Impact of a phage cocktail targeting Escherichia coli and Enterococcus faecalis as members of a gut bacterial consortium in vitro and in vivo.

Escherichia coli and Enterococcus faecalis have been implicated as important players in human gut health that have been associated with the onset of inflammatory bowel disease (IBD). Bacteriophage (phage) therapy has been used for decades to target pathogens as an alternative to antibiotics, but the ability of phage to shape complex bacterial consortia in the lower gastrointestinal tract is not clearly understood. We administered a cocktail of six phages (either viable or heat-inactivated) targeting pro-inflammatory Escherichia coli LF82 and Enterococcus faecalis OG1RF as members of a defined community in both a continuous fermenter and a murine colitis model. The two target strains were members of a six species simplified human microbiome consortium (SIHUMI-6). In a 72-h continuous fermentation, the phage cocktail caused a 1.1 and 1.5 log (log10 genome copies/mL) reduction in E. faecalis and E. coli numbers, respectively. This interaction was accompanied by changes in the numbers of other SIHUMI-6 members, with an increase of Lactiplantibacillus plantarum (1.7 log) and Faecalibacterium prausnitzii (1.8 log). However, in germ-free mice colonized by the same bacterial consortium, the same phage cocktail administered twice a week over nine weeks did not cause a significant reduction of the target strains. Mice treated with active or inactive phage had similar levels of pro-inflammatory cytokines (IFN-y/IL12p40) in unstimulated colorectal colonic strip cultures. However, histology scores of the murine lower GIT (cecum and distal colon) were lower in the viable phage-treated mice, suggesting that the phage cocktail did influence the functionality of the SIHUMI-6 consortium. For this study, we conclude that the observed potential of phages to reduce host populations in in vitro models did not translate to a similar outcome in an in vivo setting, with this effect likely brought about by the reduction of phage numbers during transit of the mouse GIT.

Polymicrobial Interactions in the Cystic Fibrosis Airway Microbiome Impact the Antimicrobial Susceptibility of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most dominant pathogens in cystic fibrosis (CF) airway disease and contributes to significant inflammation, airway damage, and poorer disease outcomes. The CF airway is now known to be host to a complex community of microorganisms, and polymicrobial interactions have been shown to play an important role in shaping P. aeruginosa pathogenicity and resistance. P. aeruginosa can cause chronic infections that once established are almost impossible to eradicate with antibiotics. CF patients that develop chronic P. aeruginosa infection have poorer lung function, higher morbidity, and a reduced life expectancy. P. aeruginosa adapts to the CF airway and quickly develops resistance to several antibiotics. A perplexing phenomenon is the disparity between in vitro antimicrobial sensitivity testing and clinical response. Considering the CF airway is host to a diverse community of microorganisms or 'microbiome' and that these microorganisms are known to interact, the antimicrobial resistance and progression of P. aeruginosa infection is likely influenced by these microbial relationships. This review combines the literature to date on interactions between P. aeruginosa and other airway microorganisms and the influence of these interactions on P. aeruginosa tolerance to antimicrobials.

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.

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.

Evaluation of Phage Therapy in the Context of Enterococcus faecalis and Its Associated Diseases.

Bacteriophages (phages) or bacterial viruses have been proposed as natural antimicrobial agents to fight against antibiotic-resistant bacteria associated with human infections. Enterococcus faecalis is a gut commensal, which is occasionally found in the mouth and vaginal tract, and does not usually cause clinical problems. However, it can spread to other areas of the body and cause life-threatening infections, such as septicemia, endocarditis, or meningitis, in immunocompromised hosts. Although E. faecalis phage cocktails are not commercially available within the EU or USA, there is an accumulated evidence from in vitro and in vivo studies that have shown phage efficacy, which supports the idea of applying phage therapy to overcome infections associated with E. faecalis. In this review, we discuss the potency of bacteriophages in controlling E. faecalis, in both in vitro and in vivo scenarios. E. faecalis associated bacteriophages were compared at the genome level and an attempt was made to categorize phages with respect to their suitability for therapeutic application, using orthocluster analysis. In addition, E. faecalis phages have been examined for the presence of antibiotic-resistant genes, to ensure their safe use in clinical conditions. Finally, the domain architecture of E. faecalis phage-encoded endolysins are discussed.