Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage K8 and Its Genomic Analysis

Phage therapy requires the comprehensive understanding of the mechanisms underlying the host-phage interactions. In this work, to identify the genes related to Pseudomonas aeruginosa phage K8 receptor synthesis, 16 phage-resistant mutants were selected from a Tn5G transposon mutant library of strain PAK. The disrupted genetic loci were identified and they were related to O-specific antigen (OSA) synthesis, including gene wbpR, ssg, wbpV, wbpO, and Y880_RS05480, which encoded a putative O-antigen polymerase Wzy. The LPS profile of the Y880_RS05480 mutant was analyzed and shown to lack the O-antigen. Therefore, the data from characterization of Y880_RS05480 by TMHMM and SDS-PAGE silver staining analysis suggest that this locus might encode Wzy. The complete phage K8 genome was characterized as 93879 bp in length and contained identical 1188-bp terminal direct repeats. Comparative genomic analysis showed that phage K8 was highly homologous to members of the genus PaP1-like phages. On the basis of our genetic findings, OSA of P. aeruginosa PAK is proven to be the receptor of phage K8. The highly conserved structural proteins among the genetic closely related phages suggest that they may recognize the same receptor

Multi-Fluorescence Real-Time PCR Assay for Detection of RIF & INH Resistance of M. tuberculosis

Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH)Methods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with 4 kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC and rrs. The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay.Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6% and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9% and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2% and 100% for RIF-resistance and 97.9% and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 Mycobacterium tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate.Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates