Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction （PCR） and capillary electrophoresis （MPCE） assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catorrholis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebactefium diphthefiae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 252 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE with high specificity and sensitivity. This assay survey of respiratory pathogens. assay is a rapid, reliable, and high-throughput method has great potential in the molecular epidemiological.

Legionella dumoffii Tex-KL Mutated in an Operon Homologous to traC-traD is Defective in Epithelial Cell Invasion

Objective To understand the mechanism of invasion by Legionella dumoffii. Methods The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903 d IIlac Z. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in He La and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR. Results The transposon insertion was in a gene homologous to Salmonella typhi tra C, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the tra C gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a tra C deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain. Conclusion Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.

Isolation and Characterization of Vagococcus fluvialis from Bats

The genus Vagococcus was first described by Collinsetal.and initially consisted of a single species,V.fluvialis.This species was isolated from chicken feces and river water and first described by Hashimoto et al.[1,2].Teixeira et al.isolated V.fluvialis from human blood and peritoneal fluid,suggesting that it poses a potential threat to human health[3].

Phylogenetic Analysis of Legionella Strains and Identification of Serogroups by Lipopolysaccharide-and O-antigen-based PCR Assay

Legionella,a genus of pathogenic Gram-negative bacteria,is widely present in natural water sources and artificial water systems.A total of 65 species and>70 serogroups of Legionella have been characterized[1].Lipopolysaccharide(LPS),the main component of the outer membrane of Legionella,is not only associated with toxicity,but also provides the basis for classification in serotyping[2].