pheS Gene Research Articles

Identification, Safety Assessment, and Antimicrobial Characteristics of Cocci Lactic Acid Bacteria Isolated from Traditional Egyptian Dairy Products.

The main objective of this study is to isolate and identify lactic acid bacteria (LAB) from various Egyptian dairy products, examine their antibacterial and hemolysis potential, and ensure their safety when used as starter cultures in different dairy industries. Egyptian dairy products are often made without the use of commercial starter cultures, using raw milk and artisanal methods. The most popular traditional dairy products are Laban Rayeb and Zabady, as well as the cheese varieties of Ras, Domiati, and Karish. The microbial communities used for fermentation and the diversity of lactic acid bacteria are the most important factors that can affect the quality of these products. In order to investigate the diversity of cocci lactic acid bacteria in Egyptian dairy products, 70 samples of raw or fermented milk and cheeses were collected from traditional cheese-making factories, local markets, and farmhouses located in the Delta area of Egypt. Following this, the LAB were isolated from the samples. One hundred fifty-seven isolates of Gram-positive, catalase-negative, and cocci bacterial species were identified via rep-PCR, and some isolates were confirmed using pheS and 16S rRNA gene sequencing, as follows: Streptococcus infantarius subsp. infantarius (three isolates), Enterococcus hirae (three isolates), Enterococcus faecium (ninety-six isolates), Enterococcus faecalis (forty isolates), Enterococcus durans (six isolates), Lactococcus garvieae (one isolate), Pediococcus acidilactici (seven isolates), and Lactococcus lactis subsp. Lactis (one isolate). These findings validate that five strains have strong antibacterial activity against Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes, and one hundred thirty-four strains were safe for hemolysis. The five strains were selected as protective cultures, including Pediococcus acidilactici, Lactococcus lactis subsp. lactis, E. faecalis, and E. faecium.

In Vitro Hypoglycemic Activities of Lactobacilli and Bifidobacterium Strains from Healthy Children's Sources and Their Effect on Stimulating GLP-1 Secretion in STC-1 Cells.

A long-term use of chemical drugs cannot cure type II diabetes mellitus (T2DM) and their numerous toxic side effects can be harmful to human health. In recent years, probiotics have emerged as a natural resource to replace chemical drugs in alleviating many human ailments. Healthy children's intestines have a lot of colonized Lactobacilli and Bifidobacterium, and these beneficial bacteria can help promote overall health. The objective of this study was to isolate potential antidiabetic probiotic strains from healthy children and evaluate their application prospects. Firstly, Lactobacillus and Bifidobacterium strains were isolated from healthy children's feces and identified by the pheS or clpC genes with their respective 16S rRNA genes. Then, hydrophobicity, artificial gastrointestinal fluid tolerance, α-Glucosidase and Dipeptidyl peptidase IV (DPP-IV) inhibitory activities of isolated strains were determined, and antioxidant activities and promoting secretion of GLP-1 in STC-1 cells of candidate strains were tested. Results showed that 6 strains of Lactobacillus and Bifidobacterium were obtained from the feces of healthy children aged 3 years, respectively, including Lacticaseibacillus paracasei L-21 and L-25, Levilactobacillus brevis L-16, Lentilactobacillus buchneri L-9, Lactiplantibacillus plantarum L-8 and L-3, Bifidobacterium bifidum 11-1 and B-84, Bifidobacterium longum subsp. longum 6-1, 6-2, B42 and B53. The hydrophobicity and auto-aggregation levels of all these strains were higher than 30% and 50%, respectively, and the decrease in the number of colonies of all strains in the artificial gastrointestinal fluid was less than 2 log CFU/mL. Strains L-3, L-8, L-9, L-21, 6-1, 11-1, B53 and B84 were selected based on their high α-glucosidase inhibitory activity and DPP-IV inhibitory activity, and results of the antioxidant capacity assay showed that the remaining strains all had intense comprehensive antioxidant activity. Additionally, Lacticaseibacillus paracasei L-21 and Bifidobacterium longum subsp. longum B-53 had the most substantial prompting effect on GLP-1 secretion in the STC-1 cell line. These results indicated that Lacticaseibacillus paracasei L-21 and Bifidobacterium longum subsp. longum B-53 could be used as a potential antidiabetic strain; thus, its application as a food supplement and drug ingredient could be recommended after in vivo mitigation of type II diabetes test.

Development of a counterselectable system for rapid and efficient CRISPR-based genome engineering in Zymomonas mobilis

BackgroundZymomonas mobilis is an important industrial bacterium ideal for biorefinery and synthetic biology studies. High-throughput CRISPR-based genome editing technologies have been developed to enable targeted engineering of genes and hence metabolic pathways in the model ZM4 strain, expediting the exploitation of this biofuel-producing strain as a cell factory for sustainable chemicals, proteins and biofuels production. As these technologies mainly take plasmid-based strategies, their applications would be impeded due to the fact that curing of the extremely stable plasmids is laborious and inefficient. Whilst counterselection markers have been proven to be efficient for plasmid curing, hitherto only very few counterselection markers have been available for Z. mobilis.ResultsWe constructed a conditional lethal mutant of the pheS gene of Z. mobilis ZM4, clmPheS, containing T263A and A318G substitutions and coding for a mutated alpha-subunit of phenylalanyl-tRNA synthetase to allow for the incorporation of a toxic analog of phenylalanine, p-chloro-phenylalanine (4-CP), into proteins, and hence leading to inhibition of cell growth. We demonstrated that expression of clmPheS driven by a strong Pgap promoter from a plasmid could render the Z. mobilis ZM4 cells sufficient sensitivity to 4-CP. The clmPheS-expressing cells were assayed to be extremely sensitive to 0.2 mM 4-CP. Subsequently, the clmPheS-assisted counterselection endowed fast curing of genome engineering plasmids immediately after obtaining the desired mutants, shortening the time of every two rounds of multiplex chromosome editing by at least 9 days, and enabled the development of a strategy for scarless modification of the native Z. mobilis ZM4 plasmids.ConclusionsThis study developed a strategy, coupling an endogenous CRISPR-based genome editing toolkit with a counterselection marker created here, for rapid and efficient multi-round multiplex editing of the chromosome, as well as scarless modification of the native plasmids, providing an improved genome engineering toolkit for Z. mobilis and an important reference to develope similar genetic manipulation systems in other non-model organisms.

Genomic analysis and in vivo efficacy of Pediococcus acidilactici as a potential probiotic to prevent hyperglycemia, hypercholesterolemia and gastrointestinal infections

Lactic acid bacteria are the well acknowledged probiotics that can cure a variety of diseases. In this study, we observed the in vivo potentials of Pediococcus to treat hyperglycemia, hypercholesterolemia and gastrointestinal infections. A total of 77 Lactobacillus were isolated from the milk of 10 cows and 10 goats, four of those strains inhibited both carbohydrates-hydrolyzing enzymes, α-glucosidase, and α-amylase. They all showed antagonistic effects on pathogenic E. coli and S. Typhimurium which were confirmed by performing pathogen challenge test and visualizing on Electron microscopy. 16S rRNA gene sequence identified that all four strains belong to Pediococcus genus which were further distinguished as Pediococcus acidilactici by pheS gene sequence. Whole genome sequence analysis revealed their non-pathogenic properties for human and the presence of probiotic genes responsible for stress resistance, immunomodulation, adhesion, metal and drug resistance. In vivo trial with diabetes-induced mice ascertained that all Pediococcus acidilactici had significant potentials to reduce elevated glucose and low-density lipoprotein level in blood. Interestingly, two out of four strains were significantly more effective (p < 0.0001 each) than metformin in reducing the blood glucose level. This in vivo study demonstrated that Pediococcus acidilactici might be a promising probiotic to prevent hyperglycemia, hypercholesterolemia and gastrointestinal infections.

Resolution of inter/intraspecies variation in Weissella group requires multigene analysis and functional characterization.

Weissella confusa and Weissella cibaria strains isolated from the human- gut are considered as potential probiotics, but remain under-explored owing to their ambiguous taxonomic assignment. The present study assesses the taxonomic resolution of 11 strains belonging to W. confusa and W. cibaria species and highlights the inter- and intraspecies variations using an array of phenetic and molecular methods. Remarkable genomic variability among the strains was observed by phylogenetic analysis using concatenated housekeeping genes (pheS, gyrB, and dnaA) along with 16S rRNA gene sequence, suggesting intraspecies variations; which is also supported by the phenetic data. Analysis showed that 16S rRNA gene sequence alone could not resolve the variation, and among the tested marker genes, signals from pheS gene provide better taxonomic resolution. The biochemical and antibiotic susceptibility tests also showed considerable variations among the isolates. Additionally, 'quick' identification using mass spectroscopy-based matrix-assisted laser desorption/ionization-time of flight mass spectrawas accurate up to genus only, and not species level, for the Weissella group. The study highlights need for inclusion of functional, phenetic, and multigene phylogenetic analysis in addition to 16S rRNA gene-based identification for the Weissella group, to provide better resolution in taxonomic assignments, which is often a prerequisite for the selection of potential strains with biotechnological applications.

PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk.

The number of viable lactic acid bacteria (LAB) is a key indicator of the quality of fermented milk. Currently, the combination of propidium monoazide (PMA) and qPCR has been applied in the quantification of viable bacteria in various matrices. In this research, the PMA-qPCR method was used to detect the number of viable bacteria of each LAB species in fermented milk. By analyzing pheS gene and 16S rRNA gene sequence similarities in five species of LAB, namely Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, Streptococcus thermophilus, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, the pheS gene resolved species identities better and was thus selected to design specific primers and probes. The pheS gene was cloned into the pUC19 vector and used to construct a standard curve for absolute quantification. Standard curves for quantification were constructed for each LAB species for serial dilutions between 1011 and 106 CFU/mL, with R2 > 0.99. The number of viable bacteria in the fermented milk detected by PMA-qPCR was significantly lower than that of qPCR (P < 0.05), indicating that PMA inhibited the amplification of DNA from dead cells. This was corroborated by the results from bacterial staining and plate count experiments. The proposed PMA-qPCR method provided rapid qualitative and quantitative determination of the number of viable bacteria for each LAB species in fermented milk within 3 h.

Phylogenomic analysis of the genus Leuconostoc.

Leuconostoc is a genus of saccharolytic heterofermentative lactic acid bacteria that inhabit plant-derived matrices and a variety of fermented foods (dairy products, dough, milk, vegetables, and meats), contributing to desired fermentation processes or playing a role in food spoilage. At present, the genus encompasses 17 recognized species. In total, 216 deposited genome sequences of Leuconostoc were analyzed, to check the delineation of species and to infer their evolutive genealogy utilizing a minimum evolution tree of Average Nucleotide Identity (ANI) and the core genome alignment. Phylogenomic relationships were compared to those obtained from the analysis of 16S rRNA, pheS, and rpoA genes. All the phylograms were subjected to split decomposition analysis and their topologies were compared to check the ambiguities in the inferred phylogenesis. The minimum evolution ANI tree exhibited the most similar topology with the core genome tree, while single gene trees were less adherent and provided a weaker phylogenetic signal. In particular, the 16S rRNA gene failed to resolve several bifurcations and Leuconostoc species. Based on an ANI threshold of 95%, the organization of the genus Leuconostoc could be amended, redefining the boundaries of the species L. inhae, L. falkenbergense, L. gelidum, L. lactis, L. mesenteroides, and L. pseudomesenteroides. Two strains currently recognized as L. mesenteroides were split into a separate lineage representing a putative species (G16), phylogenetically related to both L. mesenteroides (G18) and L. suionicum (G17). Differences among the four subspecies of L. mesenteroides were not pinpointed by ANI or by the conserved genes. The strains of L. pseudomesenteroides were ascribed to two putative species, G13 and G14, the former including also all the strains presently belonging to L. falkenbergense. L. lactis was split into two phylogenetically related lineages, G9 and G10, putatively corresponding to separate species and both including subgroups that may correspond to subspecies. The species L. gelidum and L. gasicomitatum were closely related but separated into different species, the latter including also L. inhae strains. These results, integrating information of ANI, core genome, and housekeeping genes, complemented the taxonomic delineation with solid information on the phylogenetic lineages evolved within the genus Leuconostoc.

Highly Effective Markerless Genetic Manipulation of Streptococcus suis Using a Mutated PheS-Based Counterselectable Marker.

Streptococcus suis is an important zoonotic pathogen, however, an efficient markerless genetic manipulation system is still lacking for further physiological and pathological studies on this bacterium. Several techniques have been developed for markerless genetic manipulation of S. suis utilizing either a temperature-sensitive vector or a counterselectable markers (CSMs), however, at present, the efficiency of these techniques is not very satisfactory. In this study, we developed a strategy for markerless genetic manipulation of S. suis employing a CSM based on a conditionally lethal mutant allele of pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase (PheS). This mutant pheS, mPheS, was constructed by introducing site-directed mutations for a T261S/A315G double-substitution and a number of silent mutations to decrease its similarity with the endogenous wild type pheS gene (wtPheS). Additionally, five potentially strong promoters from S. suis were screened for their ability to drive high-level expression of mPheS, thus endowing the carrier strain with sufficient sensitivity to the phenylalanine analog p-chloro-phenylalanine (p-Cl-phe). Insertion of these P-mPheS cassettes into a vector or into the chromosomal locus via a linked erythromycin resistance gene revealed that mPheS allele driven by promoters P0530 and P1503 renders S. suis sensitive to as low as 0.01% (or 0.5 mM) of p-Cl-phe. This offers two potential CSMs for S. suis with p-Cl-phe as a counterselective agent. P1503-mPheS was revealed to be 100% efficient for counter-selection in S. suis by application in a precise gene deletion. Using P1503-mPheS as a CSM, a two-step insertion and excision strategy for markerless genetic manipulation of S. suis were developed, supplying a powerful tool for markerless genetic manipulation of S. suis.

The Probiotic Attributes and Anti-pseudorabies Virus Evaluation of Lactobacillus Isolates.

The emergence of pseudorabies virus (PRV) variants brings serious harm to the swine industry, and its effective treatments are limited at present. As one of the probiotics, the Lactobacillus species have beneficial characteristics of regulating the balance of intestinal flora, inhibiting the growth of pathogenic bacteria and viruses' proliferation, and improving self-immunity. In this study, Lactobacillus plantarum HN-11 and Lactobacillus casei HN-12 were selected and identified through morphology observation, Gram stain microscopy, 16S rRNA sequencing analysis, and specific amplification of the recA gene and pheS gene. All tested isolates exhibited rapid adaptation to the different conditions, excellent acid, and bile tolerance, and sensitivity to Salmonella, Staphylococcus aureus, and Escherichia coli. The antibiotic susceptibility assay displayed the isolates sensitive to most antibiotics and resistant to Lincomycin and Norfloxacin. Moreover, the supernatants of HN-11 and HN-12 inhibited PRV proliferation in ST cells. The results of animal experiments showed that supplementing the challenged mice with the supernatants of Lactobacillus isolates in advance delayed the course of the disease. PRV was detected in the heart, liver, spleen, lung, kidney, and brain tissues of dead mice in the test groups, and its copies in the lungs were significantly decreased compared with the control mice (P < 0.05). These findings proved the advantages of L. plantarum and L. casei as potential probiotic cultures, which could provide a basis for its application in microecological preparations and functional formulations.

Development of duplex qPCR targeting Carnobacterium maltaromaticum and Vagococcus salmoninarum.

In November 2018, Vagococcus salmoninarum was identified as the causative agent of a chronic coldwater streptococcosis epizootic in broodstock brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery in Wisconsin, USA. By February 2019, the epizootic spread to adjacent raceways containing broodstock lake trout (Salvelinus namaycush), whereby fish were found to be coinfected with Carnobacterium maltaromaticum and V.salmoninarum. To differentiate these two pathogens and determine the primary cause of the lake trout morbidity, a quantitative real-time PCR (qPCR) was developed targeting the C.maltaromaticum phenylalanyl-tRNA synthase alpha subunit (pheS) gene. The qPCR was combined with a V.salmoninarum qPCR, creating a duplex qPCR assay that simultaneously quantitates C.maltaromaticum and V.salmoninarum concentrations in individual lake trout tissues, and screens presumptive isolates from hatchery inspections and wild fish from national fish hatchery source waters throughout the Great Lakes basin. Vagococcus salmoninarum and C.maltaromaticum were co-detected in broodstock brook trout from two tribal hatcheries and C.maltaromaticum was present in wild fish in source waters of several national fish hatcheries. This study provides a powerful new tool to differentiate and diagnose two emerging Gram-positive bacterial pathogens.

First characterization of the probiotic potential of lactic acid bacteria isolated from Costa Rican pineapple silages.

BackgroundAgro-industrial waste from tropical environments could be an important source of lactic acid bacteria (LAB) with probiotic potential.MethodsTwelve LAB isolates were isolated from pineapple silages. The species identification was carried out considering 16S rRNA and pheS genes. Experiments to evaluate the probiotic potential of the isolates included survival under simulated gastrointestinal environment, in vitro antagonistic activity (against Salmonella spp. and Listeria monocytogenes), auto-aggregation assays, antibiotic susceptibility, presence of plasmids, adhesiveness to epithelial cells, and antagonistic activity against Salmonella in HeLa cells.ResultsLacticaseibacillus paracasei, Lentilactobacillus parafarraginis, Limosilactobacillus fermentum, and Weissella ghanensis were identified. Survival of one of the isolates was 90% or higher after exposure to acidic conditions (pH: 2), six isolates showed at least 61% survival after exposure to bile salts. The three most promising isolates, based on survivability tests, showed a strong antagonistic effect against Salmonella. However, only L. paracasei_6714 showed a strong Listeria inhibition pattern; this isolate showed a good auto-aggregation ability, was resistant to some of the tested antibiotics but was not found to harbor plasmids; it also showed a high capacity for adhesion to epithelial cells and prevented the invasion of Salmonella in HeLa cells. After further in vivo evaluations, L. paracasei_6714 may be considered a probiotic candidate for food industry applications and may have promising performance in acidic products due to its origin.

Rejection of the reclassification of Leuconostoc gasicomitatum as Leuconostoc gelidum subsp. gasicomitatum based on whole genome analysis.

In 2014, Rahkila et al. transferred Leuconostoc gasicomitatum to Leuconostoc gelidum as L. gelidum subsp. gasicomitatum comb. nov. based on a 75 % DNA-DNA hybridization value. In the present study, the taxonomic status of L. gelidum subsp. gasicomitatum is re-evaluated by a polyphasic approach, including 16S rRNA, pheS, rpoA, recA, and atpA gene sequence analyses, phylogenomic treeing, analyses of ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization), fatty acid methyl ester analysis and a phenotypic characterization. On the basis of the ANI and dDDH values, we propose to reject the proposal of Rahkila et al. to reclassify L. gasicomitatum as L. gelidum subsp. gasicomitatum.

Lactic Acid Bacteria Isolated from Fermented Doughs in Spain Produce Dextrans and Riboflavin.

Many lactic acid bacteria (LAB) produce metabolites with applications in the food industry, such as dextran-type exopolysaccharides (EPS) and riboflavin (vitamin B2). Here, 72 bacteria were isolated from sourdoughs made by Spanish bread-makers. In the presence of sucrose, colonies of 22 isolates showed a ropy phenotype, and NMR analysis of their EPS supported that 21 of them were dextran producers. These isolates were identified by their random amplified polymorphic DNA (RAPD) patterns and their rrs and pheS gene sequences as LAB belonging to four species (Weissella cibaria, Leuconostoc citreum, Leuconostoc falkenbergense and Leuconostoc mesenteroides). Six selected strains from the Leuconostoc (3) and Weissella (3) genera grew in the absence of riboflavin and synthesized vitamin B2. The EPS produced by these strains were characterized as dextrans by physicochemical analysis, and the L. citreum polymer showed an unusually high degree of branching. Quantification of the riboflavin and the EPS productions showed that the W. cibaria strains produce the highest levels (585–685 μg/and 6.5–7.4 g/L, respectively). Therefore, these new LAB strains would be good candidates for the development of fermented foods bio-fortified with both dextrans and riboflavin. Moreover, this is the first report of riboflavin and dextran production by L. falkenbergense.

Proposal of Lactococcus garvieae subsp. bovis Varsha and Nampoothiri 2016 as a later heterotypic synonym of Lactococcus formosensis Chen et al. 2014 and Lactococcus formosensis subsp. bovis comb. nov.

Currently, Lactococcus garvieae contains two subspecies: L. garvieae subsp. bovis and L. garvieae subsp. garvieae. In a study by Varsha and Nampoothiri, high pheS (99.7 %) and rpoA (99.6 %) sequence similarities indicated that L. garvieae subsp. bovis and Lactococcus formosensis probably have a close taxonomic relationship; low pheS (92.2 %) and rpoA (97.8 %) sequence similarities and relatively low DNA-DNA hybridization value (75.8 %) indicated that L. garvieae subsp. bovis and L. garvieae subsp. garvieae probably represent two different species. In the present study, the taxonomic relationships between L. garvieae subsp. bovis, L. garvieae subsp. garvieae and L. formosensis were re-examined based on sequence analyses of 16S rRNA, pheS, recA, rpoA and rpoB genes, average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) values, average amino acid identity (AAI), fatty acid methyl ester analysis and phenotypic characterization. L. garvieae subsp. bovis LMG 30663T exhibited 97.3 % ANI, 78.3 % dDDH and 96.4 % AAI values to L. formosensis NBRC 109475T, higher than the threshold for species demarcation (95-96, 70 and 95-96 %, respectively), indicating that L. garvieae subsp. bovis LMG 30663T and L. formosensis NBRC 109475T belong to the same species. L. garvieae subsp. bovis LMG 30663T had 91.2 % ANI, 43.3 % dDDH and 92.9-93.0% AAI values with the type strain of L. garvieae subsp. garvieae, indicating that they represent two different species. Because L. formosensis has been proposed and validated before L. garvieae subsp. bovis, L. garvieae subsp. bovis is transferred to L. formosensis as L. formosensis subsp. bovis comb. nov. The type strain of L. formosensis subsp. bovis is BSN307T (=DSM 100577T=MCC 2824T=KCTC 21083T=LMG 30663T). The type strain of L. formosensis subsp. formosensis is 516T (=NBRC 109475T=BCRC 80576T).

Identification and characterisation of the lactic acid bacteria associated with the traditional fermentation of dairy fermented product.

The aim of this research was to identify the key lactic acid bacteria associated with the fermentation of dairy traditional fermented products for developing starter cultures for controlled fermentation. A total of 100 lactic acid bacteria (LAB) were isolated from dairy traditional fermented products. Samples were obtained from eight producers in the South East of Nigeria. Isolates were identified by phenotypic and genotypic techniques including rep-PCR genotyping and sequencing of the 16S rRNA, pheS and rpoA genes. Isolates were characterised for antimicrobial activity against foodborne pathogens, exopolysaccharide (EPS) production and survival at low pH and in the presence of bile salts. All isolates clustered into 11 distinct rep-PCR groups and were identified as Lactobacillus fermentum (40%), Lactobacillus delbrueckii (23%), Streptococcus thermophilus (22%), Streptococcus infantarius (10%), Lactobacillus senioris (2%), Leuconostoc pseudomesenteriodes (2%) and Enterococcus thailandicus (1%). Lactobacillus fermentum showed a broad spectrum antimicrobial activity and survival at low pH, while Lactobacillus delbrueckii was able to tolerate low pH and produce EPS. All isolates survived in vitro exposure to 1% (w/v) bile salts over a 3-h period. L. fermentum, L. delbrueckii and S. thermophilus could be used to simulate the fermentation of dairy traditional fermented products.

MALDI-TOF MS insight into the biodiversity of Staka, the artisanal Cretan soured cream

Staka is a naturally fermented cream produced from sheep or mixtures of sheep and goat milk in the island of Crete, Greece. This work is the first report on Staka cream microbiota. Initially, counts of various bacterial groups were enumerated using selective media and growth conditions. All isolates (101) were subjected to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis; the spectra obtained were compared with those of an in-house reference database. While 70 isolates were considered identified at the species level, the remaining 31 were not, probably due to a lack of, or an insufficient number of, reference spectra in the MALDI-TOF MS database. Strains representative of the observed diversity among the unidentified MALDI-TOF MS spectra were therefore subjected to pheS and 16S rRNA gene sequencing analysis for further identification. The identification results revealed the presence of Lactobacillus spp., Enterococcus spp., Streptococcus thermophilus and Aerococcus viridans.

Leuconostoc falkenbergense sp. nov., isolated from a lactic culture, fermentating string beans and traditional yogurt.

In the present study, the taxonomic positions of five strains (C, 17-2, LMG 10779T, LMG 18969 and LMG 11483) of Leuconostoc pseudomesenteroides were re-evaluated by a polyphasic approach, including the analyses of 16S rRNA, pheS and rpoA gene sequences, cellular fatty acids, average nucleotide and amino acid identities (ANI and AAI), digital DNA-DNA hybridization (dDDH), and phenotypic features. Based on rpoA sequence analysis, the five strains and L. pseudomesenteroides LMG 11482T were divided into two groups: strains C, LMG 10779T and LMG 18969; strains 17-2, LMG 11483 and LMG 11482T. Each of the two groups had almost identical rpoA sequences. The rpoA sequence similarity between strain LMG 10779T and L. pseudomesenteroides LMG 11482T was 95.6 %. Strains LMG 11483 and 17-2 had 98.1 and 97.2 % ANI values, 83.5 and 73.2 % dDDH values, and a 97.0 % AAI value with L. pseudomesenteroides LMG 11482T, greater than the threshold for species demarcation, indicating that strains LMG 11483 and 17-2 belong to L. pseudomesenteroides. Strains LMG 18969 and C shared 97.1 and 98.2 % ANI values, 73.4 and 83.2 % dDDH values, and 96.9 and 96.6 % AAI values with strain LMG 10779T, greater than the threshold for species demarcation, indicating that strains LMG 10779T, LMG 18969 and C represent the same species. The ANI, dDDH and AAI values between strain LMG 10779T and the type strains of phylogenetically related species were 75.2-92.5, 20.0-48.2 and 75.3-93.9 %, respectively, below the thresholds for species demarcation, indicating that strain LMG 10779T represents a novel species within the genus Leuconostoc. On the basis of the results presented here, (i) strains 17-2 and LMG 11483 belong to L. pseudomesenteroides, and (ii) strains LMG 10779T, LMG 18969 and C are considered to represent a novel species within the genus Leuconostoc, for which the name Leuconostoc falkenbergense sp. nov. is proposed with the type strain LMG 10779T (=CCUG 27119T).

Distribution and antimicrobial activity of lactic acid bacteria associated with lychee fruits

Lychee is a popular fruit in China and southeastern Asia. Although it is very popular, the microbiota of lactic acid bacteria (LAB) associated with lychee remains poorly described. Lychee samples from seven different markets located in three cities in Taiwan were collected and a total of 104 LAB were isolated. Through RFLP analyses of 16S rDNA and rpoA genes for grouping and 16S rRNA gene sequencing, these isolates were finally divided into 6 groups (A to F). The most common genera of LAB in lychee samples were Weissella and Leuconostoc. Weissella confusa strain E was found to produce a bacteriocin active against Listeria monocytogenes and some other Gram-positive bacteria. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 3426.77 Da, which is different to other known Weissella bacteriocins. In addition, strain MB7 included in the genus Leuconostoc was identified as potential novel species or subspecies on the basis of phylogenetic analysis of 16S rRNA, rpoA and pheS gene sequences. Thus, this is the first report describing the distribution and varieties of LAB associated with lychee fruits. In addition, one potential novel LAB species or subspecies and one potential novel bacteriocin were also reported in this study.

Phylogenetic analysis of Leuconostoc and Lactobacillus species isolated from sugarcane processing streams.

High levels of gums such as dextran, produced by Leuconostoc and Lactobacillus spp., have a severe impact on factory throughput and sugar quality. This study aimed to determine the phylogenetic relationships between gum‐producing Leuconostoc and Lactobacillus bacteria which were isolated from various locations in a sugarcane processing factory at times when low‐ and high‐dextran raw sugar, respectively, were produced. Phylogenetic analysis of 16S rRNA gene sequences grouped 81 isolates with the type strains of Leuconostoc mesenteroides (subspp. mesenteroides, dextranicum, and cremoris), Leuconostoc pseudomesenteroides, Leuconostoc lactis, and Leuconostoc citreum, respectively. Forty‐three isolates clustered with the type strain of Lactobacillus fermentum. The phylogenetic relatedness of the isolates was determined by sequencing and analysis of the housekeeping genes rpoA and dnaA for Leuconostoc spp. and the pheS and tuf genes for the Lactobacillus spp. The rpoA gene proved discriminatory for the phylogenetic resolution of all of the isolated Leuconostoc spp. and the dnaA housekeeping gene was shown to be effective for isolates clustering with the type strains of Leuc. mesenteroides and Leuc. citreum. None of the loci examined permitted differentiation at the subspecies level of Leuc. mesenteroides. Single‐locus analysis, as well as the concatenation of the pheS and tuf housekeeping gene sequences, yielded identical phylogenies for the Lactobacillus isolates corresponding to L. fermentum.

Proposal of Lactobacillus kosoi Chiou et al. 2018 as a later heterotypic synonym of Lactobacillus micheneri McFrederick et al. 2018, elevation of Lactobacillus plantarum subsp. argentoratensis to the species level as Lactobacillus argentoratensis sp. nov., and Lactobacillus zhaodongensis sp. nov., isolated from traditional Chinese pickle and the intestinal tract of a honey bee (Apis mellifera).

Lactobacillus kosoi Chiou et al. 2018 and Lactobacillus micheneri McFrederick et al. 2018 are closely related, and they share 100 % 16S rRNA gene sequence similarity, 99.6 % pheS gene sequence similarity, 100 % rpoA gene sequence similarity, 97.3 % average nucleotide identity (ANI) value and 76.6 % in silico DNA-DNA hybridization (isDDH) value, indicating that they represent the same species. Fatty acid methyl esters (FAME) analysis and phenotypic characterization also indicated that L. kosoi and L. micheneri are very similar. We propose L. kosoi Chiou et al. 2018 as a later heterotypic synonym of L. micheneri McFrederick et al. 2018. The taxonomic position of Lactobacillus plantarum subsp. argentoratensis in the L. plantarum group was re-examined using a polyphasic approach, including sequence analyses of 16S rRNA, pheS, rpoA and recA genes, average nucleotide identity analysis, in silico DNA-DNA hybridization, fatty acid methyl ester analysis and phenotypic characterization. Results of 16S rRNA gene sequence analysis indicated that L. plantarum subsp. argentoratensis was closely related to L. plantarum subsp. plantarum, L. pentosus and L. paraplantarum in the L. plantarum group, sharing 99.6-99.7 % 16S rRNA gene sequence similarities. Results of pheS, rpoA and recA gene sequence analyses indicated that L. plantarum subsp. argentoratensis was most closely related to L. plantarum subsp. plantarum, having 91.8 % pheS gene sequence similarity, 98.9 % rpoA gene sequence similarity and 93.1 % recA gene sequence similarity. L. plantarum subsp. argentoratensis DSM 16365T shared 95.6 % ANI value and 62.9 % isDDH value with L. plantarum subsp. plantarum ATCC 14917T. The low isDDH value confirmed that L. plantarum subsp. argentoratensis and L. plantarum subsp. plantarum represent two different species, rather than two different subspecies in the L. plantarum group. On the basis of the data from polyphasic characterization obtained in the present study and in previous studies, L. plantarum subsp. argentoratensis is elevated to the species level and represents a novel species of the genus Lactobacillus, for which the name Lactobacillus argentoratensis sp. nov. is proposed and the type strain is DKO 22T (=CIP 108320T=DSM 16365T=JCM 16169T). Two novel Gram-stain-positive bacterial strains, designated 1206-1T and F027-1-2, were isolated from traditional pickle in Heilongjiang Province, PR China, and from the intestinal tract of a honey bee (Apis mellifera) in Hubei Province, PR China, respectively. The two bacteria were characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, average nucleotide identity analysis, in silico DNA-DNA hybridization analysis and an analysis of phenotypic features. The results of 16S rRNA gene sequence analysis indicated that strains 1206-1T and F027-1-2 were distantly related to Lactobacillus sharpeae, Lactobacillus hulanensis, Lactobacillus songhuajiangensis, Lactobacillus pantheris, Lactobacillus thailandensis, Lactobacillus camelliae, Lactobacillus jixianensis, Lactobacillus nasuensis, Lactobacillus baoqingensis, Lactobacillus manihotivorans and Lactobacillus porcinae. Strain 1206-1T exhibited 94.2-96.4 % 16S rRNA gene sequence similarities, 69.5-83.3 % pheS gene sequence similarities and 73.1-90.3 % rpoA gene sequence similarities to type strains of phylogenetically related species. ANI and isDDH values between strain 1206-1T and the type strains of phylogenetically related species were 52.7-73.7 % and 21.1-30.1 %, respectively. On the basis of the data obtained in the present study, a novel species, Lactobacillus zhaodongensis sp. nov. is proposed and the type strain is 1206-1T (=CCM 8981T=CCTCC AB 2019200T=LMG 31620T).