Genomic Characterization Provides New Insights for Detailed Phage- Resistant Mechanism for Brucella abortus.

As the causative agent of cattle brucellosis, Brucella abortus commonly exhibits smooth phenotype (by virtue of colony morphology) that is characteristically sensitive to specific Brucella phages, playing until recently a major role in taxonomical classification of the Brucella species by the phage typing approach. We previously reported the discrepancy between traditional phenotypic typing and MLVA results of a smooth phage-resistant (SPR) strain Bab8416 isolated from a 45-year-old custodial worker with brucellosis in a cattle farm. Here, we performed whole genome sequencing and further obtained a complete genome sequence of strain Bab8416 by a combination of multiple NGS technologies and routine PCR sequencing. The detailed genetic differences between B. abortus SPR Bab8416 and large smooth phage-sensitive (SPS) strains were investigated in a comprehensively comparative genomic study. The large indels between B. abortus SPS strains and Bab8416 showed possible divergence between two evolutionary branches at a far phylogenetic node. Compared to B. abortus SPS strain 9-941 (Bab9-941), the specific re-arrangement event in Bab8416 displaying a closer linear relationship with B. melitensis 16M than other B. abortus strains resulted in the truncation of c-di-GMP synthesis, and 3 c-di-GMP-metabolizing genes, were present in Bab8416 and B. melitensis 16M, but absent in Bab9-941 and other B. abortus strains, indicating potential SPR-associated key determinants and novel molecular mechanisms. Moreover, despite almost completely intact smooth LPS related genes, only one mutated OmpA family protein of Bab8416, functionally related to flagellar and efflux pump, was newly identified. Several point mutations were identified to be Bab8416 specific while a majority of them were verified to be B. abortus ST2 characteristic. In conclusion, our study therefore identifies new SPR-associated factors that could play a role in refining and updating Brucella taxonomic schemes and provides resources for further detailed analysis of mechanism for Brucella phage resistance.

Molecular typing of Leptospira interrogans strains isolated from Rattus tanezumi in Guizhou Province, Southwest of China.

OBJECTIVE: To identify and type three leptospires isolated from Rattus tanezumi in Guizhou Province by using three molecular techniques (PFGE, MLVA, and MLST), reveal the molecular characteristic of causative agents of local leptospirosis and evaluate these three molecular methods based on their detection resolution and efficiency. METHODS: Three Leptospira strains were isolated from the kidney of Rattus tanezumi and cultured with EMJH medium. PFGE, MLVA, and MLST assays were applied to type the three strains isolated from Rattus tanezumi in Guizhou Province. RESULTS: PFGE, MLVA, and MLST typing showed that the three leptospiral isolates matched with leptospiral serogroup Icterohaemorrhagiae serovar Lai. The findings of the genotyping methods were consistent. MLVA and MLST defined genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes, and the findings of molecular typing were also consistent with those of traditional techniques. CONCLUSION: Three leptospiral isolates from Guizhou Province matched with leptospiral serogroup Icterohaemorrhagiae serovar Lai, and PFGE, MLVA, and MLST, as reliable molecular techniques for identifying and typing of Leptospira interrogans, would contribute to the active surveillance, outbreak investigation and source tracking for leptospirosis in Guizhou Province.

[Establishment and application of TaqMan Real-time PCR for the detection of pathogenic Leptospira species].

OBJECTIVE: To develop and evaluate a TaqMan Real-time PCR method for the detection of pathogenic Leptospira species. METHODS: rrs gene of part fragment on 16S rRNA was used to design primers and TaqMan probe. The target gene was cloned into vector pMD19-T in order to make the standard curve and be used for quality control. To determine the specificity and specificity, DNA from Chinese Leptospira strains belonging to 15 pathogenic reference strains, 21 non-pathogenic reference strains, and 50 different serotypes of pathogenic isolates as well as 27 other micro-organisms were included in this study. Eight serial DNA dilutions from pathogenic Leptospira and DNA from 25 kidney tissues were detected by Real-time PCR and conventional PCR simultaneously. RESULTS: A Real-time PCR methodology was developed and optimised. All the pathogenic Leptospira gave a positive amplification. Non-pathogenic Leptospira and all the other micro-organisms were not amplified. The plasmid sensitivity of Real-time PCR and conventional PCR were 10 copy/µl and 10(4)copy/µl respectively. The DNA sensitivity of Real-time PCR and conventional PCR were 100 fg/µl and 1 ng/µl respectively. The kidney tissue detection of the two methods appeared to be exactly the same. CONCLUSION: This research project successfully developed a Real-time PCR methodology with better sensitivity and specificity for the identification of pathogenic Leptospira, using the rrs gene.