A postbiotic consisting of heat-treated lactobacilli has a bifidogenic effect in pure culture and in human fermented faecal communities.

The gut microbiota has a significant impact on host health. Dietary interventions using probiotics, prebiotics and postbiotics have the potential to alter microbiota composition and function. Other therapeutic interventions such as antibiotics and faecal microbiota transplantation have also been shown to significantly alter the microbiota and its metabolites. Supplementation of a faecal fermentation model of the human gut with a postbiotic product Lactobacillus LB led to changes in microbiome composition (i.e. increase in beneficial bifidobacteria) and associated metabolic changes (i.e. increased acid production). Lactobacillus LB is a heat-treated preparation of cellular biomass and a fermentate generated by Limosilactobacillus fermentum CNCM MA65/4E-1b (formerly known as Lactobacillus fermentum CNCM MA65/4E-1b) and Lactobacillus delbrueckii ssp. delbrueckii CNCM MA65/4E-2z, medically relevant strains used to produce antidiarrheal preparations. In pure culture, Lactobacillus LB also stimulates the growth of a range of bifidobacterial species and strains. Lactobacillus LB-like preparations generated using other Lactobacillaceae, including commercially available probiotic bacteria, did not have the same impact on a model strain (Bifidobacterium longum subsp. infantis ATCC 15697). This bifidogenic activity is heat- and enzyme-stable and cannot be attributed to lactose, which is a major constituent of Lactobacillus LB. L fermentum CNCM MA65/4E-1b is largely responsible for the observed activity and there is a clear role for compounds smaller than 1 kDa.Importance In general, disruptions to the gut microbiota are associated with multiple disorders in humans. The presence of high levels of Bifidobacterium spp. in the human gut is commonly considered to be beneficial. Bifidobacteria can be supplemented in the diet (as probiotics) or those bifidobacteria already present in the gut can be stimulated by the consumption of prebiotics such as inulin. We demonstrate that Lactobacillus LB (a product consisting of two heat-killed lactic acid bacteria and their metabolites) can stimulate the growth of bifidobacteria in human fermented faecal communities and in pure culture. Given the heat-treatment applied during the production process, there is no risk of the lactic acid bacteria colonising (or causing bacteraemia) in vulnerable consumers (infants, immunocompromised, etc). Lactobacillus LB has the potential to affect human health by selectively promoting the growth of beneficial bacteria.

Non-antibiotic microbial solutions for bovine mastitis - live biotherapeutics, bacteriophage, and phage lysins.

Bovine mastitis is a disease with a multi-etiological nature, defined as an inflammation of the udder. The main treatment for mastitis is the administration of antibiotics - usually directly to the udder. There is an urgent need for novel therapies to treat and prevent the disease, given the widespread emergence of antibiotic resistance and concomitant problems in the treatment of human and animal infections. We provide an overview of treatments for bovine mastitis, with emphasis on probiotics, bacteriocins, bacteriophages (phages), and phage endolysins. Probiotics have in recent years proved to be particularly efficacious in bovine mastitis treatment and prevention. In this case, the mode of action is most likely to be due to stimulation of the host immune response which clears the mastitis pathogen. Bacteriocins have the potential to be incorporated into teat washes and wipes, thus preventing pathogen spread on the farm. Phage therapy is limited by the inability of some phages to replicate in raw milk, as reported for some staphylococcal phages, and by their narrow host specificity. The use of phage endolysins is more promising, by enabling the development of broad host range potent antimicrobials, but additional research is required in terms of efficacy, safety and production.

Analysis of Germination Capacity and Germinant Receptor (Sub)clusters of Genome-Sequenced Bacillus cereus Environmental Isolates and Model Strains.

Spore germination of 17 Bacillus cereus food isolates and reference strains was evaluated using flow cytometry analysis in combination with fluorescent staining at a single-spore level. This approach allowed for rapid collection of germination data under more than 20 conditions, including heat activation of spores, germination in complex media (brain heart infusion [BHI] and tryptone soy broth [TSB]), and exposure to saturating concentrations of single amino acids and the combination of alanine and inosine. Whole-genome sequence comparison revealed a total of 11 clusters of operons encoding germinant receptors (GRs): GerK, GerI, and GerL were present in all strains, whereas GerR, GerS, GerG, GerQ, GerX, GerF, GerW, and GerZ (sub)clusters showed a more diverse presence/absence in different strains. The spores of tested strains displayed high diversity with regard to their sensitivity and responsiveness to selected germinants and heat activation. The two laboratory strains, B. cereus ATCC 14579 and ATCC 10987, and 11 food isolates showed a good germination response under a range of conditions, whereas four other strains (B. cereus B4085, B4086, B4116, and B4153) belonging to phylogenetic group IIIA showed a very weak germination response even in BHI and TSB media. Germination responses could not be linked to specific (combinations of) GRs, but it was noted that the four group IIIA strains contained pseudogenes or variants of subunit C in their gerL cluster. Additionally, two of those strains (B4086 and B4153) carried pseudogenes in the gerK and gerRI (sub)clusters that possibly affected the functionality of these GRs. IMPORTANCE: Germination of bacterial spores is a critical step before vegetative growth can resume. Food products may contain nutrient germinants that trigger germination and outgrowth of Bacillus species spores, possibly leading to food spoilage or foodborne illness. Prediction of spore germination behavior is, however, very challenging, especially for spores of natural isolates that tend to show more diverse germination responses than laboratory strains. The approach used has provided information on the genetic diversity in GRs and corresponding subclusters encoded by B. cereus strains, as well as their germination behavior and possible associations with GRs, and it provides a basis for further extension of knowledge on the role of GRs in B. cereus (group member) ecology and transmission to the host.

Somatic cell count as an indicator of subclinical mastitis and increased inflammatory response in asymptomatic lactating women.

Subclinical mastitis is an asymptomatic inflammatory condition that can be difficult to define and diagnose. In the dairy industry, subclinical mastitis is diagnosed by milk somatic cell counts (SCCs) of ≥250,000 cells mL-1. In this pilot study, we assessed the efficacy of this index to identify human subclinical mastitis by comparing SCC levels with the inflammatory response [interleukin-8 (IL-8) levels] in 37 samples from asymptomatic and 10 clinical mastitis (CM) lactating women. The milk microbiota was determined by 16S rRNA gene sequencing. The SCC of CM samples ranged from 310,000 to 6,600,000 cells mL-1. However, 14 of 37 (37.8%) asymptomatic samples had high SCC (250,000-460,000 cells mL-1), indicating subclinical mastitis. SCC levels significantly (P < 0.001) and positively correlated with milk IL-8 levels reflecting the escalating inflammatory response across subclinical and clinical mastitis samples. Samples with an SCC of ≥250,000 cells mL-1 showed significant increases in IL-8 responses when compared with milk samples from healthy women. The milk microbiome of CM samples was dominated by streptococcal and staphylococcal species (89.9% combined median relative abundance). In contrast, the combined median streptococcal/staphylococcal relative levels were 75.4% and 66.3% in milks from asymptomatic (subclinical mastitis) and healthy groups, respectively. The Streptococcus genus was increased in samples with an SCC of ≥250,000, although this should be interpreted with caution. Thus, the index of ≥250,000 somatic cells mL-1 could be a reliable indicator of subclinical mastitis in humans and should aid future studies investigating the impact of subclinical mastitis on maternal health, breastfeeding behaviors, infant health, and development. IMPORTANCE: This pilot study suggests that SCC at a level of (greater than or equal to) 250,000 cells mL-1, as used in the dairy industry, is a suitable index to identify asymptomatic subclinical mastitis in lactating women since it reflects a significant increase in the inflammatory response compared to milk samples from healthy women. Using this index should aid studies into the short- and long-term consequences of subclinical mastitis for mother and infant.

Cross-sectional observational study protocol: missing microbes in infants born by caesarean section (MiMIC): antenatal antibiotics and mode of delivery.

INTRODUCTION: The intestinal microbiome in early life plays a major role in infant health and development. Factors like antibiotic exposure, breast/formula feeding and mode of delivery are known to affect the microbiome. The increasing occurrence of caesarean section (C-section) deliveries and antibiotic exposure warrants further insight into the potential missing microbes in those infants. The study objective is to study the effect of maternal antibiotic administration during pregnancy and/or C-section mode of delivery on the development of the infant's intestinal microbiome until the age of 2 years. METHODS AND ANALYSIS: A single site, cross-sectional observational study of C-section and vaginally delivered infants being either exposed to maternal antibiotic treatment or not during the third trimester of pregnancy. Throughout the nine visits, stool, urine, saliva, hair, breast milk and vaginal swabs will be collected from either mother and/or infant for microbiome and metabolomic analysis. ETHICS AND DISSEMINATION: The protocol was approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals. The trial has been registered at ClinicalTrials.gov.The findings from this study will be disseminated in peer-reviewed journals, during scientific conferences, and directly to the study participants. Sequencing data will be deposited in public databases. TRIAL REGISTRATION NUMBER: NCT04134819.

Vancomycin and nisin A are effective against biofilms of multi-drug resistant Staphylococcus aureus isolates from human milk.

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments.

Oral Administration of Heat-Treated Lactobacilli Modifies the Murine Microbiome and Reduces Citrobacter Induced Colitis.

Significant evidence supports a relationship between the gut microbiome, inflammation, host response, and health, including the finding that a number of disorders are associated with disruption of the microbiome. In these disorders, a number of dietary interventions (including prebiotics, live probiotics, or heat-killed microbes) have been proposed to be curative or preventative agents. The use of heat-killed microbes has a number of benefits over living organisms, including reduced infection risk in vulnerable individuals, extended shelf life and the potential for use in combination with antimicrobial agents. We previously reported that murine chow supplemented with 5% ADR-159, a heat-treated fermentate generated by two Lactobacillus strains, altered both behavior and the microbiome of male mice. Now we show that ADR-159 fed female mice also display a similar microbiome shift as determined by 16S rDNA analysis. In particular, we observed a reduction of levels of Turicibacter and Clostridium sensu stricto. These subtle changes in the bacterial component of the microbiome were mirrored by changes in the virome. Extended consumption of the ADR-159 diet had no negative effect on general health and lipocalin 2 levels (LCN2; a proxy for inflammation), but we observed increased IL-17f and decreased IL-12α expression in the colon and decreased short chain fatty acid levels in the ADR-159 fed animals. Four weeks into the diet, half of the animals were dosed with Citrobacter to determine the effect of ADR-159 on infection and on pathogen induced colitis. Overall, our results suggest that while the ADR-159 diet does not prevent Citrobacter infection, it had an effect on Citrobacter-induced inflammation. In contrast to animals fed standard chow, ADR-159 fed animals did not show a reduction of small intestine length and increase of colon crypt depth, which occurred in control mice. These microbiological, histological, and immunological results provide evidence to support the impact of heat-treated microorganisms and their metabolites on the murine microbiome and health.

Diverse Bacteriocins Produced by Strains From the Human Milk Microbiota.

Microbial colonization of the infant gut is a convoluted process dependent on numerous contributing factors, including age, mode of delivery and diet among others that has lifelong implication for human health. Breast milk also contains a microbiome which acts as a source of colonizing bacteria for the infant. Here, we demonstrate that human milk harbors a wide diversity of bacteriocin-producing strains with the potential to compete among the developing gut microbiota of the infant. We screened 37 human milk samples and found isolates with antimicrobial activity and distinct cross-immunity profiles. From these isolates, we detected 73 putative gene clusters for bacteriocins of all known sub-classes, including 16 novel prepeptides. More specifically, we detected two novel lantibiotics, four sactibiotics and three class IIa bacteriocins with an unusual modification of the pediocin box that is composed of YDNGI instead of the highly conserved motif YGNGV. Moreover, we identified a novel class IIb bacteriocin, four novel class IIc and two class IId bacteriocins. In conclusion, human milk contains a variety of bacteriocin-producing strains which may provide them a competitive advantage in the colonization of the infant gut and suggests that the milk microbiota is a source of antimicrobial potential.

Heat-killed lactobacilli alter both microbiota composition and behaviour.

Recently it has been proposed to expand the definition of psychobiotics (beneficial bacteria (probiotics) or support for such bacteria (prebiotics) that positively impact mental health) to any exogenous influence whose effect on the brain is bacterially-mediated. This definition would include inactivated microorganisms with anxiolytic and antidepressant effects. The use of inactivated microorganisms has several advantages over living organisms, including no risk of infection in vulnerable individuals and ease of use in terms of storage and delivery options. It has been reported that consumption of inactivated microorganisms can affect behaviour, particularly in chronic or prolonged stress situations, but effects on healthy populations have not been investigated to the same extent. Also, only limited data is available on the effects of inactivated microorganisms on the microbiota of healthy individuals (animal or human). Therefore, we investigated the effect of feeding a standard mouse chow which incorporates ADR-159, a heat-killed fermentate generated by two Lactobacillus strains, on the behaviour and microbiota of healthy mice. Prolonged consumption of ADR-159 diet had no adverse effect on anthropometrics or general health, but the ADR-159 fed animals demonstrated increased sociability and lower baseline corticosterone levels (stress hormone). The diet also led to subtle but significant changes in the microbiota, with less abundant taxa being most affected. The behavioural, biochemical and microbiological results provide a new light on the impact of inactivated microorganisms and their metabolites on the social behaviour and microbiota of healthy mice.

Recovery of Heat Treated Bacillus cereus Spores Is Affected by Matrix Composition and Factors with Putative Functions in Damage Repair.

The ability of spores to recover and grow out after food processing is affected by cellular factors and by the outgrowth conditions. In the current communication we studied the recovery and outgrowth of individually sorted spores in BHI and rice broth media and on agar plates using flow cytometry. We show that recovery of wet heat treated Bacillus cereus ATCC 14579 spores is affected by matrix composition with highest recovery in BHI broth or on rice agar plates, compared to BHI agar plates and rice broth. Data show that not only media composition but also its liquid or solid state affect the recovery of heat treated spores. To determine the impact of factors with putative roles in recovery of heat treated spores, specific genes previously shown to be highly expressed in outgrowing heat-treated spores were selected for mutant construction. Spores of nine B. cereus ATCC 14579 deletion mutants were obtained and their recovery from wet heat treatment was evaluated using BHI and rice broth and agar plates. Deletion mutant spores showed different capacity to recover from heat treatment compared to wild type with the most pronounced effect for a mutant lacking BC5242, a gene encoding a membrane protein with C2C2 zinc finger which resulted in over 95% reduction in recovery compared to the wild type in BHI broth. Notably, similar relative performance of wild type and mutants was observed using the other recovery conditions. We obtained insights on the impact of matrix composition and state on recovery of individually sorted heat treated spores and identified cellular factors with putative roles in this process. These results may provide leads for future developments in design of more efficient combined preservation treatments.

Identification of CdnL, a Putative Transcriptional Regulator Involved in Repair and Outgrowth of Heat-Damaged Bacillus cereus Spores.

Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional values. Consequently subpopulations of spores may emerge that are sublethally damaged rather than inactivated. Such spores may germinate, repair damage, and eventually grow out leading to uncontrolled spoilage and safety issues. To gain insight into both the behaviour of damaged Bacillus cereus spores, and the process of damage repair, we assessed the germination and outgrowth performance using OD595 measurements and microscopy combined with genome-wide transcription analysis of untreated and heat-treated spores. The first two methods showed delayed germination and outgrowth of heat-damaged B. cereus ATCC14579 spores. A subset of genes uniquely expressed in heat-treated spores was identified with putative roles in the outgrowth of damaged spores, including cdnL (BC4714) encoding the putative transcriptional regulator CdnL. Next, a B. cereus ATCC14579 cdnL (BC4714) deletion mutant was constructed and assessment of outgrowth from heat-treated spores under food relevant conditions showed increased damage compared to wild type spores. The approach used in this study allows for identification of candidate genes involved in spore damage repair. Further identification of cellular parameters and characterisation of the molecular processes contributing to spore damage repair may provide leads for better control of spore outgrowth in foods.

Linking Bacillus cereus Genotypes and Carbohydrate Utilization Capacity.

We characterised carbohydrate utilisation of 20 newly sequenced Bacillus cereus strains isolated from food products and food processing environments and two laboratory strains, B. cereus ATCC 10987 and B. cereus ATCC 14579. Subsequently, genome sequences of these strains were analysed together with 11 additional B. cereus reference genomes to provide an overview of the different types of carbohydrate transporters and utilization systems found in B. cereus strains. The combined application of API tests, defined growth media experiments and comparative genomics enabled us to link the carbohydrate utilisation capacity of 22 B. cereus strains with their genome content and in some cases to the panC phylogenetic grouping. A core set of carbohydrates including glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose could be used by all strains, whereas utilisation of other carbohydrates like xylose, galactose, and lactose, and typical host-derived carbohydrates such as fucose, mannose, N-acetyl-galactosamine and inositol is limited to a subset of strains. Finally, the roles of selected carbohydrate transporters and utilisation systems in specific niches such as soil, foods and the human host are discussed.

Bacterial Spores in Food: Survival, Emergence, and Outgrowth.

Spore-forming bacteria are ubiquitous in nature. The resistance properties of bacterial spores lie at the heart of their widespread occurrence in food ingredients and foods. The efficacy of inactivation by food-processing conditions is largely determined by the characteristics of the different types of spores, whereas food composition and storage conditions determine the eventual germination and outgrowth of surviving spores. Here, we review the current knowledge on variation in spore resistance, in germination, and in the outgrowth capacity of spores relevant to foods. This includes novel findings on key parameters in spore survival and outgrowth obtained by gene-trait matching approaches using genome-sequenced Bacillus spp. food isolates, which represent notorious food spoilage and pathogenic species. Additionally, the impact of strain diversity on heat inactivation of spores and the variability therein is discussed. Knowledge and quantification of factors that influence variability can be applied to improve predictive models, ultimately supporting effective control of spore-forming bacteria in foods.

Influence of food matrix on outgrowth heterogeneity of heat damaged Bacillus cereus spores.

Spoilage of heat treated foods can be caused by the presence of surviving spore-formers. It is virtually impossible to prevent contamination at the primary production level as spores are ubiquitous present in the environment and can contaminate raw products. As a result spore inactivation treatments are widely used by food producing industries to reduce the microbial spore loads. However consumers prefer mildly processed products that have less impact on its quality and this trend steers industry towards milder preservation treatments. Such treatments may result in damaged instead of inactivated spores, and these spores may germinate, repair, and grow out, possibly leading to quality and safety issues. The ability to repair and grow out is influenced by the properties of the food matrix. In the current communication we studied the outgrowth from heat damaged Bacillus cereus ATCC 14579 spores on Anopore membrane, which allowed following outgrowth heterogeneity of individual spores on broccoli and rice-based media as well as standard and mildly acidified (pH 5.5) meat-based BHI. Rice, broccoli and BHI pH 5.5 media resulted in delayed outgrowth from untreated spores, and increased heterogeneity compared to BHI pH 7.4, with the most pronounced effect in rice media. Exposure to wet heat for 1 min at 95 °C caused 2 log inactivation and approximately 95% of the spores in the surviving fraction were damaged resulting in substantial delay in outgrowth based on the time required to reach a maximum microcolony size of 256 cells. The delay was most pronounced for heat-treated spores on broccoli medium followed by spores on rice media (both untreated and treated). Interestingly, the increase in outgrowth heterogeneity of heat treated spores on BHI pH 7.4 was more pronounced than on rice, broccoli and BHI pH 5.5 conceivably reflecting that conditions in BHI pH 7.4 better support spore damage repair. This study compares the effects of three main factors, namely heat treatment, pH of BHI and the effect of food matrix highlighting the impact of different (model) food recovery media on outgrowth efficiency and heterogeneity of non-heat-treated and heat-damaged B. cereus spores.