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Antimicrobial peptide presenting potential strain-specific real time polymerase chain reaction assay for detecting the probiotic Lactobacillus reuteri KUB-AC5 in chicken intestine.

ABSTRACT: The gene coding for antimicrobial peptides produced by probiotic Lactobacillus reuteri KUB-AC5 located on the cloned DNA fragment I-C46 containing 2 open reading frames I-C46-F2.1 and I-C46-F2.2 were designed for strain-specific primers P1 and P2, respectively, and assessed by real-time quantitative polymerase chain reaction. According to the obtained results, primer P1 has limited strain specificity. Primer P2 exhibited high efficacy and specificity at annealing temperature of 70°C while P1 annealed at 57°C causing nonspecific bands. Hence, P2 was selected for quantitative polymerase chain reaction assay by isothermal annealing and extension reaction at high temperature of 70°C resulting in linearity for its DNA sequences ranging from 102 to 107 target copy numbers per assay, and displaying a detection limit of 6.17 log cfu/g of cecal digesta. Using spike testing, this system was able to detect 7.88 ± 0.06 to 11.78 ± 0.06 log copy number/g of digesta, higher than cultivation assay at about 1 log cfu/g, with good correlation of 0.99. These results suggested possible detection of strain KUB-AC5 in the gastrointestinal tract of chicken to evaluate the efficacy and persistence of a probiotic strain which requires correct inclusion rates in the feed.

SUBMITTER: Sobanbua S

PROVIDER: S-EPMC7587613 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Antimicrobial peptide presenting potential strain-specific real time polymerase chain reaction assay for detecting the probiotic Lactobacillus reuteri KUB-AC5 in chicken intestine.

Sobanbua S S Dolkittikul S S Nakphaichit M M Keawsompong S S Nitisinprasert S S

Poultry science 20191230 1

The gene coding for antimicrobial peptides produced by probiotic Lactobacillus reuteri KUB-AC5 located on the cloned DNA fragment I-C46 containing 2 open reading frames I-C46-F2.1 and I-C46-F2.2 were designed for strain-specific primers P1 and P2, respectively, and assessed by real-time quantitative polymerase chain reaction. According to the obtained results, primer P1 has limited strain specificity. Primer P2 exhibited high efficacy and specificity at annealing temperature of 70°C while P1 ann ...[more]

PMID: 32416839

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Project description:BackgroundExpression systems for lactic acid bacteria have been developed for metabolic engineering applications as well as for food-grade recombinant protein production. But the industrial applications of lactic acid bacteria as cell factories have been limited due to low biomass formation resulted in low efficiency of biomanufacturing process. Limosilactobacillus reuteri KUB-AC5 is a safe probiotic lactic acid bacterium that has been proven as a gut health enhancer, which could be developed as a mucosal delivery vehicle for vaccines or therapeutic proteins, or as expression host for cell factory applications. Similar to many lactic acid bacteria, its oxygen sensitivity is a key factor that limits cell growth and causes low biomass production. The aim of this study is to overcome the oxidative stress in L. reuteri KUB-AC5. Several genes involved in oxidative and anti-oxidative stress were investigated, and strain improvement for higher cell densities despite oxidative stress was performed using genetic engineering.ResultsAn in-silico study showed that L. reuteri KUB-AC5 genome possesses an incomplete respiratory chain lacking four menaquinone biosynthesis genes as well as a complete biosynthesis pathway for the production of the precursor. The presence of an oxygen consuming enzyme, NADH oxidase (Nox), leads to high ROS formation in aerobic cultivation, resulting in strong growth reduction to approximately 25% compared to anaerobic cultivation. Recombinant strains expressing the ROS scavenging enzymes Mn-catalase and Mn-superoxide dismutase were successfully constructed using the pSIP expression system. The Mn-catalase and Mn-SOD-expressing strains produced activities of 873 U/ml and 1213 U/ml and could minimize the ROS formation in the cell, resulting in fourfold and sevenfold higher biomass formation, respectively.ConclusionsExpression of Mn-catalase and Mn-SOD in L. reuteri KUB-AC5 successfully reduced oxidative stress and enhanced growth. This finding could be applied for other lactic acid bacteria that are subject to oxidative stress and will be beneficial for applications of lactic acid bacteria for cell factory applications.

Project description:Probiotics and commensal intestinal microbes suppress mammalian cytokine production and intestinal inflammation in various experimental model systems. Limited information exists regarding potential mechanisms of probiotic-mediated immunomodulation in vivo. In this report, we demonstrate that specific probiotic strains of Lactobacillus reuteri suppress intestinal inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model. Only strains that possess the hdc gene cluster, including the histidine decarboxylase and histidine-histamine antiporter genes, can suppress colitis and mucosal cytokine (interleukin-6 [IL-6] and IL-1? in the colon) gene expression. Suppression of acute colitis in mice was documented by diminished weight loss, colonic injury, serum amyloid A (SAA) protein concentrations, and reduced uptake of [(18)F]fluorodeoxyglucose ([(18)F]FDG) in the colon by positron emission tomography (PET). The ability of probiotic L. reuteri to suppress colitis depends on the presence of a bacterial histidine decarboxylase gene(s) in the intestinal microbiome, consumption of a histidine-containing diet, and signaling via the histamine H2 receptor (H2R). Collectively, luminal conversion of l-histidine to histamine by hdc(+) L. reuteri activates H2R, and H2R signaling results in suppression of acute inflammation within the mouse colon.Probiotics are microorganisms that when administered in adequate amounts confer beneficial effects on the host. Supplementation with probiotic strains was shown to suppress intestinal inflammation in patients with inflammatory bowel disease and in rodent colitis models. However, the mechanisms of probiosis are not clear. Our current studies suggest that supplementation with hdc(+) L. reuteri, which can convert l-histidine to histamine in the gut, resulted in suppression of colonic inflammation. These findings link luminal conversion of dietary components (amino acid metabolism) by gut microbes and probiotic-mediated suppression of colonic inflammation. The effective combination of diet, gut bacteria, and host receptor-mediated signaling may result in opportunities for therapeutic microbiology and provide clues for discovery and development of next-generation probiotics.

Dataset Information

Antimicrobial peptide presenting potential strain-specific real time polymerase chain reaction assay for detecting the probiotic Lactobacillus reuteri KUB-AC5 in chicken intestine.

Publications

Antimicrobial peptide presenting potential strain-specific real time polymerase chain reaction assay for detecting the probiotic Lactobacillus reuteri KUB-AC5 in chicken intestine.

Similar Datasets

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