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September 22, 2019  |  

Complete genome sequence of Enterococcus durans KLDS6.0933, a potential probiotic strain with high cholesterol removal ability

Enterococci are commensal bacteria in the mammalian gastrointestinal tract which play an important role in the production of various fermented foods. Thus, certain enterococcal strains are commonly used as probiotics to confer health benefits to human and animals. Enterococcus durans KLDS6.0933 is a potential probiotic strain with high cholesterol removal ability, which was isolated from traditional naturally fermented cream in Inner Mongolia of China. To better understand the genetic basis of the probiotic properties of this strain, the whole-genome sequence was performed using the PacBio RSII platform.

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September 22, 2019  |  

The energy-coupling factor transporter module EcfAA’T, a novel candidate for the genetic basis of fatty acid-auxotrophic small-colony variants of Staphylococcus aureus.

Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA’T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA’T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid-auxotrophic staphylococcal SCVs.

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September 22, 2019  |  

A novel bacteriocin BMP11 and its antibacterial mechanism on cell envelope of Listeria monocytogenes and Cronobacter sakazakii

Listeria monocytogenes and Cronobacter sakazakii are notorious pathogens involved in numerous foodborne outbreaks after ingested contaminated food. Bacteriocins are natural food preservatives, some of which have antimicrobial activity comparable with antibiotics. In this study, a plasmid encoded novel bacteriocin BMP11 produced by Lactobacillus crustorum MN047 was innovatively identified by combining complete genome and LC-MS/MS. The BMP11 was found to have rich a-helix conformation after prediction. Moreover, the antimicrobial activity of BMP11 was verified after its heterologous expression in E. coli with 1280 and 640 AU/mL against L. monocytogenes and C. sakazakii, respectively. After purification by anion-exchange chromatography and HPLC, BMP11 had MIC values of 0.3–38.4?µg/mL against tested foodborne pathogens. Further, it was found that BMP11 had bactericidal action mode with concomitant cell lysis to pathogens by growth curve and time-kill kinetics. The results of scanning electron microscope (SEM) and transmission electron microscope (TEM) indicated that BMP11 destroyed the integrity of cell envelope of pathogens with cell wall perforation and cell membrane permeabilization. The destruction of cell envelope integrity was further verified by propidium iodide (PI) uptake and lactic dehydrogenase (LDH) release. BMP11 increased inner-membrane permeability of C. sakazakii in a concentration-dependent manner. Meanwhile, BMP11 exhibited antibiofilm formation activity. In addition, BMP11 inhibited the growth of L. monocytogenes in milk. Therefore, BMP11 had promising potential as antimicrobial to control foodborne pathogens in dairy products.

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September 22, 2019  |  

Genome alterations associated with improved transformation efficiency in Lactobacillus reuteri.

Lactic acid bacteria (LAB) are one of the microorganisms of choice for the development of protein delivery systems for therapeutic purposes. Although there are numerous tools to facilitate genome engineering of lactobacilli; transformation efficiency still limits the ability to engineer their genomes. While genetically manipulating Lactobacillus reuteri ATCC PTA 6475 (LR 6475), we noticed that after an initial transformation, several LR 6475 strains significantly improved their ability to take up plasmid DNA via electroporation. Our goal was to understand the molecular basis for how these strains acquired the ability to increase transformation efficiency.Strains generated after transformation of plasmids pJP067 and pJP042 increased their ability to transform plasmid DNA about one million fold for pJP067, 100-fold for pSIP411 and tenfold for pNZ8048. Upon sequencing of the whole genome from these strains, we identified several genomic mutations and rearrangements, with all strains containing mutations in the transformation related gene A (trgA). To evaluate the role of trgA in transformation of DNA, we generated a trgA null that improved the transformation efficiency of LR 6475 to transform pSIP411 and pJP067 by at least 100-fold, demonstrating that trgA significantly impairs the ability of LR 6475 to take-up plasmid DNA. We also identified genomic rearrangements located in and around two prophages inserted in the LR 6475 genome that included deletions, insertions and an inversion of 336 Kb. A second group of rearrangements was observed in a Type I restriction modification system, in which the specificity subunits underwent several rearrangements in the target recognition domain. Despite the magnitude of these rearrangements in the prophage genomes and restriction modification systems, none of these genomic changes impacted transformation efficiency to the level induced by trgA.Our findings demonstrate how genetic manipulation of LR 6475 with plasmid DNA leads to genomic changes that improve their ability to transform plasmid DNA; highlighting trgA as the primary driver of this phenotype. Additionally, this study also underlines the importance of characterizing genetic changes that take place after genome engineering of strains for therapeutic purposes.

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September 22, 2019  |  

Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum.

Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.

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September 22, 2019  |  

Genomic characterization of Lactobacillus delbrueckii TUA4408L and evaluation of the antiviral activities of its extracellular polysaccharides in porcine intestinal epithelial cells.

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor ?B (NF-?B) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-ß, Myxovirus resistance gene A (MxA) and RNaseL.

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September 22, 2019  |  

Genomic characterization and probiotic potency of Bacillus sp. DU-106, a highly effective producer of L-lactic acid isolated from fermented yogurt.

Bacillus sp. DU-106, a newly isolated member of Bacillus cereus group, exhibits the predominant ability to produce L-lactic acid. The probiotic potency of test strain revealed its survivability at acidic pH, bile salts and viability in simulated gastric juice in vitro. The acute oral toxicity test indicated its no toxicity to laboratory mice in vivo. We further determined the complete genome of strain DU-106 to understand genetic basis as a potential probiotic. It has a circular chromosome and three plasmids for a total genome 5,758,208 bp in size with a G + C content of 35.10%. Genes associated with lactate synthesis were found in the DU-106 genome. We also annotated various stress-related, bile salt resistance, and adhesion-related domains in this strain, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival under gastrointestinal tract. Moreover, strain DU-106 genome lacks the virulence genes encodes cereulide synthetase, enterotoxin FM, and cytotoxin K. These phenotypic and genomic probiotic potencies facilitate its potential candidate as probiotic starter in food industry.

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September 22, 2019  |  

Complete genome sequencing and analysis of endophytic Sphingomonas sp. LK11 and its potential in plant growth.

Our study aimed to elucidate the plant growth-promoting characteristics and the structure and composition of Sphingomonas sp. LK11 genome using the single molecule real-time (SMRT) sequencing technology of Pacific Biosciences. The results revealed that LK11 produces different types of gibberellins (GAs) in pure culture and significantly improves soybean plant growth by influencing endogenous GAs compared with non-inoculated control plants. Detailed genomic analyses revealed that the Sphingomonas sp. LK11 genome consists of a circular chromosome (3.78 Mbp; 66.2% G+C content) and two circular plasmids (122,975 bps and 34,160 bps; 63 and 65% G+C content, respectively). Annotation showed that the LK11 genome consists of 3656 protein-coding genes, 59 tRNAs, and 4 complete rRNA operons. Functional analyses predicted that LK11 encodes genes for phosphate solubilization and nitrate/nitrite ammonification, which are beneficial for promoting plant growth. Genes for production of catalases, superoxide dismutase, and peroxidases that confer resistance to oxidative stress in plants were also identified in LK11. Moreover, genes for trehalose and glycine betaine biosynthesis were also found in LK11 genome. Similarly, Sphingomonas spp. analysis revealed an open pan-genome and a total of 8507 genes were identified in the Sphingomonas spp. pan-genome and about 1356 orthologous genes were found to comprise the core genome. However, the number of genomes analyzed was not enough to describe complete gene sets. Our findings indicated that the genetic makeup of Sphingomonas sp. LK11 can be utilized as an eco-friendly bioresource for cleaning contaminated sites and promoting growth of plants confronted with environmental perturbations.

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September 22, 2019  |  

Lactobacillus rhamnosus LRB mediated inhibition of oral streptococci.

Lactobacillus rhamnosus is a lactic acid bacterium with a diverse ecological habitat. We recently isolated a L. rhamnosus strain (LRB) from a healthy baby-tooth that had naturally fallen out. We determined the whole genome sequence of LRB and found that the isolate is closely genetically related to an intestinal isolate, L. rhamnosus GG (ATCC 53103). However, the LRB genome had lost about a 75-kb segment and undergone a genomic rearrangement. We assessed LRB’s capacity to survive in the gut environment, at least temporarily. We found that LRB, like the intestinal isolate ATCC 53103, showed resistance to low pH but sensitive to bile salt. Surprisingly, we found that this oral isolate LRB showed strong antimicrobial activity against a variety of oral streptococci including Streptococcus mutans. The production of antimicrobial activity is dependent on media composition since some media supported the production while others did not. The production of antimicrobial activity is also dependent on growth temperature, with optimal production at 37°C. The antimicrobial activity was not restricted to streptococci, but effective against a variety of organisms, including ESKAPE pathogens.© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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September 22, 2019  |  

Comparative genome analysis and evaluation of probiotic characteristics of Lactobacillus plantarum strain JDFM LP11.

In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, attachment to porcine intestinal epithelial cells (IPEC-J2), and antimicrobial activity. The genetic characteristics of strain JDFM LP11 were explored by performing whole genome sequencing (WGS) using a PacBio system. The circular draft genome have a total length of 3,206,883 bp and a total of 3,021 coding sequences were identified. Phylogenetically, three genes, possibly related to survival and metabolic activity in the porcine host, were identified. These genes encode p60, lichenan permease IIC component, and protein TsgA, which are a putative endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. Our findings suggest that understanding the functional and genetic characteristics of L. plantarum strain JDFM LP11, with its candidate genes for gut health, could provide new opportunities and insights into applications in the animal food and feed additive industries.

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September 22, 2019  |  

Update on Tetracycline Susceptibility of Pediococcus acidilactici Based on Strains Isolated from Swiss Cheese and Whey.

Bacterial strains used as starter cultures in the production of fermented foods may act as reservoirs for antibiotic resistance (AbR) genes. To avoid the introduction of such genes into the food chain, the presence of acquired AbR in bacterial strains added to food must be tested. Standard protocols and microbiological cut-off values have been defined to provide practitioners with a basis for evaluating whether their bacterial isolates harbor an acquired resistance to a given antibiotic. Here, we tested the AbR of 24 strains of Pediococcus acidilactici by using the standard protocol and microbiological cut-off values recommended by the European Food Safety Authority. Phenotypic data were complemented by searching for known AbR genes using an in silico analysis of whole genomes. The majority (54.2%) of the strains were able to grow at a tetracycline concentration above the defined cut-off, even though only one strain carried a known tetracycline resistance gene, tetM. The same strain also carried the AbR gene of an erythromycin resistance methylase, ermA, and displayed resistance toward clindamycin and erythromycin. Our results bolster the scarce data on the sensitivity of P. acidilactici to tetracycline and suggest that the microbiological cut-off recommended by the European Food Safety Authority for this antibiotic should be amended.

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September 22, 2019  |  

Complete genome sequencing of Lactobacillus plantarum ZLP001, a potential probiotic that enhances intestinal epithelial barrier function and defense against pathogens in pigs.

The mammalian gastrointestinal tract is a heterogeneous ecosystem with the most abundant, and one of the most diverse, microbial communities. The gut microbiota, which may contain more than 100 times the number of genes in the human genome, endows the host with beneficial functional features, including colonization resistance, nutrient metabolism, and immune tolerance (Bäckhed, 2005). Dysbiosis of gut microbiota may result in serious adverse consequences for the host, such as neurological disorders, cancer, obesity, malnutrition, inflammatory dysregulation, and susceptibility to pathogens

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September 22, 2019  |  

Molecular characteristics and comparative genomics analysis of a clinical Enterococcus casseliflavus with a resistance plasmid.

The aim of this work was to investigate the molecular characterization of a clinical Enterococcus casseliflavus strain with a resistance plasmid.En. casseliflavus EC369 was isolated from a patient in a hospital in southern China. The minimum inhibitory concentration was found by means of the agar dilution method to determine the antimicrobial susceptibilities of the strains. Whole-genome sequencing and comparative genomics analysis were performed to analyze the mechanism of antibiotic resistance and the horizontal gene transfer of the resistance gene-related mobile genetic elements.En. casseliflavus EC369 showed resistance to erythromycin, kanamycin, and streptomycin, but was susceptible to vancomycin, ampicillin, and streptothricin and other antimicrobials. There were six resistance genes (aph3′, ant6, bla, sat4, and two ermBs) carried by a transposon identified on the plasmid pEC369 and a complete resistance gene cluster of vancomycin and a tet (M) gene encoded on the chromosome. This is the first complete plasmid sequence reported in clinically isolated En. casseliflavus. The plasmid with the greatest sequence identity with pEC369 was the plasmid of Enterococcus sp. FDAARGOS_375, followed by the plasmids of Enterococcus faecium strains F12085 and pRE25, whereas the sequence with the greatest identity to the resistance genes carrying a transposon of pEC369 was on the chromosome of Staphylococcus aureus strain GD1677.The resistance profiles of En. casseliflavus EC369 might contribute to the resistance genes encoded on the plasmid. The fact that the most similar sequence to the transposon carrying resistance genes of pEC369 was encoded in the chromosome of a S. aureus strain provides insights into the mechanism of dissemination of multidrug resistance between bacteria of different species or genera through horizontal gene transfer.

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September 22, 2019  |  

Genomic and metatranscriptomic analyses of Weissella koreensis reveal its metabolic and fermentative features during kimchi fermentation

The genomic and metabolic features of Weissella koreensis, one of the major lactic acid bacteria in kimchi, were investigated through genomic, metabolic, and transcriptomic analyses for the genomes of strains KCTC 3621T, KACC 15510, and WiKim0080. W. koreensis strains were intrinsically vancomycin-resistant and harbored potential hemolysin genes that were actively transcribed although no hemolysin activity was detected. KEGG and reconstructed fermentative metabolic pathways displayed that W. koreensis strains commonly employ the heterolactic pathway to produce d-lactate, ethanol, acetate, CO2, d-sorbitol, thiamine, and folate from various carbohydrates including d-glucose, d-mannose, d-lactose, l-malate, d-xylose, l-arabinose, d-ribose, N-acetyl-glucosamine, and gluconate, and strains KCTC 3621T and WiKim0080 additionally have metabolic pathways of d-galacturonate and d-glucoronate. Phenotypic analyses showed that all strains did not ferment d-galactose, probably due to the lack of d-galactose transporting system, and strains KCTC 3621T and WiKim0080 fermented d-fructose, indicating the presence of d-fructose transporting system. Fermentative features of W. koreensis were investigated through kimchi transcriptional analysis, suggesting that W. koreensis is mainly responsible for kimchi fermentation with the production of various fermentative metabolites during late fermentation period. This was the first study to investigate the genomic and metabolic features of W. koreensis, which may provide better understandings on kimchi fermentation.

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September 22, 2019  |  

Complete genome sequence of Leuconostoc citreum EFEL2700, a host strain for transformation of pCB vectors.

Leuconostoc citreum is an important lactic acid bacterium used as a starter culture for producing kimchi, the traditional Korean fermented vegetables. An efficient host strain for plasmid transformation, L. citreum EFEL2700, was isolated from kimchi, and it has been frequently used for genetic engineering of L. citreum. In this study, we report the whole genome sequence of the strain and its genetic characteristics. Genome assembly yielded 5 contigs (1 chromosome and 4 plasmids), and the complete genome contained 1,923,830 base pairs (bp) with a G?+?C content of 39.0%. Average nucleotide identity analysis showed high homology (= 99%) to the reference strain L. citreum KM 20. The smallest plasmid (4.3 kbp) was used as an Escherichia coli shuttle vector (pCB) for heterologous gene expression, and L. citreum EFEL2700 showed the highest transformation efficiency, 6.7?×?104 CFU µg-1 DNA. Genetic analysis of the genome enabled the construction of primary metabolic pathway showing a typical hetero-type lactic acid fermentation. Notably, no core genes for primary metabolism were observed in plasmid 4 and it could be eliminated to create an efficient host for gene transformation. This report will facilitate the understanding and application of L. citreum EFEL2700 as a food-grade microbial cell factory.Copyright © 2018. Published by Elsevier B.V.

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