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.

Recent guidelines indicate that oral probiotics, living microorganisms able to confer a health benefit on the host, should be safe for human consumption, when administered in a sufficient amount, and resist acid and bile to exert their beneficial effects (e.g., metabolic, immunomodulatory, anti-inflammatory, competitive). This study evaluated quantitative and qualitative aspects and the viability in simulated gastric and intestinal juices of commercial probiotic formulations available in Italy. Plate counting and MALDI-TOF mass spectrometry were used to enumerate and identify the contained organisms. In vitro studies with two artificial gastric juices and pancreatin–bile salt solution were performed to gain information on the gastric tolerance and bile resistance of the probiotic formulations. Most preparations satisfied the requirements for probiotics and no contaminants were found. Acid resistance and viability in bile were extremely variable depending on the composition of the formulations in terms of contained species and strains. In conclusion, this study indicates good microbiological quality but striking differences in the behavior in the presence of acids and bile for probiotic formulations marketed in Italy.

Besides sporulation, Bacillus cereus can undergo a differentiation process in which short swimmer cells become elongated and hyperflagellated swarmer cells that favor migration of the bacterial community on a surface. The functionally enigmatic flagellar protein FlhF, which is the third paralog of the signal recognition particle (SRP) GTPases Ffh and FtsY, is required for swarming in many bacteria. Previous data showed that FlhF is involved in the control of the number and positioning of flagella in B. cereus. In this study, in silico analysis of B. cereus FlhF revealed that this protein presents conserved domains that are typical of SRPs in many organisms and a peculiar N-terminal basic domain. By proteomic analysis, a significant effect of FlhF depletion on the amount of secreted proteins was found with some proteins increased (e.g., B component of the non-hemolytic enterotoxin, cereolysin O, enolase) and others reduced (e.g., flagellin, L2 component of hemolysin BL, bacillolysin, sphingomyelinase, PC-PLC, PI-PLC, cytotoxin K) in the extracellular proteome of a ΔflhF mutant. Deprivation of FlhF also resulted in significant attenuation in the pathogenicity of this strain in an experimental model of infection in Galleria mellonella larvae. Our work highlights the multifunctional role of FlhF in B. cereus, being this protein involved in bacterial flagellation, swarming, protein secretion, and pathogenicity.

On the worldwide market, a great number of probiotic formulations are available to consumers as drugs, dietary supplements, and functional foods. For exerting their beneficial effects on host health, these preparations should contain a sufficient amount of the indicated living microbes and be pathogen-free to be safe. Therefore, the contained microbial species and their amount until product expiry are required to be accurately reported on the labels. While commercial formulations licensed as drugs are subjected to rigorous quality controls, less stringent regulations are generally applied to preparations categorized as dietary supplements and functional foods. Many reports indicated that the content of several probiotic formulations does not always correspond to the label claims in terms of microbial identification, number of living organisms, and purity, highlighting the requirement for more stringent quality controls by manufacturers. The main focus of this review is to provide an in-depth overview of the microbiological quality of probiotic formulations commercialized worldwide. Many incongruences in the compositional quality of some probiotic formulations available on the worldwide market were highlighted. Even if manufacturers carry at least some of the responsibility for these inconsistencies, studies that analyze probiotic products should be conducted following recommended and up-to-date methodologies.

Spores of several Bacillus species have long history of consumption and safe use as probiotics and a variety of formulations containing these organisms are available in the global market. Considering the difficulties in the identification of Bacillus species and the poor microbiological quality of many probiotic formulations, we used three up-to-date methodological approaches for analyzing the content of ten formulations marketed in Italy and labeled to contain Bacillus spores. We compared the performance of biochemical tests based on the BCL Vitek2 card and MALDI-TOF mass spectrometry, using 16S rDNA sequencing as the reference technique. The BCL card performed well in identifying all Bacillus probiotic strains as well as the Bruker’s MALDI Biotyper. Nevertheless, the MALDI score values were sometimes lower than those indicated by the manufacturer for correct species identification. Contaminant bacteria ( Lysinibacillus fusiformis , Acinetobacter baumannii , Bacillus cereus , Brevibacillus choshinensis , Bacillus licheniformis , Bacillus badius ) were detected in some formulations. Characterization of the B . cereus contaminant showed the potential pathogenicity of this strain. Microbial enumeration performed by the plate count method revealed that the number of viable cells contained in many of the analyzed products differed from the labeled amount. Overall, our data show that only two of the ten analyzed formulations qualitatively and quantitatively respect what is on the label. Since probiotic properties are most often strain specific, molecular typing of isolates of the two most common Bacillus species, B . clausii and B . coagulans , was also performed. In conclusion, the majority of the analyzed products do not comply with quality requirements, most likely leading to reduced/absent efficacy of the preparation and representing a potential infective risk for consumers.