Clinical trials and animal studies on the gut microbiota are often limited by the difficult access to the gut, restricted possibility of in vivo monitoring, and ethical issues. An easily accessible ...and monitorable in vitro model of the gut microbiota represents a valid tool for a wider comprehension of the mechanisms by which microbes interact with the host and with each other. Herein, we present a novel and reliable system for culturing the human gut microbiota in vitro. An electrospun gelatin structure was biofabricated as scaffold for microbial growth. The efficiency of this structure in supporting microbial proliferation and biofilm formation was initially assessed for five microbes commonly inhabiting the human gut. The human fecal microbiota was then cultured on the scaffolds and microbial biofilms monitored by confocal laser and scanning electron microscopy and quantified over time. Metagenomic analyses and Real-Time qPCRs were performed to evaluate the stability of the cultured microbiota in terms of qualitative and quantitative composition. Our results reveal the three-dimensionality of the scaffold-adhered microbial consortia that maintain the bacterial biodiversity and richness found in the original sample. These findings demonstrate the validity of the developed electrospun gelatin-based system for in vitro culturing the human gut microbiota.
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.
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.
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.
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.
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.,
,
, diarrheagenic
, non-typhoidal
,
and
,
, and
) on intestinal microbial communities is summarized, with specific emphasis on results derived from investigations employing animal and in vitro models.
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.
In this research, nine probiotic strains (
NR, OC, SIN, and T,
ATCC 7050,
DSM 16604,
DSM 17938,
ATCC 53103, and
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).
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
and
additionally able to produce propionic and butyric acids. Quantitative differences in the secretion of SCFAs were also evidenced.
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.
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.
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
study, 21 leading probiotic formulations commercialized worldwide were evaluated for their microbial content and survivability in simulated GI conditions.
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
model composed of different simulated gastric and intestinal fluids was adopted.
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.
This
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.
Microorganisms with probiotic properties are eliciting an increasing interest as coadjuvants in the prevention and treatment of obesity through modulation of the gut microbiota. In this study, a ...probiotic formulation based on
SF68 was administered to mice fed with a high-fat diet (HFD) to evaluate its efficacy in reducing body mass gain and in modulating the intestinal bacterial composition. Both stool and ileum samples were collected from untreated and treated mice and absolute abundances of specific
constituting the gut microbial
were evaluated. SF68 administration significantly reduced the HFD-induced weight gain. In these animals, the microbial gut composition shifted toward an enrichment in microbes positively correlated with mucus thickness, lower inflammation, lower glycemia levels, and SCFA production (i.e.,
,
, and
), as well as a depletion in bacterial phyla having a key role in obesity (i.e.,
,
). Our results demonstrate the efficacy of
SF68 in adjusting the composition of the dysbiotic microbiota of HFD-fed animals, thus ameliorating clinical conditions and exerting anti-obesity effects.
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