Pontibacter harenae sp. nov., isolated from the soil of a Populus euphratica forest.

A Gram-negative, rod-shaped, aerobic and light pink-pigmented bacterium, designated XAAS-A31T, was isolated from the soil of a Populus euphratica forest located near Hotan City, Xinjiang, PR China. Polyphasic, taxonomic and phylogenomic analyses were used to determine the taxonomy position of the strain. Phylogenetic analysis based on 16S rRNA gene sequence analysis indicated that XAAS-A31T belongs to the genus Pontibacter, family Hymenobacteraceae, and shows highest sequence similarity to Pontibacter silvestris XAAS-R86T (96.2 %). The digital DNA-DNA hybridization (22.0 %-19.2 %) and orthologous average nucleotide identity (74.1 %-72.7 %) values relative to closest validly published Pontibacter species were lower than the recommended thresholds of 70 and 96 %, respectively. The cells grew at 4-40 °C (optimum, 28-30 °C), at pH 6.5-8.5 (pH 7.0-7.5) and with 0-8% NaCl (0.5-2.0% NaCl). The main respiratory quinone of XAAS-A31T is MK-7, and the principal cellular fatty acids are iso-C15 : 0, iso-C17 : 0 3OH and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B). The major polar lipids are phosphatidylethanolamine, three unidentified amino-phospholipids, one unidentified glycolipid and eight unidentified lipids. The genome length of strain XAAS-A31T is 5.48 Mbp with a DNA G+C content of 44.2 mol% and 4013 protein-coding genes. Phenotypic and genotypic data suggested that XAAS-A31T represents a novel Pontibacter species, for which we propose the name Pontibacter harenae sp. nov. and type strain XAAS-A31T (=CCTCC AB 2017162T=KCTC 62049T).

Screening and Identification of a Streptomyces Strain with Quorum-Sensing Inhibitory Activity and Effect of the Crude Extracts on Virulence Factors of Pseudomonas aeruginosa.

Quorum-sensing (QS) is involved in numerous physiological processes in bacteria, such as biofilm formation, sporulation, and virulence formation. Therefore, the search for new quorum-sensing inhibitors (QSI) is a promising strategy that opens up a new perspective for controlling QS-mediated bacterial pathogens. To explore new QSIs, a strain named Streptomyces sp. D67 with QS inhibitory activity was isolated from the soil of the arid zone around the Kumutag Desert in Xinjiang. Phylogenetic analyses demonstrated that strain D67 shared the highest similarity with Streptomyces ardesiacus NBRC 15402T (98.39%), which indicated it represented a potential novel species in the Streptomyces genus. The fermentation crude extracts of strain D67 can effectively reduce the violacein production produced by Chromobacterium violaceum CV026 and the swarming and swimming abilities of Pseudomonas aeruginosa. It also has significant inhibitory activity on the production of virulence factors such as biofilm, pyocyanin, and rhamnolipids of P. aeruginosa in a significant concentration-dependent manner, but not on protease activity. A total of 618 compounds were identified from the fermentation crude extracts of strain D67 by LC-MS, and 19 compounds with significant QS inhibitory activity were observed. Overall, the strain with QS inhibitory activity was screened from Kumutag Desert in Xinjiang for the first time, which provided a basis for further research and development of new QSI.

Bacterial Community Composition and Isolation of Actinobacteria from the Soil of Flaming Mountain in Xinjiang, China.

In this work, bacterial community composition and actinobacteria resources were explored in extremely hot and hyper-arid areas of Flaming Mountain. This was achieved through a combination of PCR amplicon sequencing of bacterial 16S rRNA gene and cultivation-dependent isolation and characterization efforts. According to the high-throughput sequencing results and soil characteristics, 11 kinds of media were firstly designed to isolate actinobacteria, following the screening and identification of related strains. The results showed that a total of 2994 operational taxonomic units (OTUs) were obtained, involving 22 phyla, 77 orders and 121 genera. Among them, actinobacteria with the relative abundance of 8% ranked third, accounting for 33 genera and 47 species. A total of 132 strains distributed by eight families and 11 genera of actinobacteria were isolated from 11 media, of which six strains were potential new species. Furthermore, the functional characteristics of isolated strains were preliminarily evaluated. The results showed that the obtained strains generally had tolerance against heat, salt and alkali. Fifty-two strains had antibacterial activity, 69 strains could produce hydrolases, and 12.4% of the strains had quorum sensing inhibitory activity. The present study has laid a solid foundation for further understanding the bacterial diversity and exploiting actinobacteria resources in the Flaming Mountain area.

A comparative investigation on the role and interaction of EsxA and EsxB in host immune response.

Staphylococcus aureus (S. aureus) is a frequent and major cause of bovine mastitis; it poses a tremendous economic burden to dairy industries of numerous countries. Early-secretion antigen-6 secretion system (ESS) has been viewed as an essential virulence and pathogenic factor of S. aureus. EsxA and EsxB are small acidic proteins secreted by ESS and identified as potential T-cell antigens of S. aureus. Unlike those of Mycobacterium tuberculosis (M. tuberculosis), the EsxA and EsxB of S. aureus do not form a dimer. Instead, EsxA dimerizes with itself or EsaC. Therefore, the interaction of EsxA and EsxB remains incompletely understood. In this study, to explore their interactions, EsxA and EsxB were expressed and used for immunization, alone or in combination, of murine infection models. Both components can interact with each other. Through the analysis of the immune response by immunological method, EsxB could significantly enhance the EsxA-specific IgG2a antibody level and increase the proliferation proportion of CD8+ T cells. These results indicate that when vaccinated with EsxA, EsxB can play a critical role in stimulating T helper 1 immunity by activating IgG2a and CD8+ T cells. We further show that vaccination with the combination of EsxA and EsxB resulted in enhanced stimulation of TLR-4 and improved protection against S. aureus. The findings may help us better understand the role of EsxB in the virulence and pathogenesis of S. aureus.

[Comparison of immunogenicity between recombinant flagellins C and B of Salmonella abortus equi].

To evaluate and compare of the immunogenicity differences of flagellins FliC and FljB of Salmonella abortus equi, and lay the experimental foundation for the further utilization of the two recombinant proteins, FliC and FljB recombinant proteins were induced, expressed and purified. The purified FliC and FljB were used to immunize mice separately. The antibody level, titer and subtype of mice serum were detected after immunization. Immune-related receptors and histopathological changes were observed in immunized mice after challenged. The recombinant proteins FliC and FljB were successfully induced and expressed. Proteins of about 52 kDa and 42 kDa were purified. High levels of specific IgG antibodies were induced in mice immunized with these two proteins, the antibody level of FljB-immunized group was higher than that of FliC-immunized group, and IgG1 was the dominant subtype of antibody. The challenge protection rate of the FljB-immunized group was 87.5%, higher than that of FliC immunized group. Bacterial loads and observation pathological of FljB-immunized group were better than that of FliC immunized group, the levels of TCR2, TCR4, MHC-I and TCR induced by FljB-immunized group were higher than those of FliC-immunized group. The of immune response induced by FljB group was better than that of FliC group.

Analysis of the Genetic Diversity in Methicillin-Resistant Staphylococcus aureus Isolates from Bovine Subclinical Mastitis Case in Xinjiang, China.

BACKGROUND: Diseases caused by livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) are an important global public health concern, and MRSA is increasingly being isolated in bovine milk. However, information on the genotype and antimicrobial resistance of MRSA in bovine milk in Xinjiang is limited. The objective of this study was to determine the antimicrobial-susceptible phenotypes and genotypes of the circulating MRSA clone isolated in bovine mastits milk samples in Xinjiang, China. METHODS: Fifty six MRSA isolates collected from milk of bovine mastitis were investigated by multilocus sequence typing (MLST), staphylococcal protein A (spa) typing, and a minimum inhibitory concentration test with 21 antimicrobial agents. RESULTS: Antibiotic resistance results showed that 47.4% of the isolates were resistant to 16 or more antibiotics. Twelve MLST types were defined in this study, and ST398 (n = 7) and ST2393 (n = 2) were found to be the most prevalent types. Seven spa types (t034, t269, t4030, t114, t35, t189, and t7589) were identified, of which t034 (n = 7), t189 (n = 3), and t4030 (n = 3) were predominant. Here, 3 MRSA ST188 is reported among human MRSA isolates in China, and this is the first time that it is reported in bovine MRSA strains. CONCLUSIONS: The antimicrobial susceptibility of MRSA in this area exhibited multidrug resistance, and clonal complexes CC398 and CC188, which have been reported among human MRSA isolates, do occur in Xinjiang dairy cows. This study provides a foundation for further MRSA monitoring.

[Expression and immunogenicity analysis of EsxA protein of Staphylococcus aureus isolated from cow milk].

To study the immunogenicity of EsxA protein of Staphylococcus aureus, the EsxA-pET-28a recombinant plasmid was constructed and the expression product was analyzed by SDS-PAGE and Western blotting after the positive recombinant plasmid was induced by IPTG. Mice were immunized with purified EsxA protein and then the IgG, IgG1 and IgG2a antibody were detected with indirect ELISA. Then the histopathological examination, bacteria loading and immune protection of immunized mice were studied after challenge with S. aureus. The recombinant protein EsxA was successfully induced and expressed. After immunization the EsxA specific antibody titer could reach 1:900. Bacteria loading and pathological damage of liver, spleen and kidney were reduced after immunization with EsxA in the immunized mice. The protection rate of immunized mice was 75%. In conclusion, EsxA protein has good immunogenicity.