Anti-Staphylococcal Biofilm Effects of a Liposome-Based Formulation Containing Citrus Polyphenols.

Biofilms are surface-associated microbial communities embedded in a matrix that is almost impenetrable to antibiotics, thus constituting a critical health threat. Biofilm formation on the cornea or ocular devices can lead to serious and difficult-to-treat infections. Nowadays, natural molecules with antimicrobial activity and liposome-based delivery systems are proposed as anti-biofilm candidates. In this study, the anti-biofilm activity of a formulation containing citrus polyphenols encapsulated in liposomes was evaluated against Staphylococcus aureus and Staphylococcus epidermidis, the most common agents in ocular infections. The formulation activity against planktonic staphylococci was tested by broth microdilution and sub-inhibitory concentrations were used to evaluate the effect on biofilm formation using the crystal violet (CV) assay. The eradicating effect of the preparation on mature biofilms was investigated by the CV assay, plate count, and confocal laser scanning microscopy. The product was bactericidal against staphylococci at a dilution of 1:2 or 1:4 and able to reduce biofilm formation even if diluted at 1:64. The formulation also had the ability to reduce the biomass of mature biofilms without affecting the number of cells, suggesting activity on the extracellular matrix. Overall, our results support the application of the used liposome-encapsulated polyphenols as an anti-biofilm strategy to counter biofilm-associated ocular infections.

Hospital distribution, seasonality, time trends and antifungal susceptibility profiles of all Aspergillus species isolated from clinical samples from 2015 to 2022 in a tertiary care hospital.

BACKGROUND: Aspergillus species cause a variety of serious clinical conditions with increasing trend in antifungal resistance. The present study aimed at evaluating hospital epidemiology and antifungal susceptibility of all isolates recorded in our clinical database since its implementation. METHODS: Data on date of isolation, biological samples, patients' age and sex, clinical settings, and antifungal susceptibility tests for all Aspergillus spp. isolated from 2015 to 2022 were extracted from the clinical database. Score test for trend of odds, non-parametric Mann Kendall trend test and logistic regression analysis were used to analyze prevalence, incidence, and seasonality of Aspergillus spp. isolates. RESULTS: A total of 1126 Aspergillus spp. isolates were evaluated. A. fumigatus was the most prevalent (44.1%) followed by A. niger (22.3%), A. flavus (17.7%) and A. terreus (10.6%). A. niger prevalence increased over time in intensive care units (p-trend = 0.0051). Overall, 16 (1.5%) were not susceptible to one azole compound, and 108 (10.9%) to amphotericin B, with A. niger showing the highest percentage (21.9%). The risk of detecting A. fumigatus was higher in June, (OR = 2.14, 95% CI [1.16; 3.98] p = 0.016) and reduced during September (OR = 0.48, 95% CI [0.27; 0.87] p = 0.015) and October as compared to January (OR = 0.39, 95% CI [0.21; 0.70] p = 0.002. A. niger showed a reduced risk of isolation from all clinical samples in the month of June as compared to January (OR = 0.34, 95% CI [0.14; 0.79] p = 0.012). Seasonal trend for A. flavus showed a higher risk of detection in September (OR = 2.7, 95% CI [1.18; 6.18] p = 0.019), October (OR = 2.32, 95% CI [1.01; 5.35] p = 0.048) and November (OR = 2.42, 95% CI [1.01; 5.79] p = 0.047) as compared to January. CONCLUSIONS: This is the first study to analyze, at once, data regarding prevalence, time trends, seasonality, species distribution and antifungal susceptibility profiles of all Aspergillus spp. isolates over a 8-year period in a tertiary care center. Surprisingly no increase in azole resistance was observed over time.

A Millifluidic Chamber for Controlled Shear Stress Testing: Application to Microbial Cultures.

In vitro platforms such as bioreactors and microfluidic devices are commonly designed to engineer tissue models as well as to replicate the crosstalk between cells and microorganisms hosted in the human body. These systems promote nutrient supply and waste removal through culture medium recirculation; consequently, they intrinsically expose cellular structures to shear stress, be it a desired mechanical stimulus to drive the cell fate or a potential inhibitor for the model maturation. Assessing the impact of shear stress on cellular or microbial cultures thus represents a crucial step to define proper environmental conditions for in vitro models. In this light, the aim of this study was to develop a millifluidic device enabling to generate fully controlled shear stress profiles for quantitatively probing its influence on tissue or bacterial models, overcoming the limitations of previous reports proposing similar devices. Relying on this millifluidic tool, we present a systematic methodology to test how adherent cellular structures react to shear forces, which was applied to the case of microbial biofilms as a proof of concept. The results obtained suggest our approach as a suitable testbench to evaluate culture conditions in terms of shear stress faced by cells or microorganisms.

Impact of Bacillus cereus on the Human Gut Microbiota in a 3D In Vitro Model.

In vitro models for culturing complex microbial communities are progressively being used to study the effects of different factors on the modeling of in vitro-cultured microorganisms. In previous work, we validated a 3D in vitro model of the human gut microbiota based on electrospun gelatin scaffolds covered with mucins. The aim of this study was to evaluate the effect of Bacillus cereus, a pathogen responsible for food poisoning diseases in humans, on the gut microbiota grown in the model. Real-time quantitative PCR and 16S ribosomal RNA-gene sequencing were performed to obtain information on microbiota composition after introducing B. cereus ATCC 14579 vegetative cells or culture supernatants. The adhesion of B. cereus to intestinal mucins was also tested. The presence of B. cereus induced important modifications in the intestinal communities. Notably, levels of Proteobacteria (particularly Escherichia coli), Lactobacillus, and Akkermansia were reduced, while abundances of Bifidobacterium and Mitsuokella increased. In addition, B. cereus was able to adhere to mucins. The results obtained from our in vitro model stress the hypothesis that B. cereus is able to colonize the intestinal mucosa by stably adhering to mucins and impacting intestinal microbial communities as an additional pathogenetic mechanism during gastrointestinal infection.

Antibacterial and Antibiofilm Effects of Lactobacilli Strains against Clinical Isolates of Pseudomonas aeruginosa under Conditions Relevant to Cystic Fibrosis.

Therapy of lung infections sustained by Pseudomonas aeruginosa in cystic fibrosis (CF) patients is challenging due to the presence of a sticky mucus in the airways and the ability of the bacterium to form biofilm, which exhibits increased antibiotic tolerance. A lung-directed bacteriotherapy through the airway administration of probiotics could represent an alternative approach to probiotic diet supplementation to improve the benefits and clinical outcomes of this kind of intervention in CF patients. This study aims to evaluate the ability of probiotic strains to grow in artificial sputum medium (ASM), mimicking the CF lung microenvironment, and to affect the planktonic and biofilm growth of CF clinical strains of P. aeruginosa in the same conditions. The results demonstrate that Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum (LP) can grow in ASM. LP inhibited the planktonic growth of P. aeruginosa, while both lactobacilli reduced the pre-formed biofilm of P. aeruginosa. Interestingly, LP was demonstrated to reduce the amount of polysaccharides in the extracellular matrix of P. aeruginosa biofilms and to potentiate the antibiofilm effects of tobramycin. Overall, the results indicated that LP is a promising candidate as an adjuvant in the antimicrobial therapy of P. aeruginosa infections in CF patients.

Use of Saccharomyces boulardii CNCM I-745 as therapeutic strategy for prevention of nonsteroidal anti-inflammatory drug-induced intestinal injury.

BACKGROUND AND PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) can be associated with severe adverse digestive effects. This study examined the protective effects of the probiotic Saccharomyces boulardii CNCM I-745 in a rat model of diclofenac-induced enteropathy. EXPERIMENTAL APPROACH: Enteropathy was induced in 40-week-old male rats by intragastric diclofenac (4 mg·kg-1 BID for 14 days). S. boulardii CNCM I-745 (3 g·kg-1 BID by oral gavage) was administered starting 14 days before (preventive protocol) or along with (curative protocol) diclofenac administration. Ileal damage, inflammation, barrier integrity, gut microbiota composition and toll-like receptors (TLRs)-nuclear factor κB (NF-κB) pathway were evaluated. KEY RESULTS: Diclofenac elicited intestinal damage, along with increments of myeloperoxidase, malondialdehyde, tumour necrosis factor and interleukin-1ß, overexpression of TLR2/4, myeloid differentiation primary response 88 (Myd88) and NF-κB p65, increased faecal calprotectin and butyrate levels, and decreased blood haemoglobin levels, occludin and butyrate transporter monocarboxylate transporter 1 (MCT1) expression. In addition, diclofenac provoked a shift of bacterial taxa in both faecal and ileal samples. Treatment with S. boulardii CNCM I-745, in both preventive and curative protocols, counteracted the majority of these deleterious changes. Only preventive administration of the probiotic counteracted NSAID-induced decreased expression of MCT1 and increase in faecal butyrate levels. Occludin expression, after probiotic treatment, did not significantly change. CONCLUSIONS AND IMPLICATIONS: Treatment with S. boulardii CNCM I-745 prevents diclofenac-induced enteropathy through anti-inflammatory and antioxidant activities. Such effects are likely to be related to increased tissue butyrate bioavailability, through an improvement of butyrate uptake by the enteric mucosa.

Development of an In Vitro Model of the Gut Microbiota Enriched in Mucus-Adhering Bacteria.

Culturing the gut microbiota in in vitro models that mimic the intestinal environment is increasingly becoming a promising alternative approach to study microbial dynamics and the effect of perturbations on the gut community. Since the mucus-associated microbial populations in the human intestine differ in composition and functions from their luminal counterpart, we attempted to reproduce in vitro the microbial consortia adhering to mucus using an already established three-dimensional model of the human gut microbiota. Electrospun gelatin structures supplemented or not with mucins were inoculated with fecal samples and compared for their ability to support microbial adhesion and growth over time, as well as to shape the composition of the colonizing communities. Both scaffolds allowed the establishment of long-term stable biofilms with comparable total bacterial loads and biodiversity. However, mucin-coated structures harbored microbial consortia especially enriched in Akkermansia, Lactobacillus, and Faecalibacterium, being therefore able to select for microorganisms commonly considered mucosa-associated in vivo. IMPORTANCE These findings highlight the importance of mucins in shaping intestinal microbial communities, even those in artificial gut microbiota systems. We propose our in vitro model based on mucin-coated electrospun gelatin structures as a valid device for studies evaluating the effects of exogenous factors (nutrients, probiotics, infectious agents, and drugs) on mucus-adhering microbial communities.

Analysis of the microbial content of probiotic products commercialized worldwide and survivability in conditions mimicking the human gut environment.

Introduction: Probiotics are living microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Adequate number of living microbes, the presence of specific microorganisms, and their survival in the gastrointestinal (GI) environment are important to achieve desired health benefits of probiotic products. In this in vitro study, 21 leading probiotic formulations commercialized worldwide were evaluated for their microbial content and survivability in simulated GI conditions. Methods: Plate-count method was used to determine the amount of living microbes contained in the products. Culture-dependent Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry and culture-independent metagenomic analysis through 16S and 18S rDNA sequencing were applied in combination for species identification. To estimate the potential survivability of the microorganisms contained in the products in the harsh GI environment, an in vitro model composed of different simulated gastric and intestinal fluids was adopted. Results: The majority of the tested probiotic products were concordant with the labels in terms of number of viable microbes and contained probiotic species. However, one product included fewer viable microbes than those displayed on the label, one product contained two species that were not declared, and another product lacked one of the labeled probiotic strains. Survivability in simulated acidic and alkaline GI fluids was highly variable depending on the composition of the products. The microorganisms contained in four products survived in both acidic and alkaline environments. For one of these products, microorganisms also appeared to grow in the alkaline environment. Conclusion: This in vitro study demonstrates that most globally commercialized probiotic products are consistent with the claims described on their labels with respect to the number and species of the contained microbes. Evaluated probiotics generally performed well in survivability tests, although viability of microbes in simulated gastric and intestinal environments showed large variability. Although the results obtained in this study indicate a good quality of the tested formulations, it is important to stress that stringent quality controls of probiotic products should always be performed to provide optimal health benefits for the host.

Dietary Supplementation with the Probiotic SF68 Reinforces Intestinal Epithelial Barrier in Obese Mice by Improving Butyrate Bioavailability.

SCOPE: Modifications in intestinal microbiota and its metabolites, the short-chain fatty acids (SCFA) are main factors altering intestinal epithelial barrier integrity and eliciting the onset of a meta-inflammation observed in obesity. The present study is aimed at evaluating the efficacy of Enterococcus faecium (SF68) administration in counteracting the impairment of gut barrier and enteric inflammation in a model of diet-induced obesity, characterizing the molecular mechanisms underlying such beneficial effects. METHODS AND RESULTS: Male C57BL/6J mice, fed with standard diet (SD) or high-fat diet (HFD), are treated with SF68 (108  CFU day-1 ). After 8 weeks, plasma interleukin (IL)-1ß and lipopolysaccharide binding protein (LBP) are measured, analysis of fecal microbiota composition and butyrate content as well as intestinal malondialdehyde, myeloperoxidase, mucins, tight junction protein, and butyrate transporter expression are investigated. After 8 weeks, SF68 administration counteracts the body weight gain in HFD mice, reducing plasma IL-1ß and LBP. In parallel, SF68 treatment acts against the intestinal inflammation in HFD-fed animals and improves the intestinal barrier integrity and functionality in obese mice via the increase in tight junction protein and intestinal butyrate transporter (sodium-coupled monocarboxylate transporter 1 ) expression. CONCLUSIONS: Supplementation with SF68 reduces intestinal inflammation and reinforces the enteric epithelial barrier in obese mice, improving the transport and utilization of butyrate.

HPLC-MS-MS quantification of short-chain fatty acids actively secreted by probiotic strains.

Introduction: Short-chain fatty acids (SCFAs) are the main by-products of microbial fermentations occurring in the human intestine and are directly involved in the host's physiological balance. As impaired gut concentrations of acetic, propionic, and butyric acids are often associated with systemic disorders, the administration of SCFA-producing microorganisms has been suggested as attractive approach to solve symptoms related to SCFA deficiency. Methods: In this research, nine probiotic strains (Bacillus clausii NR, OC, SIN, and T, Bacillus coagulans ATCC 7050, Bifidobacterium breve DSM 16604, Limosilactobacillus reuteri DSM 17938, Lacticaseibacillus rhamnosus ATCC 53103, and Saccharomyces boulardii CNCM I-745) commonly included in commercial formulations were tested for their ability to secrete SCFAs by using an improved protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS). Results: The developed method was highly sensitive and specific, showing excellent limits of detection and quantification of secreted SCFAs. All tested microorganisms were shown to secrete acetic acid, with only B. clausii and S. boulardii additionally able to produce propionic and butyric acids. Quantitative differences in the secretion of SCFAs were also evidenced. Discussion: The experimental approach described in this study may contribute to the characterization of probiotics as SCFA-producing organisms, a crucial stage toward their application to improve SCFA deficiency.

Prevalence, Virulence Potential, and Growth in Cheese of Bacillus cereus Strains Isolated from Fresh and Short-Ripened Cheeses Sold on the Italian Market.

This study investigated B. cereus presence in 122 samples belonging to 34 typologies of fresh or short-ripened cheeses made from cow, sheep, goat, or buffalo pasteurized milk, and sold on the Italian market. B. cereus was isolated at a prevalence of 9.8%, with a marked variability among cheese categories, and at low counts (always below 2.26 Log CFU/g). Twelve isolates were identified by MALDI-TOF analysis and typified by RAPD PCR as belonging to different B. cereus strains. All the strains were tested for the production of hemolysin BL, phosphatidylcholine-specific phospholipase C, proteases, and biofilm formation, and for the presence of chromosomal toxin-encoding genes (sph, plcA, cytK, entFM, bcet, nheA, nheB, nheC). Overall, 92% of strains harbored bcet, 75% the three genes nheA, nheB, and nheC, as well as plcA and sph, 67% entFM, and 33% cytK. All strains showed biofilm-forming ability. A chemical-physical characterization of the cheeses was also performed to show their suitability as substrates for B. cereus growth, showing high heterogeneity in terms of pH, aw, salt content, and concentration of organic acids. Finally, the ability to support spore germination and vegetative cell growth of a selected cheese was investigated in spores-inoculated samples maintained at 10 °C and 15 °C, showing the inhibitory effect of low storage temperatures.

Sensitization of KPC and NDM Klebsiella pneumoniae To Rifampicin by the Human Lactoferrin-Derived Peptide hLF1-11.

A synergistic effect of non-bactericidal concentrations of the human lactoferrin (hLF)-derived peptide hLF1-11 and rifampicin against multidrug-resistant KPC (Klebsiella pneumoniae carbapenemase)-producing K. pneumoniae has been previously shown. The present study focuses on the mechanism(s) underlying this synergistic effect. The contribution of hLF1-11 and rifampicin to the synergistic effect was evaluated by killing assays with KPC K. pneumoniae cells incubated with hLF1-11 and, after washing, with rifampicin, or vice versa. Cell membrane permeability and polarization upon exposure to hLF1-11 and/or rifampicin were evaluated by ethidium bromide (EtBr) and DiBAC4(3) (bis-1,3-dibutylbarbituric acid trimethine oxonol) permeability, respectively. The effect of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an uncoupler of oxidative phosphorylation, was also evaluated. KPC K. pneumoniae cells were effectively killed after prior exposure to rifampicin for 30 to 60 min followed by treatment with hLF1-11, while no antibacterial activity was observed when cells were incubated with hLF1-11 first and then with rifampicin. EtBr accumulation increased upon exposure to hLF1-11 or the combination of hLF1-11 and rifampicin, but not upon exposure to rifampicin alone. Moreover, hLF1-11 induced a dose-dependent membrane depolarization. As expected, the antibacterial activity of hLF1-11 alone or combined with rifampicin was significantly reduced in the presence of CCCP. Furthermore, hLF1-11 and rifampicin were synergistic also against a colistin-resistant NDM (New Delhi metallo-ß-lactamase)-producing K. pneumoniae strain. The results suggest that rifampicin was accumulated by KPC cells during the 30-to-60-min incubation and that the addition of hLF1-11 sensitized bacterial cells to rifampicin by inducing a transient loss of membrane potential and increased cell membrane permeability, thus facilitating the entrance and retention of rifampicin into the cytoplasm. IMPORTANCE The present study describes a synergistic effect between rifampicin, an impermeable hydrophobic antibiotic with an intracellular target, and an hLF1-11, an antimicrobial peptide derived from human lactoferrin, against multidrug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae has recently caused an outbreak in Tuscany, Italy, thus pressing the need for the development of new treatment options. The mechanisms underlying such a synergistic effect have been studied. The results suggest that the synergistic effect was due to the transient loss of membrane potential induced by hLF1-11 and the subsequent increase in cell membrane permeability which allowed rifampicin to enter the bacterial cell. Therefore, it is likely that a sub-inhibitory concentration of hLF1-11 can efficiently permeabilize K. pneumoniae cells to rifampicin, allowing the antibiotic to reach its intracellular target. These results encourage further exploration of possible applications of this synergistic combination in the treatment of K. pneumoniae infections.

Animal and In Vitro Models as Powerful Tools to Decipher the Effects of Enteric Pathogens on the Human Gut Microbiota.

Examining the interplay between intestinal pathogens and the gut microbiota is crucial to fully comprehend the pathogenic role of enteropathogens and their broader impact on human health. Valid alternatives to human studies have been introduced in laboratory practice to evaluate the effects of infectious agents on the gut microbiota, thereby exploring their translational implications in intestinal functionality and overall health. Different animal species are currently used as valuable models for intestinal infections. In addition, considering the recent advances in bioengineering, futuristic in vitro models resembling the intestinal environment are also available for this purpose. In this review, the impact of the main human enteropathogens (i.e., Clostridioides difficile, Campylobacter jejuni, diarrheagenic Escherichia coli, non-typhoidal Salmonella enterica, Shigella flexneri and Shigella sonnei, Vibrio cholerae, and Bacillus cereus) on intestinal microbial communities is summarized, with specific emphasis on results derived from investigations employing animal and in vitro models.

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 ß-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 ß-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.

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.

Ciclopirox Hydroxypropyl Chitosan (CPX-HPCH) Nail Lacquer and Breathable Cosmetic Nail Polish: In Vitro Evaluation of Drug Transungual Permeation Following the Combined Application.

BACKGROUND: Onychomycosis produces nail chromatic alterations that lead patients to mask them with cosmetic enamels. Objectives: Evaluate drug transungual permeation and antimycotic activity against selected strains after application of CPX-HPCH nail lacquer (NL) on the nail pre-covered with breathable cosmetic polish. METHODS: CPX transungual permeation after applying CPX-HPCH NL once or twice a day on bovine hoof membranes pre-covered with a breathable cosmetic nail polish was compared to that obtained applying CPX-HPCH NL directly on the membrane. The relevant experimental permeates underwent an in vitro susceptibility test. RESULTS: After CPX-HPCH NL application once a day, the drug transungual flux in the presence of cosmetic product tended to decrease while maintaining the antifungal activity. Two daily applications of CPX-HPCH NL on the membrane pre-covered with cosmetic polish exhibited the same permeation profile as daily application of the medicated lacquer directly on the nail as well as the same microbiological activity. CONCLUSIONS: The breathable cosmetic nail polish can be applied on the nail affected by onychomycosis in association with CPX-HPCH NL to mask the imperfections. The application of CPX-HPCH NL twice a day appears to be a good solution to obtain the same results as for a daily application without the presence of the cosmetic layer.

Current Progress and Future Perspectives on the Use of Bacillus clausii

Bacillus clausii is a probiotic that benefits human health. Its key characteristics include the ability to form spores; the resulting tolerance to heat, acid, and salt ensures safe passage through the human gastrointestinal tract with no loss of cells. Although B. clausii has been widely used for many decades, the beneficial properties of other probiotics, such as Lactobacillus spp. and Bifidobacterium spp., are better disseminated in the literature. In this review, we summarize the physiological, antimicrobial, and immunomodulatory properties of probiotic B. clausii strains. We also describe findings from studies that have investigated B. clausii probiotics from the perspective of quality and safety. We highlight innovative properties based on biochemical investigations of non-probiotic strains of B. clausii, revealing that B. clausii may have further health benefits in other therapeutic areas.

Characterization of a Bacillus cereus strain associated with a large feed-related outbreak of severe infection in pigs.

AIMS: Bacillus cereus is often responsible for foodborne diseases and both local and systemic infections in humans. Cases of infection in other mammals are rather rare. In this study, we report a B. cereus feed-related outbreak that caused the death of 6234 pigs in Italy. METHODS AND RESULTS: Massive doses of a Gram-positive, spore-forming bacterium were recovered from the animal feed, faeces of survived pigs and intestinal content of dead ones. The B. cereus MM1 strain was identified by MALDI-TOF MS and typified by RAPD-PCR. The isolate was tested for the production of PC-PLC, proteases, hemolysins and biofilm, for motility, as well as for the presence of genes encoding tissue-degrading enzymes and toxins. Antimicrobial resistance and pathogenicity in Galleria mellonella larvae were also investigated. Our results show that the isolated B. cereus strain is swimming-proficient, produces PC-PLC, proteases, hemolysins, biofilm and carries many virulence genes. The strain shows high pathogenicity in G. mellonella larvae. CONCLUSIONS: The isolated B. cereus strain demonstrates an aggressive profile of pathogenicity and virulence, being able to produce a wide range of determinants potentially hazardous to pigs' health. SIGNIFICANCE AND IMPACT OF STUDY: This study highlights the proficiency of B. cereus to behave as a devastating pathogen in swine if ingested at high doses and underlines that more stringent quality controls are needed for livestock feeds and supplements.

Comparison of different microbiological procedures for the diagnosis of Pneumocystis jirovecii pneumonia on bronchoalveolar-lavage fluid.

BACKGROUND: The current diagnostic gold standard for Pneumocystis jirovecii is represented by microscopic visualization of the fungus from clinical respiratory samples, as bronchoalveolar-lavage fluid, defining "proven" P. jirovecii pneumonia, whereas qPCR allows defining "probable" diagnosis, as it is unable to discriminate infection from colonization. However, molecular methods, such as end-point PCR and qPCR, are faster, easier to perform and interpret, thus allowing the laboratory to give back the clinician useful microbiological data in a shorter time. The present study aims at comparing microscopy with molecular assays and beta-D-glucan diagnostic performance on bronchoalveolar-lavage fluids from patients with suspected Pneumocystis jirovecii pneumonia. Bronchoalveolar-lavage fluid from eighteen high-risk and four negative control subjects underwent Grocott-Gomori's methenamine silver-staining, end-point PCR, RT-PCR, and beta-D-glucan assay. RESULTS: All the microscopically positive bronchoalveolar-lavage samples (50%) also resulted positive by end-point and real time PCR and all, but two, resulted positive also by beta-D-glucan quantification. End-point PCR and RT-PCR detected 10 (55%) and 11 (61%) out of the 18 samples, respectively, thus showing an enhanced sensitivity in comparison to microscopy. All RT-PCR with a Ct < 27 were confirmed microscopically, whereas samples with a Ct ≥ 27 were not. CONCLUSIONS: Our work highlights the need of reshaping and redefining the role of molecular diagnostics in a peculiar clinical setting, like P. jirovecii infection, which is a rare but also severe and rapidly progressive clinical condition affecting immunocompromised hosts that would largely benefit from a faster diagnosis. Strictly selected patients, according to the inclusion criteria, resulting negative by molecular methods could be ruled out for P. jirovecii pneumonia.

Study of the Adhesion of the Human Gut Microbiota on Electrospun Structures.

Although the adhesion of bacteria on surfaces is a widely studied process, to date, most of the works focus on a single species of microorganisms and are aimed at evaluating the antimicrobial properties of biomaterials. Here, we describe how a complex microbial community, i.e., the human gut microbiota, adheres to a surface to form stable biofilms. Two electrospun structures made of natural, i.e., gelatin, and synthetic, i.e., polycaprolactone, polymers were used to study their ability to both promote the adhesion of the human gut microbiota and support microbial growth in vitro. Due to the different wettabilities of the two surfaces, a mucin coating was also added to the structures to decouple the effect of bulk and surface properties on microbial adhesion. The developed biofilm was quantified and monitored using live/dead imaging and scanning electron microscopy. The results indicated that the electrospun gelatin structure without the mucin coating was the optimal choice for developing a 3D in vitro model of the human gut microbiota.