Environmental and Nutritional Parameters Modulating Genetic Expression for Virulence Factors of Clostridioides difficile.

Clostridioides difficile infections (CDIs) continue to be a persistent healthcare concern despite newer antibiotic treatments, enhanced infection control practices, and preventive strategies focused on restoring the protective intestinal microbial barrier. Recent strides in gene sequencing research have identified many genes regulating diverse virulence factors for CDIs. These genes may be over- or under-expressed when triggered by various environmental and nutritional factors. The aims of this paper are to review the important genes involved in C. difficile pathogenesis and to identify modifiable environmental, nutritional, and other factors that may trigger the expression of these genes and thus offer new strategies to prevent CDIs.

Effect of a Probiotic Beverage Enriched with Cricket Proteins on the Gut Microbiota: Composition of Gut and Correlation with Nutritional Parameters.

The health and balance of the gut microbiota are known to be linked to diet composition and source, with fermented products and dietary proteins potentially providing an exceptional advantage for the gut. The purpose of this study was to evaluate the effect of protein hydrolysis, using a probiotic beverage enriched with either cricket protein (CP) or cricket protein hydrolysates (CP.Hs), on the composition of the gut microbiota of rats. Taxonomic characterization of the gut microbiota in fecal samples was carried out after a 14-day nutritional study to identify modifications induced by a CP- and CP.H-enriched fermented probiotic product. The results showed no significant differences (p > 0.05) in the diversity and richness of the gut microbiota among the groups fed with casein (positive control), CP-enriched, and fermented CP.H-enriched probiotic beverages; however, the overall composition of the microbiota was altered, with significant modifications in the relative abundance of several bacterial families and genera. In addition, fermented CP.H-enriched probiotic beverages could be related to the decrease in the number of potential pathogens such as Enterococcaceae. The association of gut microbiota with the nutritional parameters was determined and the results showed that digestibility and the protein efficiency ratio (PER) were highly associated with the abundance of several taxa.

Recommendations to Improve Quality of Probiotic Systematic Reviews With Meta-Analyses.

Importance: Systematic reviews and meta-analyses often report conflicting results when assessing evidence for probiotic efficacy, partially because of the lack of understanding of the unique features of probiotic trials. As a consequence, clinical decisions on the use of probiotics have been confusing. Objective: To provide recommendations to improve the quality and consistency of systematic reviews with meta-analyses on probiotics, so evidence-based clinical decisions can be made with more clarity. Evidence Review: For this consensus statement, an updated literature review was conducted (January 1, 2020, to June 30, 2022) to supplement a previously published 2018 literature search to identify areas where probiotic systematic reviews with meta-analyses might be improved. An expert panel of 21 scientists and physicians with experience on writing and reviewing probiotic reviews and meta-analyses was convened and used a modified Delphi method to develop recommendations for future probiotic reviews. Findings: A total of 206 systematic reviews with meta-analysis components on probiotics were screened and representative examples discussed to determine areas for improvement. The expert panel initially identified 36 items that were inconsistently reported or were considered important to consider in probiotic meta-analyses. Of these, a consensus was reached for 9 recommendations to improve the quality of future probiotic meta-analyses. Conclusions and Relevance: In this study, the expert panel reached a consensus on 9 recommendations that should promote improved reporting of probiotic systematic reviews with meta-analyses and, thereby, assist in clinical decisions regarding the use of probiotics.

Transcriptome analysis of the Clostridioides difficile response to a specific lactobacilli probiotic formulation: explanations for its mechanisms of action.

AIMS: Clostridioides difficile infections (CDI) are a major cause of morbidity and mortality in hospitalized patients. A probiotic formulation (Bio-K+) comprised of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacti. rhamnosus CLR2 strains have been shown to reduce the incidence of CDI and antibiotic-associated diarrhea (AAD). This research aims to therefore elucidate the mechanism of action of the three probiotic strained against C. difficile R20291, independently of the acidification of the environment. . METHODS AND RESULTS: Antitoxin activity was evaluated using ELISA method and the expression of C. difficile genes was evaluated using transcriptomic analysis in co-culture assays conducted in a bioreactor allowing precise control of the pH. The fermentation results demonstrated a decrease for toxin A and many genes directly related to C. difficile virulence were underexpressed in the co-cultures. CONCLUSIONS: The lactobacilli tested could have a role in the motility, the quorum sensing (QS), the survival of the spores, and the germination potential of the spores, which are essential elements for the virulence of C. difficile. .

Screening and Characterization of Some Lactobacillaceae for Detection of Cholesterol-Lowering Activities.

Dyslipidemia, specifically abnormal levels of low-density lipoprotein cholesterol (LDL-C), is an important risk factor of cardiovascular disease. Evidence showing the promising abilities of probiotics to lower total cholesterol or LDL-C has, however, not yet convinced experts to recommend probiotic bacteria as treatment for blood lipid management. Therefore, there are opportunities for the development of new efficient cholesterol-lowering probiotics. Bile salt hydrolase (BSH) and feruloyl esterase (FAE) are bacterial enzymes proposed to explain the cholesterol-lowering capacity of some bacteria and have both been shown to be responsible for lipid reduction in vivo. Here, in order to select for cholesterol-lowering bacteria, 70 strains related to Lactobacillaceae were screened for BSH and FAE activities. Based on this two-way screening approach, two bacteria were selected and assessed for their capacity to assimilate cholesterol in vitro, another suggested mechanism. Lactobacillus acidophilus CL1285 showed BSH and FAE activity as well as capacity to assimilate cholesterol in vitro. Lactiplantibacillus plantarum CHOL-200 exhibited BSH activity and ability to assimilate cholesterol. These properties observed in vitro make both strains good probiotic candidates for the management of dyslipidemia. Further investigation is needed to assess their ability to reduce blood cholesterol in human trial.

Physico-chemical Properties and Sensorial Appreciation of a New Fermented Probiotic Beverage Enriched with Pea and Rice Proteins.

OBJECTIVES: The purpose of this study was to evaluate the physico-chemical stability, the sensorial properties, and the microbial quality of a fermented beverage enriched with pea and rice proteins (PRF) during storage at 4 °C. To investigate the effect of the protein enrichment and fermentation, the PRF beverage quality was compared with non-fermented and non-enriched beverages. METHODS: The beverage was supplemented with a 50/50 mixture of pea and rice protein concentrate to 13% concentration. Following inoculation with 108 CFU/mL of lactic acid bacteria, it was incubated at 37 °C for 14 h. RESULTS: Results showed that the enrichment with protein induced an increase in pH, titratable acidity and viscosity of the PR products, while the fermentation led to a decrease of pH and viscosity. However, a significant increase of the viscosity of PRF from 39 to 57 cP was observed during the 143 days of storage (P ≤ 0.05). The PRF beverage contained significantly more peptides < 200 Da than the non-fermented one (PRNF) and these small peptides were also released during the storage. Despite the physico-chemical modifications, the sensorial properties of the PRF product were appreciated over the storage, particularly for the texture. Furthermore, the beverage maintained a high concentration of viable probiotics during the entire storage with 8.4 log colony form unit (CFU)/mL after 143 days. CONCLUSION: Applying probiotics and the mixture of rice and pea proteins in the fermented beverage can enhance nutritional and nutraceutical value of the product.

Effect of Physical and Enzymatic Pre-Treatment on the Nutritional and Functional Properties of Fermented Beverages Enriched with Cricket Proteins.

The aim of this study was to evaluate the effects of γ-irradiation (IR), ultrasound (US), and combined treatments of ultrasound followed by γ-irradiation (US-IR), ultrasound followed by enzymatic hydrolysis with and without centrifugation (US-E and US-EWC, respectively), and ultrasound followed by γ-irradiation and enzymatic hydrolysis (US-IRE), on the digestibility and the nutritional value of fermented beverages containing probiotics. Results showed that US (20 min), IR (3 kGy) and US-IR (tUS = 20 min, dose = 3 kGy) treatments raised protein solubility from 11.5 to 21.5, 24.3 and 29.9%, respectively. According to our results, these treatments were accompanied by the increased amount of total sulfhydryl groups, surface hydrophobicity and changes to the secondary structure of the proteins measured by Fourier-transform infrared spectroscopy (FTIR). Fermented probiotic beverages, non-enriched (C) and enriched with untreated (Cr) or treated cricket protein with combined treatments were also evaluated for their in vitro protein digestibility. Results showed that the soluble fraction of US-IRE fermented beverage had the highest digestibility (94%) as compared to the whole fermented tested beverages. The peptides profile demonstrated that US-IRE had a low proportion of high molecular weight (MW) peptides (0.7%) and the highest proportion of low MW peptides by over 80% as compared to the other treatments.

In vitro protein digestibility and physico-chemical properties of lactic acid bacteria fermented beverages enriched with plant proteins.

The objective of this study was to develop probiotic beverages, enriched with plant proteins, with high nutritional value. A rice-based beverage fermented with a specific probiotic formulation comprised Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R and Lactobacillus rhamnosus CLR2 has been enriched with a combination of pea and rice proteins (PR) or pea and hemp proteins (PH) at 13 and 11% total protein, respectively. These protein associations have been selected because their amino acid ratio was >1, as recommended by the FAO. The beverage enriched with protein significantly increased its viscosity by more than 10 times thanks to the enrichment, while the fermentation reduced it by 50% for PR and 20% for PH. In vitro protein digestibility results showed that the protein enrichment and the fermentation treatment significantly increased digestibility values of the beverages with value of 72.7% for fermented PR beverage and 61.4% for unenriched fermented control beverage (p ≤ 0.05). Peptide profiles of PR and PH enriched beverages indicated that the fermentation led to a reduced level of high molecular weight (HMW) peptides of about 60% and an increase of low molecular weight (LMW) peptides by over 50%. Therefore, both the fermentation and the enrichment in protein increased the nutritional value of the rice-based beverages. PRACTICAL APPLICATION: Good quality of probiotics formulation and high-protein products are in increasing demand and plant proteins as an alternative of animal protein are popular. This study has permit to develop rice-based commercial probiotic beverages enriched in a combination of pea and rice or pea and hemp proteins in order to obtain a complete protein in terms of amino acids composition. The lactic acid fermentation and the enrichment with a plant protein combination led to a better protein digestibility of beverage.

Protein quality of a probiotic beverage enriched with pea and rice protein.

The aim of this study was to evaluate the effect of the fermentation of a probiotic beverage enriched with pea and rice proteins (PRF) on its protein quality. The protein quality was determined as the protein efficiency ratio (PER), net protein ratio (NPR), and the apparent (AD) and the true digestibility (TD) evaluated in vivo. The probiotic beverage was incorporated to a rat diet at a final concentration of 10% protein, for the evaluation of the PER, the NPR, the AD, and the TD. The protein digestibility amino acid score was also calculated. Results showed that the fermentation of beverage enriched with PRF had no effect on the TD but significantly increased the PER and the NPR (P ≤ 0.05) from 1.88 to 2.32 and from 1.66 to 2.30, respectively. Thus, the fermentation increased the protein quality of the PRF probiotic beverage. In addition, to determine if the beverage constitute in a good carrier matrix for the probiotics, the level of alive probiotics in the feces was evaluated and showed a concentration of 7.4 log CFU/g. PRACTICAL APPLICATION: Plant proteins are often of lower quality compared to animal proteins. Lactic acid fermentation of pea and rice protein has allowed to reach the same protein quality as casein. A plant-based fermented beverage with high protein quality and enriched with probiotics was developed.

Potential role of probiotics in reducing Clostridioides difficile virulence: Interference with quorum sensing systems.

Opportunistic pathogenic bacteria may cause disease after the normally protective microbiome is disrupted (typically by antibiotic exposure). Clostridioides difficile is one such pathogen having a severe impact on healthcare facilities and increasing costs of medical care. The search for new therapeutic strategies that are not reliant on additional antibiotic exposures are currently being explored. One such strategy is to disrupt the production of C. difficile virulence factors by interfering with quorum sensing (QS) systems. QS has been well studied in other bacteria, but our understanding in C. difficile is not so well understood. Some probiotic strains or combinations of strains have been shown to be effective in the treatment or primary prevention of C. difficile infections and may possess multiple mechanisms of action. One mechanism of probiotics might be the inhibition of QS, but their role has not been clearly defined yet. A literature search was conducted using standard databases (PubMed, Google Scholar) from database inception to August 2020. The objective of this paper is to update our understanding of how QS leads to toxin production by C. difficile, which is important in pathogenesis, and how QS inhibitors or probiotics may disrupt this pathway. We found two main QS systems for C. difficile (Agr and Lux systems) that are involved in C. difficile pathogenesis by regulating toxin production, motility and adherence. Probiotics and other QS inhibitors targeting QS systems may represent important new directions of therapy and prevention of CDI.

The Acid-Dependent and Independent Effects of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 on Clostridioides difficile R20291.

Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea which is life threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 has demonstrated a strong inhibitory effect on the growth of several nosocomial C. difficile strains by production of antimicrobial metabolites during fermentation. Though there are several lactobacilli shown to inhibit C. difficile growth by processes relying on acidification, this probiotic has demonstrated potency for CDI prevention among hospitalized patients. Here, we describe the acid-dependent and independent mechanisms by which these strains impair the cytotoxicity of a hypervirulent strain, C. difficile R20291 (CD). These bacteria were co-cultured in a series of experiments under anaerobic conditions in glucose-rich and no-sugar medium to inhibit or stimulate CD toxin production, respectively. In glucose-rich medium, there was low CD toxin production, but sufficient amounts to cause cytotoxic damage to human fibroblast cells. In co-culture, there was acidification by the lactobacilli resulting in growth inhibition as well as ≥ 99% reduced toxin A and B production and no observable cytotoxicity. In the absence of glucose, CD produced much more toxin. In co-culture, the lactobacilli did not acidify the medium and CD growth was unaffected; yet, the amount of detected toxin A and B was decreased by 20% and 41%, respectively. Despite the high concentration of toxin, cells exposed to the supernatant from the co-culture were able to survive. These results suggest that in addition to known acid-dependent effects, the combination of L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 can interfere with CD pathogenesis without acidification: (1) reduced toxin A and B production and (2) toxin neutralization. This might explain the strain specificity of this probiotic in potently preventing C. difficile-associated diarrhea in antibiotic-treated patients compared with other probiotic formulae.

The synergistic effect of cell wall extracted from probiotic biomass containing Lactobacillus acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 on the anticancer activity of cranberry juice-HPLC fractions.

Anticancer effects were evaluated on three HPLC fractions obtained from a concentrated cranberry juice and cell wall constituents extracted from a probiotic biomass containing Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, and Lactobacillus rhamnosus CLR2. The samples were tested at increasing concentrations for the antiproliferative assay using HT-29 cells' line and for the quinone reductase (QR) assay using Hepa-1c1c7 murine hepatoma cells. Fraction 1 (F1) which is highly concentrated with phenolic acids inhibited the growth of the HT-29 cells' line with IC50 values of 14.80 µg Gallic acid equivalent (GAE)/ml. The fraction 3 (F3) which is highly concentrated in flavonols had potency as QR inducer. Furthermore, the results showed that all cranberry fractions combined with cell wall constituents extracted from the probiotic biomass were more effective in inhibiting the growth of HT-29 as compared to the cranberry fractions tested alone, indicating a possible synergy effect between these bio-functional compounds. PRACTICAL APPLICATIONS: This study strongly evidenced that cranberry juice fractions combined with cell wall constituents extracted from the probiotic biomass can be used as a potent preventive functional compound against colorectal cancer. Therefore, this research proposes a natural dietary compound to prevent mutagenesis and carcinogenesis of colorectal cancer. Furthermore, the industry can formulae products containing probiotic and phenolic compounds as colon cancer cell growth preventive and anticancer products.

Importance of Molecular Methods to Determine Whether a Probiotic is the Source of Lactobacillus Bacteremia.

There has been an increasing interest in the use of probiotic products for the prevention of Clostridium difficile infection (CDI). Bio-K+(®) is a commercial probiotic product comprising three strains of lactobacilli--Lactobacillus acidophilus CL1285(®), Lact. casei LBC80R(®) and Lact. rhamnosus CLR2(®)--that have been applied to prevent CDI. Generally considered as safe, lactobacilli have potential to cause bacteremia, endocarditis and other infections. The source of Lactobacillus bacteremia can be normal human flora or lactobacilli-containing probiotic. The aim of this study was to assess whether probiotic lactobacilli caused bacteremia and to show the value of molecular identification and typing techniques to determine probiotic and patient strain relatedness. We report an episode of Lactobacillus bacteremia in a 69-year-old man admitted to a hospital with severe congestive heart failure. During his hospitalization, he required long-term antibiotic therapy. Additionally, the patient received Bio-K+(®) probiotic as part of a quality improvement project to prevent CDI. Subsequently, Lactobacillus bacteremia occurred. Two independent blinded laboratory evaluations, using pulse field gel electrophoresis, 16S rRNA gene sequencing and DNA fingerprint analysis (rep-PCR), were performed to determine whether the recovered Lact. acidophilus originated from the probiotic product. Ultimately, the patient strain was identified as Lact. casei and both laboratories found no genetic relation between the patient's strain and any of the probiotic lactobacilli. This clinical case of lactobacillus bacteremia in the setting of probiotic exposure demonstrates the value of using discriminatory molecular methods to clearly determine whether there were a link between the patient's isolate and the probiotic strains.

Influence of Environmental Factors on Bacteriocin Production by Human Isolates of Lactococcus lactis MM19 and Pediococcus acidilactici MM33.

The influence of temperature, initial pH, and carbon and nitrogen sources on bacteriocin secreted by Lactococcus lactis MM19 (MM19) and Pediococcus acidilactici MM33 (MM33) was evaluated. It was found that 30 and 45 °C were the growth temperatures for higher nisin and pediocin production by MM19 and MM33, respectively. The initial pH values for higher production of nisin and pediocin were 9 and 6, respectively. Glucose and wheat peptone E430 were found as suitable carbon and nitrogen sources, respectively, for highest nisin production by MM19 at 30 °C and initial pH of 9. In these conditions, nisin production could be increased by 6.7 times as compared to the control medium (de Man, Rogosa, and Sharpe--MRS broth). Similarly, fructose and pea peptone were suitable carbon and nitrogen sources, respectively, for highest production of pediocin by MM33 at 45 °C and initial pH of 6. In these conditions, pediocin production by MM33 was increased by three times as compared to the control medium (tryptone-glucose-yeast extract-TGE broth).

Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, and Lactobacillus rhamnosus CLR2 (Bio-K+): Characterization, Manufacture, Mechanisms of Action, and Quality Control of a Specific Probiotic Combination for Primary Prevention of Clostridium difficile Infection.

A specific probiotic formulation composed of Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, and Lactobacillus rhamnosus CLR2 (Bio-K+) has been marketed in North America since 1996. The strains and the commercial products have been evaluated for safety, identity, gastrointestinal survival, and stability throughout shelf life. The capacity of both the fermented beverages and the capsules to reduce incidences of antibiotic-associated diarrhea and Clostridium difficile infection (CDI) has been demonstrated in human clinical trials. Individual strains and the finished products have shown antimicrobial activity against C. difficile and toxin A/B neutralization capacity in vitro. The use of this specific probiotic formulation as part of a bundle of preventive measures to control CDI in healthcare settings is discussed.

Pathway to Prevention of Nosocomial Clostridium difficile Infection.

BACKGROUND: To address the significant morbidity and mortality rates associated with nosocomial Clostridium difficile-associated diarrhea (CDAD), a series of recommendations and a pathway to prevention were developed. METHODS: An expert panel of infectious disease (ID) specialists participated in a modified Delphi process with specific objectives: (1) conduct a review for CDAD and prevention; (2) develop statements based upon panel members' opinions; (3) hold a panel meeting during the 2012 IDWeek; and (4) review the final recommendations and prevention pathway prior to submission for publication. RESULTS: The panel voted on (1) antibiotic stewardship (7 of 8 panelists); (2) reduction of other potentially modifiable risk factors (variable); (3) utilization of specific probiotics to prevent C. difficile overgrowth (8/8); (4) staff education regarding CDAD preventive measures (8/8); (5) appropriate hand hygiene for everyone (7/8); (6) environmental cleaning (8/8); (7) medical equipment disinfection (7/8); (8) early detection of CDAD in symptomatic patients (7/8); (9) usage of protective clothing/gloves (8/8); (10) proper measures during outbreak (8/8); and (11) surveillance to monitor efficacy data of preventive measures (8/8). CONCLUSIONS: The panel members agreed with 11 of 17 recommendations presented. The additional recommendations by the panel were proton pump inhibitor use as a risk factor and the use of adjunctive therapy with specific probiotic, as it was approved by Health Canada for the risk reduction of CDAD in hospitalized patients.

Response of Bacillus cereus to gamma-irradiation in combination with carvacrol or mild heat treatment.

Carvacrol and mild heat treatment were tested for their efficiency to increase the radiosensitivity of Bacillus cereus in broth. The bacterium was treated with gamma-irradiation alone or in combination with carvacrol at its minimal inhibitory concentration or mild heat treatment for 10 min at 45 degrees C. The effects of this combination of treatments were studied on various parameters: the bacterial viability, the modifications of the cell morphology with scanning electron microscopy (SEM), the cellular fatty acids composition of the membrane quantified by gas chromatography, the intracellular and extracellular adenosine 5'-triphosphate (ATP) concentrations, and the DNA degradation. Combined treatments resulted in additive or synergistic effects as compared to gamma-irradiation alone. A significant modification (P < or = 0.05) of the fatty acid composition and unsaturation ratios was observed. Pretreatment with mild heat or carvacrol before irradiation disturbed the membrane integrity of B. cereus and induced a significant decrease (P < or = 0.05) of the intracellular ATP concentration. SEM observations revealed that the cell membrane was more severely affected with combined treatment than irradiation alone. The electrophoresis analysis showed that DNA degradation by combined treatments was greater than the gamma-irradiation alone.

Probiotic Lactobacillus acidophilus and L. casei mix sensitize colorectal tumoral cells to 5-fluorouracil-induced apoptosis.

To assess the potential of Lactobacillus acidophilus and Lactobacillus casei strains to increase the apoptosis of a colorectal cancer cell line in the presence of 5-fluorouracil (5-FU), LS513 colorectal cancer cells were treated for 48 h with increasing concentrations of these lactic acid bacteria (LAB) in the presence of 100 mu g/ml of 5-FU. In the presence of 10(8) CFU/ml of live LAB, the apoptotic efficacy of the 5-FU increased by 40%, and the phenomenon was dose dependent. Moreover, irradiation-inactivated LAB caused the same level of induction, whereas microwave-inactivated LAB reduced the apoptotic capacity of the 5-FU. When cells were treated with a combination of live LAB and 5-FU, a faster activation of caspase-3 protein was observed, and the p21 protein seems to be downregulated. These results suggest that live L. acidophilus and L. casei are able to increase the apoptosis-induction capacity of 5-FU. The mechanisms of action are still not elucidated, and more research is needed to understand them. This is the first set of experiments demonstrating that some strains of LAB can enhance the apoptosis-induction capacity of the 5-FU. Based on these results, it is possible to speculate that LAB or probiotics could be used as an adjuvant treatment during anticancer chemotherapy.