Dataset Information

Isolation and Comparative Genomic Analysis of Reuterin-Producing Lactobacillus reuteri From the Chicken Gastrointestinal Tract.

ABSTRACT: Lactobacillus reuteri is a natural inhabitant of selected animal and human gastrointestinal tract (GIT). Certain strains have the capacity to transform glycerol to 3-hydroxypropionaldehyde (3-HPA), further excreted to form reuterin, a potent antimicrobial system. Reuterin-producing strains may be applied as a natural antimicrobial in feed to prevent pathogen colonization of animals, such as in chicken, and replace added antimicrobials. To date, only seven L. reuteri strains isolated from chicken have been characterized which limits phylogenetic studies and host-microbes interactions characterization. This study aimed to isolate L. reuteri strains from chicken GIT and to characterize their reuterin production and antimicrobial resistance (AMR) profiles using phenotypic and genetic methods. Seventy strains were isolated from faces, crops and ceca of six chicken from poultry farms and samples from slaughterhouse. Twenty-five strains were selected for further characterization. Draft genomes were generated for the new 25 isolates and integrated into a phylogenetic tree of 40 strains from different hosts. Phylogenetic analysis based on gene content as well as on core genomes showed grouping of the selected 25 L. reuteri chicken isolates within the poultry/human lineage VI. Strains harboring pdu-cob-cbi-hem genes (23/25) produced between 156 mM ± 11 and 330 mM ± 14 3-HPA, from 600 mM of glycerol, in the conditions of the test. All 25 chicken strains were sensitive to cefotaxime (MIC between 0.016 and 1 ?g/mL) and penicillin (MIC between 0.02 and 4 ?g/mL). Akin to the reference strains DSM20016 and SD2112, the novel isolates were resistant to penicillin, possibly associated with identified point mutations in ponA, pbpX, pbpF and pbpB. All strains resistant to erythromycin (4/27) carried the ermB gene, and it was only present in chicken strains. All strains resistant to tetracycline (5/27) harbored tetW gene. This study confirms the evolutionary history of poultry/human lineage VI and identifies pdu-cob-cbi-hem as a frequent trait but not always present in this lineage. L. reuteri chicken strains producing high 3-HPA yield may have potential to prevent enteropathogen colonization of chicken.

SUBMITTER: Greppi A

PROVIDER: S-EPMC7326114 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Isolation and Comparative Genomic Analysis of Reuterin-Producing Lactobacillus reuteri From the Chicken Gastrointestinal Tract.

Greppi Anna A Asare Paul T PT Schwab Clarissa C Zemp Niklaus N Stephan Roger R Lacroix Christophe C

Frontiers in microbiology 20200604

Lactobacillus reuteri is a natural inhabitant of selected animal and human gastrointestinal tract (GIT). Certain strains have the capacity to transform glycerol to 3-hydroxypropionaldehyde (3-HPA), further excreted to form reuterin, a potent antimicrobial system. Reuterin-producing strains may be applied as a natural antimicrobial in feed to prevent pathogen colonization of animals, such as in chicken, and replace added antimicrobials. To date, only seven L. reuteri strains isolate ...[more]

PMID: 32670217

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Project description:BACKGROUND:Lactobacillus reuteri is a gut symbiont of a wide variety of vertebrate species that has diversified into distinct phylogenetic clades which are to a large degree host-specific. Previous work demonstrated host specificity in mice and begun to determine the mechanisms by which gut colonisation and host restriction is achieved. However, how L. reuteri strains colonise the gastrointestinal (GI) tract of pigs is unknown. RESULTS:To gain insight into the ecology of L. reuteri in the pig gut, the genome sequence of the porcine small intestinal isolate L. reuteri ATCC 53608 was completed and consisted of a chromosome of 1.94 Mbp and two plasmids of 138.5 kbp and 9.09 kbp, respectively. Furthermore, we generated draft genomes of four additional L. reuteri strains isolated from pig faeces or lower GI tract, lp167-67, pg-3b, 20-2 and 3c6, and subjected all five genomes to a comparative genomic analysis together with the previously completed genome of strain I5007. A phylogenetic analysis based on whole genomes showed that porcine L. reuteri strains fall into two distinct clades, as previously suggested by multi-locus sequence analysis. These six pig L. reuteri genomes contained a core set of 1364 orthologous gene clusters, as determined by OrthoMCL analysis, that contributed to a pan-genome totalling 3373 gene clusters. Genome comparisons of the six pig L. reuteri strains with 14?L. reuteri strains from other host origins gave a total pan-genome of 5225 gene clusters that included a core genome of 851 gene clusters but revealed that there were no pig-specific genes per se. However, genes specific for and conserved among strains of the two pig phylogenetic lineages were detected, some of which encoded cell surface proteins that could contribute to the diversification of the two lineages and their observed host specificity. CONCLUSIONS:This study extends the phylogenetic analysis of L. reuteri strains at a genome-wide level, pointing to distinct evolutionary trajectories of porcine L. reuteri lineages, and providing new insights into the genomic events in L. reuteri that occurred during specialisation to their hosts. The occurrence of two distinct pig-derived clades may reflect differences in host genotype, environmental factors such as dietary components or to evolution from ancestral strains of human and rodent origin following contact with pig populations.

Project description:Over the last years the demand for pre-washed, fresh-cut, and minimally-processed (MP) produce has increased. MP fresh vegetable are rapidly spoiled, whereas there is consumers' concern about chemical disinfection treatments such as with chlorine. A promising antimicrobial is reuterin, a broad-spectrum-antimicrobial compound produced by food-grade Lactobacillus reuteri from glycerol. In aqueous solution, reuterin is a dynamic system consisting of 3-hydroxypropionaldehyde (3-HPA), its hydrate, its dimer as well as acrolein, which was recently identified as the main antimicrobial component of the system. Here, we tested the use of reuterin containing similar 3-HPA levels but different acrolein concentrations for decontaminating and preserving fresh-cut lettuce. Crude reuterin (CR) was produced by biotransformation of 600 mM glycerol using L. reuteri DSM 20016T. CR preparations were further incubated for 16 h at 50°C to produce enhanced reuterin (ER) with raised concentration of acrolein. Fresh-cut iceberg lettuce (Lactuca sativa) was washed using CR (1.5-1.9 mM acrolein) and ER (7.2-21.9 mM acrolein) solutions at 4°C, or sodium hypochloride (250 mg/L) and tap water, and compared with unwashed lettuce. Washed lettuce samples were packed under modified atmosphere (2% O2, 5% CO2, and 93% N2) and stored for 13 days at 4°C. Application of ER containing 12.1, 20.9, or 21.9 mM acrolein reduced the initial viable plate counts of Enterobacteriaceae (by 2.1-2.8 log CFU/g), and yeasts and molds (by 1.3-2.0 log CFU/g) when compared with unwashed samples. In contrast, reuterin solutions containing 7.2 mM acrolein, sodium hypochlorite and tap water only showed very limited and transient, or no effects on the cell loads of lettuce after washing and during storage. Visual assessment of leaves washed with ER showed acrolein concentration-dependent discoloration noticeable already after 3 days of storage for the highest acrolein concentrations. Discoloration became severe for all ER treatments after 7 days, while the other treatments preserved the aspect of washed lettuce. Our data show the predominant role of acrolein as the main antimicrobial component of the reuterin system for food biopreservation. Reuterin preparations with enhanced acrolein concentration of 12.1 mM and higher were effective to reduce plate counts of Enterobacteriaceae and yeasts and molds washed lettuce until day 7 but induced pronounced discoloration of lettuce.

Project description:Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two ?-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ?lacS ?lacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ?lacS ?lacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that ?-galactosides may constitute alternate substrates of the GOS pathway.IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as ?-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including ?-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

Dataset Information

Isolation and Comparative Genomic Analysis of Reuterin-Producing Lactobacillus reuteri From the Chicken Gastrointestinal Tract.

Publications

Isolation and Comparative Genomic Analysis of Reuterin-Producing <i>Lactobacillus reuteri</i> From the Chicken Gastrointestinal Tract.

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