demonstrate that, at least in E. coli, the loss of motility in hns mutants results from a lack of flagellin biosynthesis, due to the in vivo repression of flagellar gene expression. In contrast, several genes involved in adaptation to low pH are strongly induced in a H-NS deficient strain, resulting in an increased resistance to acidic stress. Finally, expression profiling and phenotypic analysis suggest that, unlike H-NS, its paralogous protein StpA does not play any role in these processes.

Changes in cspL, cspP, and cspC mRNA abundance as a function of cold shock and growth phase in Lactobacillus plantarum.

An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in Lactobacillus plantarum. In addition to the two previously reported cspL and cspP genes, a third gene, cspC, has been cloned and characterized. All three genes encode small 66-amino-acid proteins with between 73 and 88% identity. Comparative Northern blot analyses showed that the level of cspL mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of cspC and cspP remain unchanged or increase only slightly (about two- to threefold). Cold induction of cspL mRNA is transient and delayed in time as a function of the severity of the temperature downshift. The cold shock behavior of the three csp mRNAs contrasts with that observed for four unrelated non-csp genes, which all showed a sharp decrease in mRNA level, followed in one case (bglH) by a progressive recovery of the transcript during prolonged cold exposure. Abundance of the three csp mRNAs was also found to vary during growth at optimal temperature (28 degrees C). cspC and cspP mRNA levels are maximal during the lag period, whereas the abundance of the cspL transcript is highest during late-exponential-phase growth. The differential expression of the three L. plantarum csp genes can be related to sequence and structural differences in their untranslated regions. It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.

The alanine racemase gene is essential for growth of Lactobacillus plantarum.

The Lactobacillus plantarum alr gene encoding alanine racemase was cloned by complementation of an Escherichia coli Alr- DadX- double mutant strain. Knockout of the alr gene abolished all measurable alanine racemase activity, and the mutant was shown to be strictly dependent on D-alanine for growth.

Cloning and characterization of cspL and cspP, two cold-inducible genes from Lactobacillus plantarum.

Two cold shock genes, cspL and cspP, have been cloned from two Lactobacillus plantarum strains. These genes, which are nonallelic, were present in all strains tested. The genes encode 66-amino-acid polypeptides related to each other and to the cold shock Csp family. Transcription of cspP rendered a single mRNA, while two cspL mRNAs were found with common 5' ends. The amounts of these transcripts increased moderately upon exposure of the cultures to cold.