The number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella inBacillus subtilishave never been identified, although theifmlocus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneousifmmutant ofB. subtilis, displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how theifmlocus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, namedswrA, that controls both swimming motility and swarming differentiation inB. subtilis. Functional analysis of theswrAoperon allowedswrAA(previously namedswrA[D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol.52:357-369, 2004]) to be the first gene identified inB. subtilisthat controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon,swrAB, is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.

Bacillus thuringiensisis being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen. While phospholipases, hemolysins, and enterotoxins are claimed to be responsible forB. thuringiensisvirulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions. This report describes the characterization of a mini-Tn10mutant ofB. thuringiensisthat is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C. The mutant strain was determined to carry the transposon insertion inflhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus. Interestingly, theflhAmutant ofB. thuringiensissynthesized flagellin but was impaired in flagellin export. Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable inB. thuringiensis, thus suggesting that the flagellar gene expression hierarchy ofB. thuringiensisdiffers from that described forBacillus subtilis. TheflhAmutant ofB. thuringiensiswas also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant. Since complementation of the mutant with a plasmid harboring theflhAgene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence inB. thuringiensismay be coordinately regulated byflhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.

Spore-forming Bacillus cereus is a common contaminant of dairy products. As the microorganism is widespread in the environment, it can contaminate milk at the time of milking, but it can also reach the dairy products in each phase of production, storage and ripening. Milk pasteurization treatment is not effective in reducing contamination and can instead act as an activator of spore germination, and a potential associated risk still exists with the consumption of some processed foods. Prevalences and concentrations of B. cereus in milk and dairy products are extremely variable worldwide: in pasteurized milk, prevalences from 2% to 65.3% were reported, with concentrations of up to 3 × 10⁵ cfu/g, whereas prevalences in cheeses ranged from 0 to 95%, with concentrations of up to 4.2 × 10⁶ cfu/g. Bacillus cereus is also well known to produce biofilms, a serious concern for the dairy industry, with up to 90% of spores that are resistant to cleaning and are easily transferred. As the contamination of raw materials is not completely avoidable, and the application of decontamination treatments is only possible for some ingredients and is limited by both commercial and regulatory reasons, it is clear that the correct application of hygienic procedures is extremely important in order to avoid and manage the circulation of B. cereus along the dairy supply chain. Future developments in interventions must consider the synergic application of different mild technologies to prevent biofilm formation and to remove or inactivate the microorganism on the equipment.

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.

The epidemiology of Bacillus cereus strains responsible for food poisoning is scantly known, mostly because the genotypic and toxigenic properties of

A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillusspores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than withBacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains ofB. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.