Understanding the probiotic potential of a healthy human vaginal flora, Lactobacillus gasseri K9: genomic and in vitro aspects

Sodium hypochlorite is more effective than 70% ethanol against biofilms of clinical isolates of Staphylococcus aureus

Most of the probiotic bacterial candidates currently available in the market consist of various strains belonging to different classes. Nevertheless, the gastrointestinal microbiome is a collection of undefined microbial agents that may contribute some medical benefits to human beings. Hence, currently, researchers from different corners are centered on investigating and identifying gastrointestinal-derived probiotic strains for the advancement of next-generation probiotics. The term next-generation probiotics refers to genetically modified microorganisms created by deleting, adding or modifying specific genes to produce a probiotic strain that modulates the metabolism, gastrointestinal health and delivered directly to the mucosa. Next-generation probiotics generated from different probiotic strains are intended to provide one or more health benefits to a host and for the efficient control and/or treatment of multiple diseases. Even though some next-generation probiotics are auspicious of the control and treatment of several chronic sicknesses, studies on human beings are still sporadic and confirmations from regulatory authorities are thus rare. Furthermore, some problems need to be resolved by releasing their broad application to the public. Probiotic strains such as Faecalibacterium prausnitzii, Prevotella copri, Bacteroides uniformis, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Clostridium butyricum, Christensenella minuta, Akkermansia muciniphila and Parabacteroides goldsteinii, have been postulated as next-generation probiotic candidates, as a result of their therapeutic or preventive effects on diseases such as colitis, obesity, diabetes and liver diseases. This review highlights the probiotic potential of next-generation probiotics and discusses the potential existing and emerging next-generation probiotics.

Simple SummaryCoagulase-negative staphylococci are considered less virulent than other variants. However, they have been increasingly recognized as an important cause of bovine mastitis. Moreover, the biofilm-forming ability appears to be important in CoNS pathogenicity, which leads more resistance to antimicrobials. This study investigated the pathogenic potential by assessing the biofilm-forming ability of CoNS isolated from bulk tank milk and analyzed the biogilm-associated resistance to antimicrobial agents. The results indicate that various CoNS isolated from bulk tank milk, not from bovine with mastitis, exhibited a high prevalence of biofilm-forming ability with a high prevalence of MDR, and also biofilm-associated genes with a high prevalence. Therefore, developing a strong monitoring and sanitation program for dairy factories is important to ensure hygienic milk production.Coagulase-negative staphylococci (CoNS) are considered less virulent as they do not produce a large number of toxic enzymes and toxins; however, they have been increasingly recognized as an important cause of bovine mastitis. In particular, the ability to form biofilms appears to be an important factor in CoNS pathogenicity, and it contributes more resistance to antimicrobials. The aim of this study was to investigate the pathogenic potential by assessing the biofilm-forming ability of CoNS isolated from normal bulk tank milk using the biofilm formation assay and to analyze the biofilm-associated resistance to antimicrobial agents using the disc diffusion method. One hundred and twenty-seven (78.4%) among 162 CoNS showed the ability of biofilm formation, and all species showed a significantly high ability of biofilm formation (p < 0.05). Although the prevalence of weak biofilm formers (39.1% to 80.0%) was significantly higher than that of other biofilm formers in all species (p < 0.05), the prevalence of strong biofilm formers was significantly higher in Staphylococcus haemolyticus (36.4%), Staphylococcus chromogenes (24.6%), and Staphylococcus saprophyticus (21.7%) (p < 0.05). Also, 4 (11.4%) among 35 non-biofilm formers did not harbor any biofilm-associated genes, whereas all 54 strong or moderate biofilm formers harbored 1 or more of these genes. The prevalence of MDR was significantly higher in biofilm formers (73.2%) than in non-formers (20.0%) (p < 0.05). Moreover, the distribution of MDR in strong or moderate biofilm formers was 81.5%, which was significantly higher than in weak (67.1%) and non-formers (20.0%) (p < 0.05). Our results indicated that various CoNS isolated from bulk tank milk, not from bovine with mastitis, have already showed a high ability to form biofilms, while also displaying a high prevalence of MDR.

Simple SummaryEnteric infections, such as Salmonella spp., are common in the poultry sector. Even though the European Union has banned the use of growth-promoting antibiotics; many countries continue to use these synthetic medications, leading to the emergence of antibiotic resistance (especially cephalosporin and fluoroquinolones) in non-typhoidal Salmonella, limiting treatment options. Probiotics are beneficial bacteria that reside in the intestine and help improve the host’s health; they are also one of the most popular antibiotic alternatives. As a result, we set out to collect lactic acid bacteria from the poultry gut that had never been fed a medicated diet and conduct in vitro probiotic studies. L. reuteri PFS4, E. faecium PFS13, and E. faecium PFS14 were screened as potential probiotic candidates. The obtained strains show good aggregation, mucin adherence, antibiofilm, and anti-salmonella activities. More research is now being conducted to determine the strain’s efficacy in commercial poultry.Non-typhoidal Salmonella (NTS) can cause infection in poultry, livestock, and humans. Although the use of antimicrobials as feed additives is prohibited, the previous indiscriminate use and poor regulatory oversight in some parts of the world have resulted in increased bacterial resistance to antimicrobials, including cephalosporins and fluoroquinolones, which are among the limited treatment options available against NTS. This study aimed to isolate potential probiotic lactic acid bacteria (LAB) strains from the poultry gut to inhibit fluoroquinolone and cephalosporin resistant MDR Salmonella Typhimurium and S. Enteritidis. The safety profile of the LAB isolates was evaluated for the hemolytic activity, DNase activity, and antibiotic resistance. Based on the safety results, three possible probiotic LAB candidates for in vitro Salmonella control were chosen. Candidate LAB isolates were identified by 16S rDNA sequencing as Lactobacillus reuteri PFS4, Enterococcus faecium PFS13, and Enterococcus faecium PFS14. These strains demonstrated a good tolerance to gastrointestinal-related stresses, including gastric acid, bile, lysozyme, and phenol. In addition, the isolates that were able to auto aggregate had the ability to co-aggregate with MDR S. Typhimurium and S. Enteritidis. Furthermore, LAB strains competitively reduced the adhesion of pathogens to porcine mucin Type III in co-culture studies. The probiotic combination of the selected LAB isolates inhibited the biofilm formation of S. Typhimurium FML15 and S. Enteritidis FML18 by 90% and 92%, respectively. In addition, the cell-free supernatant (CFS) of the LAB culture significantly reduced the growth of Salmonella in vitro. Thus, L. reuteri PFS4, E. faecium PFS13, and E. faecium PFS 14 are potential probiotics that could be used to control MDR S. Typhimurium and S. Enteritidis in poultry. Future investigations are required to elucidate the in vivo potential of these probiotic candidates as Salmonella control agents in poultry and animal feed.

Enterococcus faecalis is a bacterial pathogen associated with both endodontic and systemic infections. The biofilm formation ability of E. faecalis plays a key role in its virulence and drug resistance attributes. The formation of E. faecalis biofilms on implanted medical devices often results in treatment failure. In the present study, we report protein markers associated with the biofilm formation ability of E. faecalis using iTRAQ-based quantitative proteomics approach. In order to elucidate the biofilm-associated protein markers, we investigated the proteome of strong and weak biofilm-forming E. faecalis clinical isolates in comparison with standard American Type Culture Collection (ATCC) control strains. Comparison of E. faecalis strong and weak biofilm-forming clinical isolates with ATCC control strains showed that proteins associated with shikimate kinase pathway and sulfate transport were up-regulated in the strong biofilm former, while proteins associated with secondary metabolites, cofactor biosynthesis, and tetrahydrofolate biosynthesis were down-regulated. In the weak biofilm former, proteins associated with nucleoside and nucleotide biosynthesis were up-regulated, whereas proteins associated with sulfate and sugar transport were down-regulated. Further pathway and gene ontology analyses revealed that the major differences in biofilm formation arise from differences in metabolic activity levels of the strong and weak biofilm formers, with higher levels of metabolic activity observed in the weak biofilm former. The differences in metabolic activity could therefore be a major determinant of the biofilm ability of E. faecalis The new markers identified from this study can be further characterized in order to understand their exact role in E. faecalis biofilm formation ability. This, in turn, can lead to numerous therapeutic benefits in the treatment of this oral and systemic pathogen. The data has been deposited to the ProteomeXchange with identifier PXD006542.

This study was conducted to determine the biofilm formation of coagulase negative Staphylococcus species (CoNS) isolated from patients with catheter related blood stream infection (CRBSI) and colonized central venous catheters (CVC) and their antibiotic susceptibility patterns and in situ biofilm formation of CVC tips. Eighty-two CoNS isolated from intensive care unit (ICU) patients with CRBSI (n​=​8) or colonized CVC (n​=​74) were included. Species identification and antibiotic susceptibility test were done. All isolates were screened for biofilm formation using crystal violet and 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assays and categorized as strong or moderate biofilm formers. CVC tips were subjected to crystal violet stain and scanning electron microscopy (SEM) to detect in-situ biofilm formation. Staphylococcus haemolyticus (n​=​34; 41%) was the commonest to cause both CRBSI and CVC colonization. All 82 CoNS produced biofilms. Among them 77 (93.90%) were strong biofilm formers including all from CRBSI patients and 05 (6.10%) were moderate biofilm formers as detected by both methods. SEM showed bacteria adhered to surfaces of CVC tips with microbial-aggregates embedded in extracellular matrix. Mean crystal violet absorbance of CVC from CRBSI patients (0.6628) was significantly higher than colonized CVC (mean value 0.5592) (p​=​0.030). S.haemolyticus showed higher resistance to cloxacillin compared to other CoNS (p​=​0.039). Majority of CoNS isolated were strong biofilm formers. In-situ biofilm formation on CVC tips were significantly evident in CRBSI patients compared to CVC colonized patients. S.haemolyticus is the commonest to cause both CRBSI and CVC colonization and shows significantly higher cloxacillin resistance rate.

Advances in gut microbiota research have triggered interest in developing colon butyrate producers as niche-specific next-generation probiotics, targeted at increasing colon butyrate production and countering disease-associated microbiota alterations. Crucial steps in the development of next-generation probiotics are the design of formulations with a reasonable shelf life as well as the safety demonstration of an intervention in healthy volunteers. One such potential next-generation butyrate-producing probiotic is Butyricicoccus pullicaecorum 25-3T, with demonstrated safety in in vitro as well as animal models. Here, we examined the strain's safety, tolerability, and impact on microbiota composition and metabolic activity in healthy volunteers in a randomized, double-blind, placebo-controlled crossover study in 30 healthy volunteers. The study design consisted of two 4-week intervention periods (108 CFU B. pullicaecorum [treatment] or maltodextrin [placebo] per day) with a 3-week washout in between. We assessed adverse events, blood parameters (primary endpoints), and fecal microbiota composition and metabolite profiles (secondary endpoints). The number of reported adverse events during the B. pullicaecorum treatment was similar to that of placebo intervention, as were observed changes in blood chemistry parameters, bowel habits, and fecal calprotectin concentrations. Administration of the strain did not induce any disruptive effect in microbiota composition or metabolic activity. In this first human intervention trial with a butyrate-producing Clostridium cluster IV isolate, we demonstrated B. pullicaecorum 25-3T administration to be both safe and well tolerated by healthy participants. This safety study paves the way for the further development of the strain as a next-generation probiotic. IMPORTANCE This study is the first to determine the safety and tolerance in humans of a butyrate-producing Clostridium cluster IV next-generation probiotic. Advances in gut microbiota research have triggered interest in developing colon butyrate producers as next-generation probiotics. Butyricicoccus pullicaecorum 25-3T is one such potential probiotic, with demonstrated safety in vitro as well as in animal models. Here, we produced an encapsulated B. pullicaecorum formulation that largely preserved its viability over an 8-month storage period at 4°C. Administration of this formulation to healthy volunteers allowed us to establish the intervention as safe and well tolerated. The probiotic intervention did not cause disruptive alterations in the composition or metabolic activity of health-associated microbiota. The results presented pave the way for the exploration of the impact of the strain on microbiota alterations in a clinical setting.

To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species. Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2.82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2.84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers. These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk. This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.

Exploring the multifaceted probiotic potential of Lactiplantibacillus plantarum NMGL2, investigating its antimicrobial resistance profiles and bacteriocin production.

Background: This study investigates the biofilm formation capabilities of Lactobacillus species isolated from fermented cassava and corn products. Understanding biofilm formation is crucial for evaluating the probiotic potential of these species, as biofilm-forming ability influences their survival and functionality in host environments. Methods: Nine bacterial isolates, including Lactobacillus fermentum, L. ghanensis, L. delbrueckii, L. plantarum, Lactococcus lactis, L. reuteri, Lysinibacillus sphaericus, Bacillus cereus, and B. pacificus, were assessed for biofilm production using the microtiter plate assay. After crystal violet staining, optical density (OD) values were measured at 570 nm spectrophotometrically. Based on OD values, isolates were classified into four categories: no biofilm, weak, moderate, and strong biofilm formation. Statistical analyses, including two-stage least squares regression, were employed to evaluate biofilm formation trends and predictors. Results: The predictive regression model was highly significant (R² = 0.987, F = 122.618, p &lt; 0.0001). Biofilm formation strength varied, with the highest mean percentage observed in the moderate group (31.29%), followed by weak (27.41%), strong (20.46%), and no biofilm (20.05%). Among the isolates, Lactobacillus fermentum exhibited the highest rate of strong biofilm formation (46.1%), while Lysinibacillus sphaericus showed none. Moreover, The highest biofilm formation was observed at 37°C (31.29%), followed by 25°C (27.41%), and 45°C (20.46%). Similarly, biofilm formation was highest at pH 6.5 (30.41%), followed by pH 7.5 (25.39%) and pH 4.5 (20.05%). Lactobacillus fermentum exhibited the highest strong biofilm formation (46.1%) at 37°C and pH 6.5. Conclusion: Biofilm formation in Lactobacillus species is species-specific and environmentally influenced by temperature and pH. Lactobacillus fermentum demonstrated strong biofilm formation, making it a promising candidate for probiotic applications.

BackgroundHeyndrickxia coagulans, recognized for its probiotic attributes and resilience as an endospore-forming bacterium, is increasingly studied for health supplement applications. This study aimed to evaluate the probiotic potential and safety of novel H. coagulans isolated from stingless bee honey, a new source for this bacterium, and to characterize their multifunctional probiotic properties.ResultsWe isolated two novel H. coagulans, TBRC-18260 and TBRC-18261, and conducted comprehensive in vitro analyses to assess their probiotic traits such as acid and bile salt tolerance, self-aggregation, and pathogen inhibition. Both isolates were also evaluated for safety through antibiotic susceptibility testing and hemolytic activity. Functional properties, including GABA production, antioxidant activity, were examined to establish their potential as probiotics. TBRC-18260 and TBRC-18261 exhibited core probiotic characteristics and showed excellent survivability under acidic conditions and in the presence of bile salts. They displayed strong antimicrobial activity against various pathogens and demonstrated significant GABA production and antioxidant capabilities. The safety assessments confirmed their non-hemolytic nature and susceptibility to a wide range of antibiotics.ConclusionThe novel H. coagulans isolates, TBRC-18260 and TBRC-18261, with their robust probiotic properties, antioxidant activities, and safety profiles, emerged as promising candidates for the development of functional foods and dietary supplements. This study enhances the biodiversity of available probiotics and supports the continuous search for novel strains with unique health-promoting characteristics.

Dental caries is the most prevalent chronic infectious disease globally, with Streptococcus mutans recognized as a primary causative agent due to its acidogenicity and robust biofilm-forming ability. In S. mutans biofilm formation, the role of autoinducers has been extensively studied, while the influence of other small molecules remains largely unexplored. Mutanofactins, a class of polyketide/non-ribosomal lipopeptide secondary metabolites, are emerging as potential modulators of S. mutans biofilm development. Transcriptomic analysis was conducted to examine gene expression patterns in S. mutans NMT4863 across distinct growth phases and lifestyles, aiming to identify metabolic factors influencing biofilm formation. Transcriptomic profiles were compared between cells in early-, mid-, and late-exponential-, and stationary phase, as well as between planktonic and biofilm cells. Differentially expressed genes were identified, and pathway analyses revealed significant alterations in key metabolic and regulatory pathways. Specifically, the biosynthetic mutanofactin gene cluster was analyzed via quantitative real-time polymerase chain reaction. Several genes and operons were differentially expressed across the tested growth phases, with 1,095 genes showing differential expression between stationary-phase, planktonic and biofilm cells. Pathway analysis revealed significant changes in ascorbate metabolism, carbohydrate utilization and transport systems, lipoic acid metabolism, bacterial toxin pathways, two-component regulatory systems, and secondary metabolite biosynthesis. Notably, expression of the muf gene cluster, was elevated in early exponential-phase cells relative to stationary-phase cells. Additionally, the mufCDEFGHIJ genes were identified as components of a single transcriptional unit (muf operon). MufC, a transcriptional regulator of the TetR/AcrR-family, acts as a positive regulator of the muf operon in strain NMT4863. Bioinformatic analysis pinpointed a 20-bp regulatory sequence in the muf operon promoter region (5'-AAATGAGCTATAATTCATTT-3'). Interestingly, the muf operon was found to be significantly downregulated in biofilm cells. This study provides key insights into gene expression dynamics that drive biofilm formation in S. mutans NMT4863, with a particular emphasis on the role of the muf operon. This operon is governed by the TetR/AcrR-family regulator MufC and plays a central role in biofilm development, offering a novel perspective on the molecular basis of S. mutans biofilm formation and resilience.

Probiotics play crucial roles in promoting gut health, enhancing immunity, and combating pathogenic microorganisms, with increasing interest in their applications in the food and therapeutic industries. Lactococcus lactis, a well-known lactic acid bacterium, has emerged as a promising candidate owing to its probiotic traits and safety profile. In this study, we investigated the probiotic potential and genomic profile of Lactococcus lactis SK2-659, a strain isolated from Thai fermented green mustard (Pak-kad-dong). Genomic analysis revealed numerous genes associated with probiotic traits, including stress tolerance, adhesion, and antimicrobial activity, with a particular focus on bacteriocin SK2-659, which was effective against pathogenic bacteria such as Helicobacter pylori. The bacteriocin produced by L. lactis SK2-659, identified as nisin Z, disrupts bacterial membranes via pore formation, leading to cell lysis. Metabolomic profiling further highlighted its ability to increase carbohydrate and amino acid metabolism, supporting cell growth and survival in acidic environments. Also, amino acid metabolism (elevated tryptophan, tyrosine, histidine) supports acid tolerance and immune modulation. Tryptophan metabolism produces indole derivatives, which are known to benefit gut health and immunity. Additionally, strain SK2-659 demonstrated strong tolerance to gastrointestinal conditions, high adhesion capacity to intestinal cells, and immunomodulatory effects, contributing to gut health. Safety assessments confirmed the absence of virulence factors, pathogenic traits, and antibiotic resistance genes, along with a lack of biogenic amine production, underscoring its suitability for food and health applications. These findings establish L. lactis SK2-659 as a promising probiotic candidate with potential industrial applications in functional foods, food preservation, and therapeutic products. Given its probiotic properties, antimicrobial activity, and safety profile, this strain is particularly suited for the food industry as a functional food ingredient and natural bio-preservative, as well as the healthcare and pharmaceutical industries for use in therapeutic probiotics and gut health formulations.

In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.

Background: Biofilm formation on indwelling devices is one of the most important mechanisms playing a role in device-related urinary tract infections caused by Proteus mirabilis. Increasing antibiotic resistance of microorganisms has raised questions concerning the relationship between biofilm formation and drug resistance. Objective: To determine clinical prevalence, antibiotic resistance of, and biofilm formation by P. mirabilis strains. Methods: We studied the susceptibilities of various P. mirabilis strains isolated from different clinical materials by a disc-diffusion method. Biofilm formation was determined by a crystal violet binding assay. Results: Two (13%) of 15 P. mirabilis strains were found to be strong biofilm formers (SBF). Both SBF P. mirabilis strains were isolated from urine samples from children less than 15 years old in a pediatric emergency unit. Cefixime, cefazolin, ceftriaxone, amikacin, and piperacillin/tazobactam were the most effective antibiotics against 15 P. mirabilis strains (100%), whereas SBF P. mirabilis strains were multidrug resistant or (resistant to 5 different antimicrobial classes). Both of the SBF P. mirabilis strains, but neither of the weak biofilm forming P. mirabilis strains were resistant to ampicillin and ceftazidime among ?-lactam antibiotics, or tobramycin and gentamicin among aminoglycoside antibiotics used in the present study. Conclusions: Children comprise the only patients infected with SBF P. mirabilis strains and both SBF P. mirabilis strains displayed high antimicrobial resistance in our setting. Keywords: Biofilm formation, device-related infections, multidrug resistance, Proteus mirabilis, urinary tract infection