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A case of Rickettsia sibirica subspecies sibirica BJ-90 infection in China was identified by metagenomic analysis of an eschar biopsy specimen and confirmed by nested PCR. Seroprevalence of spotted fever group Rickettsia was ≈17.4% among the local population. This report highlights the threat of rickettsioses to public health in the Qinghai-Tibet Plateau.

Nocardia is a complex and diverse genus of aerobic actinomycetes that cause complex clinical presentations, which are difficult to diagnose due to being misunderstood. To date, the genetic diversity, evolution, and taxonomic structure of the genus Nocardia are still unclear. In this study, we investigated the pan-genome of 86 Nocardia type strains to clarify their genetic diversity. Our study revealed an open pan-genome for Nocardia containing 265,836 gene families, with about 99.7% of the pan-genome being variable. Horizontal gene transfer appears to have been an important evolutionary driver of genetic diversity shaping the Nocardia genome and may have caused historical taxonomic confusion from other taxa (primarily Rhodococcus, Skermania, Aldersonia, and Mycobacterium). Based on single-copy gene families, we established a high-accuracy phylogenomic approach for Nocardia using 229 genome sequences. Furthermore, we found 28 potentially new species and reclassified 16 strains. Finally, by comparing the topology between a phylogenomic tree and 384 phylogenetic trees (from 384 single-copy genes from the core genome), we identified a novel locus for inferring the phylogeny of this genus. The dapb1 gene, which encodes dipeptidyl aminopeptidase BI, was far superior to commonly used markers for Nocardia and yielded a topology almost identical to that of genome-based phylogeny. In conclusion, the present study provides insights into the genetic diversity, contributes a robust framework for the taxonomic classification, and elucidates the evolutionary relationships of Nocardia. This framework should facilitate the development of rapid tests for the species identification of highly variable species and has given new insight into the behavior of this genus., Author summary Nocardia species can be responsible for opportunistic infections in humans, causing a variety of clinical presentations. They can also cause mycetoma in a normal host through direct inoculation. Although these species are often overlooked and misunderstood by modern medicine, they can cause life-threatening infections. However, most of our knowledge about Nocardia is based upon case reports and a few small studies of limited scope. This study provides an overview of the taxonomic and evolutionary structure of the genus Nocardia through extensive analysis of genome sequences. Our work aids the field by dissecting the genetic diversity of this species and improving the identification scheme for Nocardia species.

Bacteriophage VP1 is a typing phage used for the phage subtyping of Vibrio cholerae O1 biotype El Tor, but the molecular mechanisms of its receptor recognition and the resistance of its host to infection are mostly unknown. In this study, we aimed to identify the host receptor and its role in resistance in natural VP1-resistant strains. Generating spontaneous resistance mutations and genome sequencing mutant strains found the polyQ protein VcpQ, which carries 46 glutamine residues in its Q-rich region, to be responsible for infection by VP1. VcpQ is a membrane protein and possibly forms homotrimers. VP1 adsorbed to V. cholerae through VcpQ. Sequence comparisons showed that 72% of natural VP1-resistant strains have fewer glutamines in the VcpQ Q-rich stretch than VP1-sensitive strains. This difference did not affect the membrane location and oligomer of VcpQ but abrogated VP1 adsorption. These mutant VcpQs did not recover VP1 infection sensitivity in a V. cholerae strain with vcpQ deleted. Our study revealed that the polyQ protein VcpQ is responsible for the binding of VP1 during its infection of V. cholerae and that glutamine residue reduction in VcpQ affects VP1 adsorption to likely be the main cause of VP1 resistance in natural resistant strains. The physiological functions of this polyQ protein in bacteria need further clarification; however, mutations in the polyQ stretch may endow V. cholerae with phage resistance and enhance survival against VP1 or related phages. IMPORTANCE Receptor recognition and binding by bacteriophage are the first step for its infection of bacterial cells. In this study, we found the Vibrio cholerae subtyping phage VP1 uses a polyQ protein named VcpQ (V. cholerae polyQ protein) as the receptor for VP1 infection. Our study reveals the receptor’s recognition of phage VP1 during its adsorption and the VP1 resistance mechanism of the wild resistant V. cholerae strains bearing the mutagenesis in the receptor VcpQ. These mutations may confer the survival advantage on these resistant strains in the environment containing VP1 or its similar phages.

Background Members of the genus Proteus are mostly opportunistic pathogens that cause a variety of infections in humans. The molecular evolutionary characteristics and genetic relationships among Proteus species have not been elucidated to date. In this study, we developed a multilocus sequence analysis (MLSA) approach based on five housekeeping genes (HKGs) to delineate phylogenetic relationships of species within the genus Proteus. Results Of all 223 Proteus strains collected in the current study, the phylogenetic tree of five concatenated HKGs (dnaJ, mdh, pyrC, recA and rpoD) divided 223 strains into eleven clusters, which were representative of 11 species of Proteus. Meanwhile, the phylogenetic trees of the five individual HKGs also corresponded to that of the concatenated tree, except for recA, which clustered four strains at an independent cluster. The evaluation of inter- and intraspecies distances of HKG concatenation indicated that all interspecies distances were significantly different from intraspecies distances, which revealed that these HKG concatenations can be used as gene markers to distinguish different Proteus species. Further web-based DNA-DNA hybridization estimated by genome of type strains confirmed the validity of the MLSA, and each of eleven clusters was congruent with the most abundant Proteus species. In addition, we used the established MLSA method to identify the randomly collected Proteus and found that P. mirabilis is the most abundant species. However, the second most abundant species is P. terrae but not P. vulgaris. Combined with the genetic, genomic and phenotypic characteristics, these findings indicate that three species, P. terrae, P. cibarius and Proteus genospecies 5, should be regarded as heterotypic synonyms, and the species should be renamed P. terrae, while Proteus genospecies 5 has not been named to date. Conclusions This study suggested that MLSA is a powerful method for the discrimination and classification of Proteus at the species level. The MLSA scheme provides a rapid and inexpensive means of identifying Proteus strains. The identification of Proteus species determined by the MLSA approach plays an important role in the clinical diagnosis and treatment of Proteus infection.

The Baijiu-making microbiota has an important role in the alcohol production, flavor, and character of Baijiu. 16S rRNA gene sequencing revolutionized the understanding of Baijiu-making microbiota. In this study, nine phyla, 23 classes, 49 orders, 99 families, and 201 genera were detected in pit muds (PMs) by 16S rRNA gene sequencing. Firmicutes and Bacteroidetes predominated (>99%). At the order level, Clostridiales, Bacteroidales, and Bacillales predominated (>92%). At the genus level, Hydrogenispora, Petrimonas, Proteiniphilum, and Sedimentibacter predominated. The pure culture of Baijiu-making prokaryotes was essential to elucidating the role of these microbes in the fermentation of Baijiu. According to the theory of microbial culturomics, a culturing approach with multiple culture conditions was adopted, combining 16S rRNA gene sequencing. We identified 215 prokaryotic strains, which were assigned to 66 species, 41 genera, four phyla, and 19 potential new species. Gas conditions were key factors in culturomics. In addition, culturomics significantly increased the number of species isolated from the fermentation PM compared with previous reports. With culturomics, the diversity spectrum of culturable bacteria in the PM was increased 273.33% at the genus level. This study confirms the complementary role of culturomics in the exploration of complex microbiota.

Vibrio parahaemolyticus is an important cause of foodborne gastroenteritis globally. Thermostable direct haemolysin (TDH) and the TDH-related haemolysin are the two key virulence factors in V. parahaemolyticus. Vibrio pathogenicity islands harbour the genes encoding these two haemolysins. The serotyping of V. parahaemolyticus is based on the combination of O and K antigens. Frequent recombination has been observed in V. parahaemolyticus , including in the genomic regions encoding the O and K antigens. V. parahaemolyticus serotype O4:K12 has caused gastroenteritis outbreaks in the USA and Spain. Recently, outbreaks caused by this serotype of V. parahaemolyticus have been reported in China. However, the relationships among this serotype of V. parahaemolyticus strains isolated in different regions have not been addressed. Here, we investigated the genome variation of the V. parahaemolyticus serotype O4:K12 using the whole-genome sequences of 29 isolates. We determined five distinct lineages in this strain collection. We observed frequent recombination among different lineages. In contrast, little recombination was observed within each individual lineage. We showed that the lineage of this serotype of V. parahaemolyticus isolated in America was different from those isolated in Asia and identified genes that exclusively existed in the strains isolated in America. Pan-genome analysis showed that strain-specific and cluster-specific genes were mostly located in the genomic islands. Pan-genome analysis also showed that the vast majority of the accessory genes in the O4:K12 serotype of V. parahaemolyticus were acquired from within the genus Vibrio . Hence, we have shown that multiple distinct lineages exist in V. parahaemolyticus serotype O4:K12 and have provided more evidence about the gene segregation found in V. parahaemolyticus isolated in different continents.

Vibrio parahaemolyticus is an important cause of foodborne gastroenteritis globally. Thermostable direct haemolysin (TDH) and the TDH-related haemolysin are the two key virulence factors in V. parahaemolyticus. Vibrio pathogenicity islands harbour the genes encoding these two haemolysins. The serotyping of V. parahaemolyticus is based on the combination of O and K antigens. Frequent recombination has been observed in V. parahaemolyticus , including in the genomic regions encoding the O and K antigens. V. parahaemolyticus serotype O4:K12 has caused gastroenteritis outbreaks in the USA and Spain. Recently, outbreaks caused by this serotype of V. parahaemolyticus have been reported in China. However, the relationships among this serotype of V. parahaemolyticus strains isolated in different regions have not been addressed. Here, we investigated the genome variation of the V. parahaemolyticus serotype O4:K12 using the whole-genome sequences of 29 isolates. We determined five distinct lineages in this strain collection. We observed frequent recombination among different lineages. In contrast, little recombination was observed within each individual lineage. We showed that the lineage of this serotype of V. parahaemolyticus isolated in America was different from those isolated in Asia and identified genes that exclusively existed in the strains isolated in America. Pan-genome analysis showed that strain-specific and cluster-specific genes were mostly located in the genomic islands. Pan-genome analysis also showed that the vast majority of the accessory genes in the O4:K12 serotype of V. parahaemolyticus were acquired from within the genus Vibrio . Hence, we have shown that multiple distinct lineages exist in V. parahaemolyticus serotype O4:K12 and have provided more evidence about the gene segregation found in V. parahaemolyticus isolated in different continents.

Huangjiu is one of the Chinese unique and traditional liquor. Corn is a kind of high yield crop in China, which has the characteristics of wide distribution, low price and high starch content. Fermenting Huangjiu with corn not only enrich Huangjiu types, but also opens up a new way for the utilization of corn. The flavor compounds and microorganisms in corn wine fermentation were studied in this study. A total of 98 volatile compounds and 8 kinds of organic acids were detected. Bacillus, Weissella, Streptomyces, Aeromonas and Blautia were the dominant bacteria. The correlation analysis between flavor compounds and bacteria showed that there were 557 correlations between major flavor compounds and bacteria. Among them, Lactococcus, Virgibacillus, Sphingobacterium and Sporolactobacillus were dominant genus of flavor producing bacteria. This study may reveal the changing rule of bacteria in Huangjiu, predict the relationship between metabolites and bacteria. In addition, this study expanded the application of corn and increased the variety of Huangjiu.

The genus Shewanella comprises a group of marine-dwelling species with worldwide distribution. Several species are regarded as causative agents of food spoilage and opportunistic pathogens of human diseases. In this study, a standard multilocus sequence analysis (MLSA) based on six protein-coding genes (gyrA, gyrB, infB, recN, rpoA, and topA) was established as a rapid and accurate identification tool in 59 Shewanella type strains. This method yielded sufficient resolving power in regard to enough informative sites, adequate sequence divergences, and distinct interspecies branches. The stability of phylogenetic topology was supported by high bootstrap values and concordance with different methods. The reliability of the MLSA scheme was further validated by identical phylogenies and high correlations of genomes. The MLSA approach provided a robust system to exhibit evolutionary relationships in the Shewanella genus. The split network tree proposed twelve distinct monophyletic clades with identical G+C contents and high genetic similarities. A total of 86 tested strains were investigated to explore the population biology of the Shewanella genus in China. The most prevalent Shewanella species was Shewanella algae, followed by Shewanella xiamenensis, Shewanella chilikensis, Shewanella indica, Shewanella seohaensis, and Shewanella carassii. The strains frequently isolated from clinical and food samples highlighted the importance of increasing the surveillance of Shewanella species. Based on the combined genetic, genomic, and phenotypic analyses, Shewanella upenei should be considered a synonym of S. algae, and Shewanella pacifica should be reclassified as a synonym of Shewanella japonica. IMPORTANCE The MLSA scheme based on six housekeeping genes (HKGs) (gyrA, gyrB, infB, recN, rpoA, and topA) is well established as a reliable tool for taxonomic, evolutionary, and population diversity analyses of the genus Shewanella in this study. The standard MLSA method allows researchers to make rapid, economical, and precise identification of Shewanella strains. The robust phylogenetic network of MLSA provides profound insight into the evolutionary structure of the genus Shewanella. The population genetics of Shewanella species determined by the MLSA approach plays a pivotal role in clinical diagnosis and routine monitoring. Further studies on remaining species and genomic analysis will enhance a more comprehensive understanding of the microbial systematics, phylogenetic relationships, and ecological status of the genus Shewanella.

The genus Shewanella comprises a group of marine-dwelling species with worldwide distribution. Several species are regarded as causative agents of food spoilage and opportunistic pathogens of human diseases. In this study, a standard multilocus sequence analysis (MLSA) based on six protein-coding genes (gyrA, gyrB, infB, recN, rpoA and topA) was established as a rapid and accurate identification tool in fifty-nine type Shewanella strains. This method yielded sufficient resolving power in regard to enough informative sites, adequate sequence divergences and distinct interspecies branches. The stability of phylogenetic topology was supported by high bootstrap values and concordance with different methods. The reliability of the MLSA scheme was further validated by identical phylogenies and high correlations of genomes. The MLSA approach provided a robust system to exhibit evolutionary relationships in the Shewanella genus. The split network tree proposed twelve distinct monophyletic clades with identical G+C contents and high genetic similarities. Eighty-six tested strains were investigated to explore the population biology of the Shewanella genus in China. The most prevalent Shewanella species were Shewanella algae, Shewanella xiamenensis, Shewanella chilikensis, Shewanella indica, Shewanella seohaensis and Shewanella carassii. The strains frequently isolated from clinical and food samples highlighted the importance of increasing the surveillance of Shewanella species. Combined with the genetic, genomic and phenotypic analyses, Shewanella upenei should be considered a synonym of S. algae, and Shewanella pacifica should be reclassified as a synonym of Shewanella japonica.IMPORTANCEThe MLSA scheme based on six HKGs (gyrA, gyrB, infB, recN, rpoA and topA) is well established as a reliable tool for taxonomic, evolutionary and epidemiological analyses of the genus Shewanella in this study. The standard MLSA method allows researchers to make rapid, economical and precise identification of Shewanella strains. The robust phylogenetic network of MLSA provides profound insight into the evolutionary structure of the genus Shewanella. The population genetics of Shewanella species determined by the MLSA approach plays a pivotal role in clinical diagnosis and routine monitoring. Further studies on remaining species and genomic analysis will enhance a more comprehensive understanding of the microbial systematics, phylogenetic relationships and ecological status of the genus Shewanella.

Chinese rice wine (CRW) is the oldest kind of wine in China and is mainly fermented by wheat Qu and yeast with rice, millet, etc. This gives CRW a unique quality, but the flavor components are complex. Its formation is related to microorganisms, but the link between CRW and microorganisms is poorly understood. Here, we used two kinds of sorghum (JZ22 and JB3, of which JZ22 has a higher tannin content) as the raw materials to brew and determined the structural and functional dynamics of the microbiota by metagenomics and flavor analyses. We detected 106 (JZ22) and 109 (JB3) volatile flavor compounds and 8 organic acids. By correlation analysis, we established 687 (JZ22) and 496 (JB3) correlations between the major flavor compounds and microbes. In JZ22, Blautia, Collinsella, Bifidobacterium, Faecalibacterium and Prevotella had the most correlations with flavor production. In JB3, the top 5 genera were Stenotrophomonas, Bdellovibrio, Solibacillus, Sulfuritalea and Achromobacter. In addition, more esters were detected, and more microorganisms correlated with ester generation in JZ22. This study provides a new idea for the micro ecological diversity of CRW fermented with sorghum. This is of significance for improving the quality and broadening the CRW varieties.

Virus-mediated expression of defined transcription factor (TF) genes can effectively induce cellular reprogramming. However, sustained expression of the TFs often hinders pluripotent stem cell (PSC) differentiation into specific cell types, as each TF exerts its effect on PSCs for a defined period of time during differentiation. Here, we applied a bacterial type III secretion system (T3SS)-based protein delivery tool to directly translocate TFs in the form of protein into human PSCs. This transient protein delivery technique showed high delivery efficiency for hPSCs, and it avoids potential genetic alterations caused by the introduction of transgenes. In an established cardiomyocyte de novo differentiation procedure, five transcriptional factors, namely GATA4, MEF2C, TBX5, ESRRG and MESP1 (abbreviated as GMTEM), were translocated at various time points. By detecting the expression of cardiac marker genes (Nkx2.5 and cTnT), we found that GMTEM proteins delivered on mesoderm stage of the cardiomyocytes lineage differentiation significantly enhanced both the human ESC and iPSC differentiation into cardiomyocytes, while earlier or later delivery diminished the enhancing effect. Furthermore, all of the five factors were required to enhance the cardiac differentiation. This work provides a virus-free strategy of transient transcription factors delivery for directing human stem cell fate without jeopardizing genome integrity, thus safe for biomedical applications.

Background Leptospirosis is one of the most important neglected tropical bacterial diseases worldwide. However, there is limited information on the genetic diversity and host selectivity of pathogenic Leptospira in wild small mammal populations. Methodology/Principal findings Jiangxi Province, located in southern China, is a region highly endemic for leptospirosis. In this study, among a total of 3,531 trapped rodents dominated by Apodemus agrarius (59.7%), 330 Leptospira strains were successfully isolated from six different sites in Jiangxi between 2002 and 2015. Adding 71 local strains from humans, various kinds of livestock and wild animals in Jiangxi, a total of 401 epidemic strains were characterized using 16S rRNA gene senquencing, multilocus sequence typing (MLST) and the microscopic agglutination test (MAT). Among them, the most prevalent serogroup was Icterohaemorrhagiae (61.10%), followed by Javanica (19.20%) and Australis (9.73%); the remaining five serogroups, Canicola, Autumnalis, Grippotyphosa, Hebdomadis and Pomona, accounted for 9.97%. Species identification revealed that 325 were L. interrogans and 76 were L. borgpetersenii. Moreover, L. interrogans was the only pathogenic species in Fuliang and Shanggao and was predominant in Shangrao (95.0%); L. borgpetersenii was the most common in the remaining three sites. Twenty-one sequence types (STs) were identified. Similarly, ST1 and serogroup Icterohaemorrhagiae were most prevalent in Shangrao (86.0% and 86.4%) and Fuliang (90.4% and 90.4%), ST143 and serogroup Javanica in Shangyou (88.5% and 90.4%) and Longnan (73.1% and 73.1%), and ST105 and serogroup Australis in Shanggao (46.3% and 56.1%). Serogroup Icterohaemorhagiae primarily linked to A. agrarius (86.9%), serogroup Canicola to dogs (83.3%). There were significant differences in the distribution of leptospiral species/serogroups/STs prevalence across host species/collected locations among the 394 animal-associated strains (Fisher’s exact test, p, Author summary Leptospirosis, caused by pathogenic Leptospira spp, is one of the most widespread zoonoses. In recent years, human leptospirosis with occasionally fatal infections has been frequently reported in Jiangxi Province, a highly endemic region located in the south of China. However, there is a lack of information on circulating Leptospira strains in this province. To identify the etiological characteristics, 401 Leptospira from Jiangxi were characterized using serological and molecular typing methods. Serological typing revealed that 61.10% of the isolates belonged to serogroup Icterohaemorrhagiae. Two species, L. interrogans and L. borgpetersenii, were identified using 16S rRNA gene sequencing. A. agrarius may be the main carrier of leptospirosis in this endemic region. Furthermore, the diversity of leptospiral isolates was demonstrated using MLST analysis. ST1, as the most prevalent ST of pathogenic leptospires, was widely dispersed in China. Significant geographic variation and host diversity in the distribution of dominant species, serogroups and STs were found in Jiangxi. This study is the first to demonstrate the distribution of Leptospira in domestic and wildlife animals in Jiangxi. This retrospective study represents the longest and largest field epidemiological investigation on the etiological characteristics and genetic diversity of pathogenic Leptospira among large wild animal reservoirs and human populations in Jiangxi. A better understanding of the circulating etiological agents and epidemiology of leptospirosis will provide a good starting point for efforts to control and prevent this disease.