Type 2-resistant starch and Lactiplantibacillus plantarum NCIMB 8826 result in additive and interactive effects in diet-induced obese mice.

Little is known about how combining a probiotic with prebiotic dietary fiber affects the ability of either biotic to improve health. We hypothesized that prebiotic, high-amylose maize type 2-resistant starch (RS) together with probiotic Lactiplantibacillus plantarum NCIMB8826 (LP) as a complementary synbiotic results in additive effects on the gut microbiota in diet-induced obese mice and other body sites. Diet-induced obese C57BL/6J male mice were fed a high-fat diet adjusted to contain RS (20% by weight), LP (109 cells every 48 hours), or both (RS+LP) for 6 weeks. As found for mice fed RS, cecal bacterial alpha diversity was significantly reduced in mice given RS+LP compared with those fed LP and high-fat controls. Similarly, both RS+LP and RS also conferred lower quantities of cecal butyrate and serum histidine and higher ileal TLR2 transcript levels and adipose tissue interleukin-6 protein. As found for mice fed LP, RS+LP-fed mice had higher colonic tissue TH17 cytokines, reduced epididymal fat immune and oxidative stress responses, reduced serum carnitine levels, and increased transcript quantities of hepatic carnitine palmitoyl transferase 1α. Notably, compared with RS and LP consumed separately, there were also synergistic increases in colonic glucose and hepatic amino acids as well antagonistic effects of LP on RS-mediated increases in serum adiponectin and urinary toxin levels. Our findings show that it is not possible to fully predict outcomes of synbiotic applications based on findings of the probiotic or the prebiotic tested separately; therefore, studies should be conducted to test new synbiotic formulations.

Complete Genome Sequence of the Plantaricin-Sensitive Strain Lactobacillus plantarum NCIMB 700965.

Lactobacillus plantarum NCIMB 700965 was isolated from cheese in 1939 and is used as an indicator strain for plantaricin production. The complete genome was determined using both long (PacBio) and short (Illumina) read data resulting in a single, circular chromosome with 3,015,426 bp, a G+C content of 45%, and five plasmids.

Sensitivity to the two peptide bacteriocin plantaricin EF is dependent on CorC, a membrane-bound, magnesium/cobalt efflux protein.

Lactic acid bacteria produce a variety of antimicrobial peptides known as bacteriocins. Most bacteriocins are understood to kill sensitive bacteria through receptor-mediated disruptions. Here, we report on the identification of the Lactobacillus plantarum plantaricin EF (PlnEF) receptor. Spontaneous PlnEF-resistant mutants of the PlnEF-indicator strain L. plantarum NCIMB 700965 (LP965) were isolated and confirmed to maintain cellular ATP levels in the presence of PlnEF. Genome comparisons resulted in the identification of a single mutated gene annotated as the membrane-bound, magnesium/cobalt efflux protein CorC. All isolates contained a valine (V) at position 334 instead of a glycine (G) in a cysteine-ß-synthase domain at the C-terminal region of CorC. In silico template-based modeling of this domain indicated that the mutation resides in a loop between two ß-strands. The relationship between PlnEF, CorC, and metal homeostasis was supported by the finding that PlnEF-resistance was lost when PlnEF was applied together with high concentrations of Mg2+ , Co2+ , Zn2+ , or Cu2+ . Lastly, PlnEF sensitivity was increased upon heterologous expression of LP965 corC but not the G334V CorC mutant in the PlnEF-resistant strain Lactobacillus casei BL23. These results show that PlnEF kills sensitive bacteria by targeting CorC.

Synergy between Probiotic Lactobacillus casei and Milk to Maintain Barrier Integrity of Intestinal Epithelial Cells.

We hypothesized that Lactobacillus casei BL23 and milk work synergistically to prevent damage to epithelial barrier integrity induced by pro-inflammatory cytokines. To test this, barrier disruption was induced in polarized Caco-2 monolayers by sequential, basolateral treatment with IFN-γ and TNF-α. Apical application of either 25% v/v reconstituted skim milk (RSM) or ultra high temperature (UHT) milk (2% fat) prior to cytokine exposure reduced losses to transepithelial electrical resistance (TER). Permeability to fluorescein isothiocyanate-dextran (FD-4; 4 kDa) was also significantly reduced in the presence of 25% v/v UHT milk ( P < 0.05) but not RSM. Protection against increases in paracellular permeability was even greater when cell-free preparations of L. casei BL23 fermented UHT milk or fermented RSM were applied. The permeability coefficients of cells incubated with BL23 fermented UHT milk were equivalent to the untreated controls ( P = 0.12) and those cells also produced 247.6 ± 35.5 pg/mL IL-8, quantities significantly lower than found for cytokine-treated controls (353.9 ± 40.0 pg/mL). The benefits of the fermented milk were also confirmed by the reduced expression of TNF receptor 2 (TNFR2), myosin light-chain kinase (MLCK), and claudin-encoding genes relative to the controls. By comparison, apical application of viable L. casei onto the Caco-2 cells did not result in protection from the barrier-disruptive actions of IFN-γ and TNF-α. These results indicate that milk can maintain intestinal barrier integrity during pro-inflammatory cytokine exposure and that this is enhanced by modifications to milk matrix caused by prior incubation with L. casei BL23.

Lactobacillus plantarum bacteriocin is associated with intestinal and systemic improvements in diet-induced obese mice and maintains epithelial barrier integrity in vitro.

We investigated the Lactobacillus plantarum bacteriocin plantaricin EF (PlnEF) system for its contributions to L. plantarum mediated benefits in a mouse model of diet-induced obesity. C57BL/6J mice on a high-fat diet (HFD) were administered a rifampicin resistant mutant of L. plantarum NCMIB8826 (NICMB8826-R) or an isogenic ΔplnEFI mutant strain, LM0419, every 48 h for nine weeks. Mice fed wild-type L. plantarum, but not LM0419, reduced their consumption of the HFD starting three weeks into the study and exhibited an overall 10% reduction in weight gain. The responses were independent of glucose homeostasis, as both NCMIB8826-R and LM0419 fed mice had improved oral glucose tolerance compared to sham controls. Although bacteriocins have antibacterial properties, the ileal, cecal, and fecal microbiota and cecocolic metabolomes were unchanged between mice fed either wild-type L. plantarum or the ΔplnEFI mutant. Instead, only mice fed NCMIB8826-R showed an increased production of ZO-1 in ileal tissues. To verify a potential role for the plantaricin EF system in supporting intestinal epithelial function, synthesized PlnEF peptides were applied to Caco-2 cell monolayers challenged with TNF-α and IFN-γ. The combination of PlnE and PlnF were required to prevent sustained cytokine-induced losses to Caco-2 cell para- and transcellular permeability and elevated IL-8 levels. In conclusion, this study shows that probiotic L. plantarum ameliorates the effects of obesogenic diets through a mechanism that involves the plantaricin EF system and likely includes L. plantarum - induced fortification of the intestinal epithelium.

Detection and Quantification of Viable but Non-culturable Campylobacter jejuni.

Campylobacter can enter a viable but non-culturable (VBNC) state to evade various stresses, and this state is undetectable using traditional microbiological culturing techniques. These VBNC bacterial cells retain metabolism and demonstrate pathogenic potential due to their ability to resuscitate under favorable conditions. Rapid and accurate determination of VBNC Campylobacter is critical to further understand the induction and resuscitation of the dormancy state of this microbe in the agri-food system. Here, we integrated propidium monoazide (PMA) with real-time polymerase chain reaction (qPCR) targeting the rpoB gene to detect and quantify Campylobacter jejuni in the VBNC state. First, we optimized the concentration of PMA (20 µM) that could significantly inhibit the amplification of dead cells by qPCR with no significant interference on the amplification of viable cell DNA. PMA-qPCR was highly specific to C. jejuni with a limit of detection (LOD) of 2.43 log CFU/ml in pure bacterial culture. A standard curve for C. jejuni cell concentrations was established with the correlation coefficient of 0.9999 at the linear range of 3.43 to 8.43 log CFU/ml. Induction of C. jejuni into the VBNC state by osmotic stress (i.e., 7% NaCl) was rapid (<48 h) and effective (>10% population). The LOD of PMA-qPCR for VBNC C. jejuni exogenously applied to chicken breasts was 3.12 log CFU/g. In conclusion, PMA-qPCR is a rapid, specific, and sensitive method for the detection and quantification of VBNC C. jejuni in poultry products. This technique can give insight into the prevalence of VBNC Campylobacter in the environment and agri-food production system.

Supplementation of Lactobacillus plantarum Improves Markers of Metabolic Dysfunction Induced by a High Fat Diet.

Obesity is a prevalent chronic condition in many developed and developing nations that raises the risk for developing heart disease, stroke, and diabetes. Previous studies have shown that consuming particular probiotic strains of Lactobacillus is associated with improvement in the obese and diabetic phenotype; however, the mechanisms of these beneficial effects are not well understood. In this study, C57BL/6J male mice were fed a lard-based high fat diet for 15 weeks with Lactobacillus plantarum supplementation NCIMB8826 (Lp) between weeks 10 and 15 ( n = 10 per group). Systemic metabolic effects of supplementation were analyzed by NMR metabolomics, protein expression assays, gene transcript quantification, and 16S rRNA marker gene sequencing. Body and organ weights were not significantly different with Lp supplementation, and no microbiota community structure changes were observed in the cecum; however, L. plantarum numbers were increased in the treatment group according to culture-based and 16S rRNA gene quantification. Significant differences in metabolite and protein concentrations (serum, liver, and colon), gene expression (ileum and adipose), and cytokines (colon) were observed between groups with increases in the gene expression of tight junction proteins in the ileum and cecum and improvement of some markers of glucose homeostasis in blood and tissue with Lp supplementation. These results indicate Lp supplementation impacts systemic metabolism and immune signaling before phenotypic changes and without large-scale changes to the microbiome. This study supports the notion that Lp is a beneficial probiotic, even in the context of a high fat diet.

Sucrose metabolism alters Lactobacillus plantarum survival and interactions with the microbiota in the digestive tract.

We investigated whether sucrose metabolism by probiotic Lactobacillus plantarum influences the intestinal survival and microbial responses to this organism when administered to mice fed a sucrose-rich, Western diet. A L. plantarum mutant unable to metabolize sucrose was constructed by deleting scrB, coding for beta-fructofuranosidase, in a rifampicin-resistant strain of L. plantarum NCIMB8826. The ScrB deficient mutant survived in 8-fold higher numbers compared to the wild-type strain when measured 24 h after administration on two consecutive days. According to 16S rRNA marker gene sequencing, proportions of Faecalibacterium and Streptococcus were elevated in mice fed the L. plantarum ΔscrB mutant. Metagenome predictions also indicated those mice contained a higher abundance of lactate dehydrogenases. This was further supported by a trend in elevated fecal lactate concentrations among mice fed the ΔscrB mutant. L. plantarum also caused other changes to the fecal metabolomes including higher concentrations of glycerol in mice fed the ΔscrB mutant and increased uracil, acetate and propionate levels among mice fed the wild-type strain. Taken together, these results suggest that sucrose metabolism alters the properties of L. plantarum in the digestive tract and that probiotics can differentially influence intestinal metabolomes via their carbohydrate consumption capabilities.

Intestinal Lactobacillus in health and disease, a driver or just along for the ride?

Metagenomics and related methods have led to significant advances in our understanding of the human microbiome. Members of the genus Lactobacillus, although best understood for essential roles in food fermentations and applications as probiotics, have also come to the fore in a number of untargeted gut microbiome studies in humans and animals. Even though Lactobacillus is only a minor member of the human colonic microbiota, the proportions of those bacteria are frequently either positively or negatively correlated with human disease and chronic conditions. Recent findings on Lactobacillus species in human and animal microbiome research, together with the increased knowledge on probiotic and other ingested lactobacilli, have resulted in new perspectives on the importance of this genus to human health.

Fidaxomicin reduces early toxin A and B production and sporulation in Clostridium difficilein vitro.

PURPOSE: Fidaxomicin, a macrocyclic antibiotic, has been approved for the treatment of Clostridium difficile infection (CDI). Previous work by our group has demonstrated that some antibiotics at sub-inhibitory concentrations stimulate early toxin production and sporulation by C. difficile. Prior studies revealed that fidaxomicin, when added to late stationary-phase organisms, reduced exotoxin production and spore formation by C. difficile. However, the ability of fidaxomicin to trigger early virulence factor production and spore formation has never been investigated. METHODOLOGY: Sub-inhibitory concentrations of the RNA synthesis inhibitor fidaxomicin (1/4×, 1/8×, 1/16× MIC) were added immediately to lag-phase cultures of historical (strain 9689) and epidemic BI/NAP1/027 (strain 5325) strains of C. difficile, and their effects on sporulation and toxin A (TcdA) and toxin B (TcdB) production were compared.Results/Key findings. Even at sub-inhibitory concentrations, all doses of fidaxomicin reduced both TcdA and TcdB gene expression and protein production in the historical and epidemic C. difficile strains. Fidaxomicin also dose-dependently reduced viable spore production by the 9689 and 5325 strains. Reductions in spore formation were also observed in both strains treated with tigecycline and vancomycin. However, all concentrations of metronidazole stimulated a ~2 log increase in spore production by the 5325 isolate. CONCLUSION: The ability of fidaxomicin to suppress early exotoxin production and endospore formation by historical and epidemic strains of C. difficile may explain its clinical success in treating severe and recurrent cases of CDI disease.

Microbiota, metabolome, and immune alterations in obese mice fed a high-fat diet containing type 2 resistant starch.

SCOPE: We examined the intestinal and systemic responses to incorporating a type 2 resistant starch (RS) into a high fat diet fed to obese mice. METHODS AND RESULTS: Diet-induced obese, C57BL/6J male mice were fed an HF diet without or with 20% (by weight) high-amylose maize resistant starch (HF-RS) for 6 weeks. Serum adiponectin levels were higher with RS consumption, but there were no differences in weight gain and adiposity. With HF-RS, the expression levels of ileal TLR2 and Reg3g and cecal occludin, TLR2, TLR4, NOD1 and NOD2 were induced; whereas colonic concentrations of the inflammatory cytokine IL-17A declined. The intestinal, serum, liver, and urinary metabolomes were also altered. HF-RS resulted in lower amino acid concentrations, including lower serum branched chain amino acids, and increased quantities of urinary di/trimethylamine, 3-indoxylsulfate, and phenylacetylglycine. Corresponding to these changes were enrichments in Bacteroidetes (S24-7 family) and certain Firmicutes taxa (Lactobacillales and Erysipelotrichaceae) with the HF-RS diet. Parabacteroides and S24-7 positively associated with cecal maltose concentrations. These taxa and Erysipelotrichaceae, Allobaculum, and Bifidobacterium were directly correlated with uremic metabolites. CONCLUSION: Consumption of RS modified the intestinal microbiota, stimulated intestinal immunity and endocrine-responses, and modified systemic metabolomes in obese mice consuming an otherwise obesogenic diet.

Experimental identification and computational characterization of a novel extracellular metalloproteinase produced by Clostridium sordellii.

Clostridium sordellii is a lethal pathogen for both animals and humans. Severe capillary leakage, toxic shock syndrome, and an extreme leukemoid reaction (LR), are hallmark features of C. sordellii infections and contribute to its high mortality rate. Here we report the discovery of a previously unknown and uncharacterized metalloproteinase of C. sordellii (referred as Mcs1) that cleaves human vascular cell adhesion molecule (VCAM)-1 in vitro, an adhesion molecule critical to hematopoietic precursor retention and leukocyte diapedesis. We successfully identified the open reading frame encoding Mcs1 within the ATCC 9714 genome and developed an Δmcs1 mutant strain using the ClosTron mutagenesis technology. No VCAM-1 proteolysis was observed from exotoxins collected from mutant strain cultures. Using advanced protein structural modeling and molecular dynamics simulation techniques, the 3D molecular structure and conformational features of Mcs1 were also characterized. Our data demonstrates that Mcs1 proteolytic activity is controlled by the electrostatic interactions between Glu113 and Arg227 residues and the gating motions within its cleft region. This pilot interdisciplinary investigation provided crucial experimental evidence of the existence of Mcs1 in C. sordellii and molecular insights into its 3D structure and proteolytic activity. These findings have the potential to help advance new therapeutics and diagnostics against deadly C. sordellii infections. Follow-up in vitro and in vivo work is under way to further characterize Mcs1 enzymatic kinetics and its role in C. sordellii pathogenesis.

Resolving relationships at the animal-fungal divergence: A molecular phylogenetic study of the protist trichomycetes (Ichthyosporea, Eccrinida).

Trichomycetes is a group of microorganisms that was considered a class of fungi comprising four orders of commensal, gut-dwelling endosymbionts obligately associated with arthropods. Since molecular phylogenies revealed two of those orders (Amoebidiales and Eccrinales="protist trichos") to be closely related to members of the protist class Ichthyosporea (=Mesomycetozoea), trichomycetes have been considered an ecological association of both early-diverging fungi and protists. Understanding of the taxonomy, evolution, and diversity of the protist trichos is lacking largely due to the difficulties inherent in species collection that have contributed to undersampling and understudy. The most recent classification divides the protist trichos between two families, Amoebidiidae and Eccrinidae (suborder Trichomycina, order Eccrinida). However, there is no comprehensive molecular phylogeny available for this group and major questions about the systematics of protist trichos remain unanswered. Therefore, we generated 18S and 28S rDNA sequences for 106 protist tricho samples and combined them with publicly available Eccrinida sequences for phylogenetic analyses. We also sequenced a conserved protein-coding gene (heat-shock 70 protein) to obtain a multigene data set. We conducted ancestral state reconstruction (ASR) and Bayesian tip-association significance test (BaTS) analyses by mapping six morphological and ecological characters onto the resulting phylogenetic trees. Our results demonstrate: (1) several ecological and morphological character states (habitat, host type, host stage at time of infestation, location within host, spore production, and growth form) are significantly correlated with the phylogeny, and (2) two additional protist tricho families should be incorporated into the taxonomy to reflect phylogenetic relationships. Our data suggest that an integrated strategy that combines morphological, ecological, and molecular characters is needed to further resolve and clarify the systematics of the Eccrinida.

Health benefits of fermented foods: microbiota and beyond.

Fermented foods and beverages were among the first processed food products consumed by humans. The production of foods such as yogurt and cultured milk, wine and beer, sauerkraut and kimchi, and fermented sausage were initially valued because of their improved shelf life, safety, and organoleptic properties. It is increasingly understood that fermented foods can also have enhanced nutritional and functional properties due to transformation of substrates and formation of bioactive or bioavailable end-products. Many fermented foods also contain living microorganisms of which some are genetically similar to strains used as probiotics. Although only a limited number of clinical studies on fermented foods have been performed, there is evidence that these foods provide health benefits well-beyond the starting food materials.

Tigecycline suppresses toxin A and B production and sporulation in Clostridium difficile.

BACKGROUND: Clostridium difficile infection (CDI) is mediated by potent extracellular toxins and is spread largely via bacterial spores. We and others have shown that some antibiotics stimulate C. difficile toxin production in a strain-specific manner; however, the effects of newer anti-C. difficile antibiotics on this process remain to be investigated. METHODS: The effects of the protein synthesis inhibitor tigecycline on sporulation and toxin A and toxin B production were compared in historical (strain 9689) and hypervirulent BI/NAP1/027 (strain 5325) isolates of C. difficile in vitro. RESULTS: Tigecycline at 1/4× MIC stimulated an increased and earlier toxin A and/or B gene expression in both the historical and the hypervirulent strains, although a commensurate increase in toxin protein production was observed only in the 9689 strain. In fact, in the hypervirulent 5325 strain, toxin production was dramatically suppressed. By comparison, subinhibitory concentrations of vancomycin and metronidazole also stimulated increased protein toxin production by the historical, but not the hypervirulent, strain. In addition, tigecycline dose-dependently reduced viable spore production by both the 9689 and 5325 strains. Vancomycin treatment also suppressed spore formation in both C. difficile strains; however, metronidazole, while reducing spore formation in the 9689 strain, stimulated a near 2 log increase in spore production by the 5325 isolate. CONCLUSIONS: In summary, these findings suggest that the treatment of CDI patients with tigecycline could effectively both control disease progression and limit its spread by disrupting sporulation.