Genome-based reclassification of Lactobacillus casei: emended classification and description of the species Lactobacillus zeae.

Taxonomic relationships between Lactobacillus casei, Lactobacillus paracasei and Lactobacillus zeae have long been debated. Results of previous analyses have shown that overall genome relatedness indices (such as average nucleotide identity and core nucleotide identity) between the type strains L. casei ATCC 393T and L. zeae ATCC 15820T were 94.6 and 95.3 %, respectively, which are borderline for species definition. However, the digital DNA‒DNA hybridization value was 57.3 %, which was clearly lower than the species delineation threshold of 70 %, and hence raised the possibility that L. casei could be reclassified into two species. To re-evaluate the taxonomic relationship of these taxa, multilocus sequence analysis (MLSA) based on the concatenated five housekeeping gene (dnaJ, dnaK, mutL, pheS and yycH) sequences, phylogenomic and core genome multilocus sequence typing analyses, gene presence and absence profiles using pan-genome analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling analysis, cellular fatty acid compositions, and phenotype analysis were carried out. The results of phenotypic characterization, MLSA, whole-genome sequence-based analyses and MALDI-TOF MS profiling justified an independent species designation for the L. zeae strains, and supported an emended the description of the name of Lactobacillus zeae (ex Kuznetsov 1956) Dicks et al. 1996, with ATCC 15820T (=DSM 20178T=BCRC 17942T) as the type strain.

Prevotella hominis sp. nov., isolated from human faeces.

A strictly anaerobic predominant bacterium, designated as strain gm001T, was isolated from a freshly voided faecal sample collected from a healthy Taiwanese adult. Cells were Gram-stain-negative rods, non-motile and non-spore-forming. Strain gm001T was identified as a member of the genus Prevotella, and a comparison of 16S rRNA and hsp60 gene sequences revealed sequence similarities of 98.5 and 93.3 %, respectively, demonstrating that it was most closely related to the type strain of Prevotella copri. Phylogenomic tree analysis indicated that the gm001T cluster is an independent lineage of P. copri DSM 18205T. The average nucleotide identity, digital DNA‒DNA hybridization and average amino acid identity values between strain gm001T and P. copri DSM 18205T were 80.9, 28.6 and 83.8 %, respectively, which were clearly lower than the species delineation thresholds. The species-specific genes of this novel species were also identified on the basis of pan-genomic analysis. The predominant menaquinones were MK-11 and MK-12, and the predominant fatty acids were anteiso-C15 : 0, C15 : 0 and iso-C15 : 0. Acetate and succinate were produced from glucose as metabolic end products. Taken together, the results indicate that strain gm001T represents a novel species of the genus Prevotella, for which the name Prevotella hominis sp. nov. is proposed. The type strain is gm001T (=BCRC 81118T=JCM 33280T).

Reclassification of Micrococcus aloeverae and Micrococcus yunnanensis as later heterotypic synonyms of Micrococcus luteus.

Micrococcus aloeverae,Micrococcus endophyticus, Micrococcus luteus and Micrococcus yunnanensis are phenotypically and genotypically closely related, and together comprise the M. luteus group. In this study, the taxonomic relationships among Micrococcus aloeverae, M. luteus and M. yunnanensis were re-evaluated by using polyphasic approaches. The similarity values of the concatenated housekeeping gene (gyrB, recA and rpoB) sequences shared by the type strains of M. aloeverae, M. luteus and M. yunnanensis ranged from 98.3 to 99.4 %. The average nucleotide identity, average amino acid identity and digital DNA‒DNA hybridization values among these three taxa were greater (97.1‒98.1 %, 96.8‒98.1 % and 75.0‒83.5 %, respectively) than the thresholds for bacterial species delineation, indicating that they belong to the same species, whereas those for M. endophyticus were clearly lower than the thresholds. In addition, phenotypic and chemotaxonomic characterization results also support the synonymy of these three taxa. Therefore, we propose that M. aloeverae and M. yunnanensis should be reclassified as later heterotypic synonyms of M. luteus.

Lactobacillus suantsaii sp. nov., isolated from suan-tsai, a traditional Taiwanese fermented mustard green.

A Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strain, L88T, was isolated from suan-tsai, a traditional Taiwanese fermented mustard green. Comparative analyses of 16S rRNA, pheS and rpoA gene sequences demonstrated that strain L88Twas a member of the genus Lactobacillus. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus acidifarinae (98.2 % similarity), Lactobacillus namurensis (98.1 %), Lactobacillus zymae (98.1 %) and Lactobacillus spicheri (96.8 %) were the closest neighbours to this novel strain. The average nucleotide identity, digital DNA‒DNA hybridization and average amino acid identity values between L88T and its closest relatives were lower than 80, 30 and 90 %, respectively. Phenotypic and genotypic test results demonstrated that strain L88T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus suantsaii sp. nov., is proposed. The type strain is L88T (=BCRC 12945T=NBRC 113535T).

Inactivation of ferric uptake regulator (Fur) attenuates Helicobacter pylori J99 motility by disturbing the flagellar motor switch and autoinducer-2 production.

BACKGROUND: Flagellar motility of Helicobacter pylori has been shown to be important for the bacteria to establish initial colonization. The ferric uptake regulator (Fur) is a global regulator that has been identified in H. pylori which is involved in the processes of iron uptake and establishing colonization. However, the role of Fur in H. pylori motility is still unclear. MATERIALS AND METHODS: Motility of the wild-type, fur mutant, and fur revertant J99 were determined by a soft-agar motility assay and direct video observation. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. Single bacterial motility and flagellar switching were observed by phase-contrast microscopy. Autoinducer-2 (AI-2) production in bacterial culture supernatant was analyzed by a bioluminescence assay. RESULTS: The fur mutant showed impaired motility in the soft-agar assay compared with the wild-type J99 and fur revertant. The numbers and lengths of flagellar filaments on the fur mutant cells were similar to those of the wild-type and revertant cells. Phenotypic characterization showed similar swimming speed but reduction in switching rate in the fur mutant. The AI-2 production of the fur mutant was dramatically reduced compared with wild-type J99 in log-phase culture medium. CONCLUSIONS: These results indicate that Fur positively modulates H. pylori J99 motility through interfering with bacterial flagellar switching.

Staphylococcus hsinchuensis sp. nov., Isolated from Soymilk.

A novel coagulase-negative Staphylococcus strain (H164T) was isolated from soymilk in Taiwan. Comparative sequence analysis of the 16S rRNA gene revealed that the H164T strain is a member of the genus Staphylococcus. We used multilocus sequence analysis (MLSA) and phylogenomic analyses to demonstrate that the novel strain was closely related to Staphylococcus gallinarum, Staphylococcus nepalensis, Staphylococcus cohnii, and Staphylococcus urealyuticus. The average nucleotide identity and digital DNA-DNA hybridization values between H164T and its closest relatives were <95% and <70%, respectively. The H164T strain could also be distinguished from its closest relatives by the fermentation of d-fructose, d-maltose, d-trehalose, and d-mannitol, as well as by the activities of α-glucosidase and alkaline phosphatase. The major cellular fatty acids were C15:0 iso and C15:0 anteiso, and the predominant menaquinones were MK-7 and MK-8, respectively. The major cellular fatty acids and predominant menaquinones were C15:0 iso and C15:0 anteiso and MK-7 and MK-8, respectively. In conclusion, this strain represents a novel species, named Staphylococcus hsinchuensis sp. nov., with the type strain H164T (=BCRC 81404T = NBRC 116174T).

Micrococcus porci sp. nov., Isolated from Feces of Black Pig (Sus scrofa).

An aerobic bacterium, designated as strain KD337-16T, was isolated from the fecal samples of a black pig. It exhibited spherical, non-motile and non−spore-forming, Gram-positive cells. KD337-16T was identified as a member of the genus Micrococcus through 16S rRNA gene sequencing, and its closest relatives were found to be Micrococcus endophyticus YIM 56238T (99.5% similarity), Micrococcus luteus NCTC 2665T (99.1%), Micrococcus yunnanensis YIM 65004T (99.1%), Micrococcus aloeverae AE-6T (99.1%), Micrococcus antarcticus T2T (98.9%), and Micrococcus flavus LW4T (98.7%). Phylogenomic trees were constructed, and strain KD337-16T was found to form its own cluster as an independent lineage of M. flavus LW4T. Between KD337-16T and its close relatives, the average nucleotide identity, average amino acid identity, and digital DNA−DNA hybridization were below the respective species delineation thresholds at 82.1−86.6%, 78.1−86.1%, and 24.4−34.9%. The major cellular fatty acids and polar lipids were anteiso-C15:0 and iso-C15:0, and DPG and PG, respectively. The predominant menaquinone was MK-8(H2). Taken together, the results indicate that strain KD337-16T is a novel species of the genus Micrococcus, for which the name Micrococcus porci sp. nov. is proposed. The type strain is KD337-16T (=BCRC 81318T = NBRC 115578T).

The mutL Gene as a Genome-Wide Taxonomic Marker for High Resolution Discrimination of Lactiplantibacillus plantarum and Its Closely Related Taxa.

The current taxonomy of the Lactiplantibacillus plantarum group comprises of 17 closely related species that are indistinguishable from each other by using commonly used 16S rRNA gene sequencing. In this study, a whole-genome-based analysis was carried out for exploring the highly distinguished target genes whose interspecific sequence identity is significantly less than those of 16S rRNA or conventional housekeeping genes. In silico analyses of 774 core genes by the cano-wgMLST_BacCompare analytics platform indicated that csbB, morA, murI, mutL, ntpJ, rutB, trmK, ydaF, and yhhX genes were the most promising candidates. Subsequently, the mutL gene was selected, and the discrimination power was further evaluated using Sanger sequencing. Among the type strains, mutL exhibited a clearly superior sequence identity (61.6-85.6%; average: 66.6%) to the 16S rRNA gene (96.7-100%; average: 98.4%) and the conventional phylogenetic marker genes (e.g., dnaJ, dnaK, pheS, recA, and rpoA), respectively, which could be used to separat tested strains into various species clusters. Consequently, species-specific primers were developed for fast and accurate identification of L. pentosus, L. argentoratensis, L. plantarum, and L. paraplantarum. During this study, one strain (BCRC 06B0048, L. pentosus) exhibited not only relatively low mutL sequence identities (97.0%) but also a low digital DNA-DNA hybridization value (78.1%) with the type strain DSM 20314T, signifying that it exhibits potential for reclassification as a novel subspecies. Our data demonstrate that mutL can be a genome-wide target for identifying and classifying the L. plantarum group species and for differentiating novel taxa from known species.

Polyphasic characterization of a novel species in the Lactobacillus casei group from cow manure of Taiwan: Description of L. chiayiensis sp. nov.

Two Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strains, NCYUAST and BCRC 18859 (=NRIC 1947), were isolated from cow manure of Taiwan and coconut juice of Philippines, respectively. Comparative sequence analysis of 16S rRNA gene revealed that the novel strains were members of the genus Lactobacillus. These two strains had 100% of 16S rRNA gene sequence similarity and 98.6% of average nucleotide identity (ANI) value based on whole genome sequences. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus casei (99.6% similarity), Lactobacillus paracasei subsp. paracasei (99.1%), L. paracasei subsp. tolerans (99.1%), Lactobacillus rhmnosus (99.0%) and 'Lactobacillus zeae' (99.7%) were the closest neighbors to these novel strains. The results of phenotypic and chemotaxonomic characterization, multilocus sequence analysis (MLSA) based on the sequences of three housekeeping genes (dnaK, pheS and yycH), whole-genome sequence (WGS)-based comparison by ANI and in silico DNA-DNA hybridization (isDDH), species-specific PCR and whole-cell MALDI-TOF MS spectral pattern analyses demonstrated that the novel two strains represented a single, novel species within the L. casei group, for which the name Lactobacillus chiayiensis sp. nov., is proposed. The type strain is NCYUAST (=BCRC 81062T=NBRC 112906T).

Heteroresistance of Helicobacter pylori from the same patient prior to antibiotic treatment.

Antibiotic resistance among Helicobacter pylori strains has been increasing worldwide and has affected the efficacy of current treatments. The aim of this study was to evaluate whether treatment failure was due to the presence of antibiotic-susceptible and -resistant H. pylori simultaneously within the same host before eradication. In order to discover H. pylori with antibiotic heteroresistance in the same patient, we examined the antibiotic susceptibility of H. pylori isolated from 412 patients without H. pylori eradication. The E-test was used to determine the minimal inhibitory concentration of these strains. The results showed 19 (4.6%) of patients harbored antibiotic heteroresistant H. pylori, resistant to levofloxacin (5/19), clarithromycin (1/19) and metronidazole (16/19). Among them, three patients' isolates showed heteroresistance to two antibiotics. The genetic diversity of each isolate was evaluated by random amplified polymorphic DNA PCR and the results showed that only 1 patient' isolate (5.3%) had a different pattern while the others showed identical or similar fingerprinting patterns. Mutations in the genes responsible for antibiotic resistance were investigated by direct sequencing and compared between strains within each pair. All 5 levofloxacin-resistant isolates had mutations in GyrA at the QRDR region (N87 or D91). Strain 1571R with clarithromycin resistance had a A2042G substitution in its 23S rRNA. There were 15 metronidazole-resistant strains (100%) with isogenic variation of RdxA, and 6 strains (40%) contained FrxA variation (excluded pair 1159). These results suggest that the treatment failure of heteroresistant H. pylori mostly develops from high genomic variation of pre-existing strains through long term evolution rather than mixed infection with different strains.