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Microcin J25 Exhibits Inhibitory Activity Against Salmonella Newport in Continuous Fermentation Model Mimicking Swine Colonic Conditions.

ABSTRACT: Microcin J25 (MccJ25), a 21-amino acid bacteriocin produced by Escherichia coli (E. coli), is a potent inhibitor of Enterobacteriaceae, including pathogenic E. coli, Salmonella, and Shigella. Its lasso structure makes it highly stable and therefore of interest as a possible antimicrobial agent in foods or as an alternative to antibiotics in livestock production. In the present study, we aimed to evaluate in vitro the inhibitory activity of MccJ25 against Salmonella enterica subsp. enterica serovar Newport ATCC 6962 (Salmonella Newport) used as a model pathogen under conditions simulating those of the swine proximal colon. The growth inhibition activity of MccJ25 against Salmonella Newport was examined in lysogeny broth (LB) and in modified MacFarlane medium that allows miming the swine colonic conditions. The MccJ25 activity was further determined using the Polyfermentor intestinal model (PolyFermS), an in vitro continuous fermentation model that permits deciphering the activity of any antimicrobial molecule in real colon fermentation conditions using selected microbiota. It was set up here to simulate the porcine proximal colon fermentation. In these conditions, the inhibition activity of MccJ25 was compared to those of two antimicrobial agents, reuterin and rifampicin. The minimal inhibitory concentration (MIC) of MccJ25 was determined at 0.03 μM in LB medium, compared to 1,079 and 38 μM for reuterin and rifampicin, respectively, showing a significantly higher potency of MccJ25. Total inhibition of Salmonella Newport was observed in LB medium over 24 h of incubation at concentrations starting from the MIC. In the PolyFermS model, MccJ25 induced a significantly stronger inhibition of Salmonella Newport growth than reuterin or rifampicin. A specific and sensitive LC-MS method allowed to detect and quantify MccJ25 in the PolyFermS fermentation system, showing that MccJ25 remains stable and active against Salmonella in conditions mimicking those found in swine colon. This study paves the way for further exploring the potential of this bacteriocin as an alternative to antibiotics in livestock.

SUBMITTER: Naimi S

PROVIDER: S-EPMC7262971 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Modern large-scale agricultural practices that incorporate high density farming with subtherapeutic antibiotic dosing are considered a major contributor to the rise of antibiotic-resistant bacterial infections of humans with species of Salmonella being a leading agriculture-based bacterial infection. Microcin J25, a potent and highly stable antimicrobial peptide active against Enterobacteriaceae, is a candidate antimicrobial against multiple Salmonella species. Emerging evidence supports the hypothesis that the composition of the microbiota of the gastrointestinal tract prevents a variety of diseases by preventing infectious agents from proliferating. Reducing clearance of off-target bacteria may decrease susceptibility to secondary infection. Of the Enterobacteriaceae susceptible to microcin J25, Escherichia coli are the most abundant within the human gut. To explore the modulation of specificity, a collection of 207 mutants encompassing 12 positions in both the ring and loop of microcin J25 was built and tested for activity against Salmonella and E. coli strains. As has been found previously, mutational tolerance of ring residues was lower than loop residues, with 22% and 51% of mutations, respectively, retaining activity toward at least one target within the target organism test panel. The multitarget screening elucidated increased mutational tolerance at position G2, G3, and G14 than previously identified in panels composed of single targets. Multiple mutations conferred differential response between the different targets. Examination of specificity differences between mutants found that 30% showed significant improvements to specificity toward any of the targets. Generation and testing of a combinatorial library designed from the point-mutant study revealed that microcin J25I13T reduces off-target activity toward commensal human-derived E. coli isolates by 81% relative to Salmonella enterica serovar Enteritidis. These in vitro specificity improvements are likely to improve in vivo treatment efficacy by reducing clearance of commensal bacteria in the gastrointestinal tract of hosts.

Project description:The metabolism of lactate impacts infant gut health and may lead to acute accumulation of lactate and/or H2 associated with pain and crying of colicky infants. Because gut microbiota studies are limited due to ethical and safety concerns, in vitro fermentation models were developed as powerful tools to assess effects of environmental conditions on the gut microbiota. In this study, we established a continuous colonic fermentation model (PolyFermS), inoculated with immobilized fecal microbiota and mimicking the proximal colon of 2-month-old infants. We investigated the effects of pH and retention time (RT) on lactate metabolism and of lactate-utilizing bacteria (LUB) exhibiting little or no H2 production. We observed that a drop in pH from 6.0 to 5.0 increased the number of lactate-producing bacteria (LPB) and decreased LUB concomitantly with lactate accumulation. Increasing RT from 5 to 10 h at pH 5.0 resulted in complete lactate consumption associated with increased LUB. Supplementation with dl-lactate (60 mM) to mimic lactate accumulation promoted propionate and butyrate production with no effect on acetate production. We further demonstrated that lactate-utilizing Propionibacterium avidum was able to colonize the reactors 4 days after spiking, suggesting its ability to compete with other lactate-utilizing bacteria producing H2 In conclusion, we showed that PolyFermS is a suitable model for mimicking young infant colonic microbiota. We report for the first time pH and RT as strong drivers for composition and metabolic activity of infant gut microbiota, especially for the metabolism of lactate, which is a key intermediate product for ecology and infant health.IMPORTANCE The metabolism of lactate is important for infant gut health and may lead to acute lactate and/or H2 accumulation, pain, and crying as observed in colicky infants. Functional human studies often faced ethical challenges due to invasive medical procedures; thus, in this study, we implemented PolyFermS fermentation models to mimic the infant proximal colon, which were inoculated with immobilized fecal microbiota of two 2-month-old infants. We investigated the impact of pH, retention time, and accumulation of dl-lactate on microbiota composition and metabolic activity. We found that a drop in pH from 6.0 to 5.0 led to increased LPB and decreased LUB concomitantly with lactate accumulation. Increasing the RT resulted in complete lactate consumption associated with increased LUB. Our data highlight for the first time the impact of key abiotic factors on the metabolism of lactate, which is an important intermediate product for ecology and infant health.

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Microcin J25 Exhibits Inhibitory Activity Against Salmonella Newport in Continuous Fermentation Model Mimicking Swine Colonic Conditions.

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