FlhF Is Required for Swarming Motility and Full Pathogenicity of Bacillus cereus

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

Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate

BACKGROUND: Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media. RESULTS: Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0). CONCLUSIONS: The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits

In vitro assessment of probiotic attributes for strains contained in commercial formulations

Although probiotics are often indiscriminately prescribed, they are not equal and their effects on the host may profoundly differ. In vitro determination of the attributes of probiotics should be a primary concern and be performed even before clinical studies are designed. In fact, knowledge on the biological properties a microbe possesses is crucial for selecting the most suitable bacteriotherapy for each individual. Herein, nine strains (Bacillus clausii NR, OC, SIN, T, Bacillus coagulans ATCC 7050, Bifidobacterium breve DSM 16604, Limosilactobacillus reuteri DSM 17938, Lacticaseibacillus rhamnosus ATCC 53103, and Saccharomyces boulardii CNCM I-745) declared to be contained in six commercial formulations were tested for their ability to tolerate simulated intestinal conditions, adhere to mucins, and produce β-galactosidase, antioxidant enzymes, riboflavin, and D-lactate. With the exception of B. breve, all microbes survived in simulated intestinal fluid. L. rhamnosus was unable to adhere to mucins and differences in mucin adhesion were evidenced for L. reuteri and S. boulardii depending on oxygen levels. All microorganisms produced antioxidant enzymes, but only B. clausii, B. coagulans, B. breve, and L. reuteri synthesize β-galactosidase. Riboflavin secretion was observed for Bacillus species and L. rhamnosus, while D-lactate production was restricted to L. reuteri and L. rhamnosus. Our findings indicate that the analyzed strains possess different in vitro biological properties, thus highlighting the usefulness of in vitro tests as prelude for clinical research

In vitro resistance and evolution of resistance to tavaborole in Trichophyton rubrum

Tavaborole is currently used in the topical treatment of onychomycosis. In this study, we analyzed the in vitro emergence/evolution of resistance against tavaborole in Trichophyton rubrum When T. rubrum strains were propagated on media containing the MIC of tavaborole, spontaneous resistant mutants were isolated at a frequency of 10-8 The frequency was almost 100-fold higher following fungal growth in the presence of a sub-inhibitory tavaborole concentration (0.5-fold the MIC) for ten transfers. All collected mutants showed similar 4- to 8-fold increase in the drug minimal inhibitory concentration. No cross-resistance to other antifungals was evidenced

A new rapid method for direct antimicrobial susceptibility testing of bacteria from positive blood cultures

Background: Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections can lead to prompt appropriate antimicrobial therapy. To shorten species identification, in this study bacteria were recovered from monomicrobial blood cultures by serum separator tubes and spotted onto the target plate for direct MALDI-TOF MS identification. Proper antibiotics were selected for direct AST based on species identification. In order to obtain rapid AST results, bacteria were recovered from positive blood cultures by two different protocols: by serum separator tubes (further referred to as PR1), or after a short-term subculture in liquid medium (further referred to as PR2). The results were compared with those obtained by the method currently used in our laboratory consisting in identification by MALDI-TOF and AST by Vitek 2 or Sensititre on isolated colonies. Results: The direct MALDI-TOF method concordantly identified with the current method 97.5 % of the Gram-negative bacteria and 96.1 % of the Gram-positive cocci contained in monomicrobial blood cultures. The direct AST by PR1 and PR2 for all isolate/antimicrobial agent combinations was concordant/correct with the current method for 87.8 and 90.5 % of Gram-negative bacteria and for 93.1 and 93.8 % of Gram-positive cocci, respectively. In particular, 100 % categorical agreement was found with levofloxacin for Enterobacteriaceae by both PR1 and PR2, and 99.0 and 100 % categorical agreement was observed with linezolid for Gram-positive cocci by PR1 and PR2, respectively. There was no significant difference in accuracy between PR1 and PR2 for Gram-negative bacteria and Gram-positive cocci. Conclusions: This newly described method seems promising for providing accurate AST results. Most importantly, these results would be available in a few hours from blood culture positivity, which would help clinicians to promptly confirm or streamline an effective antibiotic therapy in patients with bloodstream infections

Role of swarming migration in the pathogenesis of bacillus endophthalmitis.

PURPOSE. Bacillus cereus causes one of the most rapidly blinding forms of bacterial endophthalmitis. Migration of B. cereus throughout the eye during endophthalmitis is a unique aspect of this disease that may contribute to intraocular virulence. This study was conducted to analyze the contribution of swarming and intraocular migration to the pathogenesis of experimental endophthalmitis. METHODS. Eyes were injected intravitreally with 100 colonyforming units (CFU) of either wild-type, nonswarming, or swarming-complemented strains of B. cereus. Pathogenicity was compared throughout the course of infection by biomicroscopy, histology, electroretinography, and bacterial and inflammatory cell quantitation. RESULTS. Wild-type, nonswarming, and swarming-complemented B. cereus strains grew to a similar number in the vitreous throughout the course of infection. Unlike the wild-type and swarming-complemented strains, the nonswarming mutant did not migrate to the anterior segment during infection. The rate of decrease in retinal responses of eyes infected with the all strains was similar, resulting in near complete elimination of retinal function by 12 hours. All Bacillus strains caused similar degrees of posterior segment inflammation and retinal destruction. However, the accumulation of inflammatory cells in the anterior chamber, hyphemae, and corneal ring abscesses did not occur in eyes infected with the nonswarming mutant. CONCLUSIONS. The deficiency in swarming had little effect on retinal function loss or the overall course or severity of experimental B. cereus endophthalmitis. However, a deficiency in swarming prevented Bacillus from migrating to the anterior segment, leading to less severe anterior segment disease

Bacillus cereus in Dairy Products and Production Plants

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

Survival and persistence of Bacillus clausii in the human gastrointestinal tract following oral administration as spore-based probiotic formulation

AIMS: This study aimed to investigate the fate of Bacillus clausii spores orally administered as lyophilized or liquid formulation to healthy volunteers. METHODS AND RESULTS: The study was a randomized, open-label, cross-over trial in which two commercial probiotic formulations containing spores of four antibiotic-resistant B. clausii strains (OC, NR, SIN, T) were given as a single dose administration. Faecal B. clausii units of each strain were counted on selective media and extrapolated for the total weight of evacuated faeces. RAPD-PCR typing was used to confirm B. clausii identification. Bacillus clausii was found alive in faeces for up to 12 days. In some volunteers, the recovered amount of OC, NR or SIN was higher than the number of administered spores. Bioequivalence among the two formulations was demonstrated. CONCLUSIONS: Bacillus clausii spores survive transit through the human gastrointestinal tract. They can undergo germination, outgrowth and multiplication as vegetative forms. Bacillus clausii strains can have different ability to survive in the intestinal environment. Bacillus clausii spores administered as liquid suspension or lyophilized form behave similarly in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes towards a better understanding of the behaviour of B. clausii spores as probiotics

Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low nutrient conditions

Bacillus cereus ATCC 14579 can respond to nutrient changes by adopting different forms of surface translocation. The B. cereus ATCC 14579 DeltaplcR mutant, but not the wild type, formed dendritic (branched) patterns on EPS [a low-nutrient medium that contains 7.0 g K(2)HPO(4), 3.0 g KH(2)PO(4), 0.1 g MgSO(4).7H(2)O, 0.1 g (NH(4))(2)SO(4), 0.01 g CaCl(2), 0.001 g FeSO(4), 0.1 g NaCl, 1.0 g glucose, and 125 mg yeast extract per liter] containing 0.7% agar. The dendritic patterns formed by sliding translocation of nonflagellated cells are enhanced under low-nutrient conditions and require sufficient production of a biosurfactant, which appears to be repressed by PlcR. The wild-type and complemented strains failed to slide on the surface of EPS agar because of the production of low levels of biosurfactant. Precoating EPS agar surfaces with surfactin (a biosurfactant produced by Bacillus subtilis) or biosurfactant purified from the DeltaplcR mutant rescued the ability of the wild-type and complemented strains to slide. When grown on a nutrient-rich medium like Luria-Bertani agar, both the wild-type and DeltaplcR mutant strains produced flagella. The wild type was hyperflagellated and elongated and exhibited swarming behavior, while the DeltaplcR mutant was multiflagellated and the cells often formed long chains but did not swarm. Thin-layer chromatography and mass spectrometry analyses suggested that the biosurfactant purified from the DeltaplcR mutant was a lipopeptide and had a mass of 1,278.1722 (m/z). This biosurfactant has hemolytic activity and inhibited the growth of several gram-positive bacteria