Beta-lactamase and integron-associated antibiotic resistance genes of Klebsiella pneumoniae isolated from Tilapia fishes (Oreochromis niloticus)

Tilapia fishes (Oreochromis niloticus) are commonly consumed and exported in Thailand. Bacterial isolation anddrug resistance from farmed tilapia fished in Thailand were previously reported. This study was purposed to studyon the distribution of human pathogenic bacteria in tilapia fishes, which were collected from Thai farms (n = 180)and fresh markets (n = 160) by identification, antibiotic susceptibility test; and conduct to identify virulence genesby molecular technique. Pathogen isolations were collected from internal organs of fish samples for identificationand test of antibiotic susceptibility according to Clinical and Laboratory Standards Institute (CLSI) criteria. blaCTX-Mand Int1 genes detection of antibiotic resistance bacteria was performed by molecular based techniques. Klebsiellapneumoniae, Edwardsiella tarda, and coagulase-negative Staphylococci were most frequent bacteria isolated fromfarming tilapia fishes, respectively. However, Escherichia coli, coagulase-negative Staphylococci, and K. pneumoniawere frequently distributed from tilapia fishes in markets of Bangkok area. Klebsiella pneumoniae, E. coli, and Proteusmirabilis were resisted to penicillin and ampicillin. Klebsiella pneumoniae is the most important isolated bacteria dueto the distribution in tilapia fishes and positive for blaCTX-M and Int1 gene detection. However, E. coli and P. mirabiliswere lack of blaCTX-M and Int1 genes, possibly there may reserve other antibiotic resistance genes

Analysis of related factors for long-term results and prognosis of personalized treatment in T790M-positive lung adenocarcinoma patients with bone metastasis / 中国肿瘤生物治疗杂志

@# Objective: To explore the related factors for efficacy and prognosis of personalized comprehensive treatment for T790Mpositive lung adenocarcinoma patients with bone metastasis. Methods: The clinical data of 68 patients undergoing personalized comprehensive treatment for T790M-positive lung adenocarcinoma with bone metastasis were retrospectively reviewed; chemotherapy, radiotherapy, molecule-targeted agents, Bevacizumab, bisphosphonate and other therapies were chosen for the patients, and the efficacy and prognosis were observed to explore the related factors. Results: Effective rate of personalized comprehensive treatment was 60.3% (41/ 68), with a median survival time of 23 months. Multiple factors showed significant effects on long-term efficacy, such as no radiotherapy, T790M mutation but no KRAS mutation, adjuvant scheme+rescue scheme in prior chemotherapy treatment, N1 stage, isolated bone metastasis, alternative treatment of osimertinib with chemotherapy, less metastasized organs and ECOG scores<2 (P<0.05). Multivariate analysis revealed that T790M mutation but no KRAS mutation (P=0.012), number of metastasized organs =0 or 1 (P=0.000), alternative treatment of osimertinib with chemotherapy (P=0.020), and isolated bone metastasis (P=0.006) were independent protective factors for long-term results of personalized comprehensive treatment for T790M-positive lung adenocarcinoma patients with bone metastasis. Conclusion: Chemotherapy combined with osimertinib, agents of bisphosphonate and other personalized comprehensive treatment prolongs survival time in T790M-positive lung adenocarcinoma patients without KRAS mutation, providing a potential therapeutic model for those patients.

M gene analysis of canine coronavirus strains detected in Korea

The purpose of this study was to investigate the genetic features of canine coronavirus (CCV) strains detected in Korea. M gene sequences obtained for isolates from 22 dogs with enteritis over a 5-year period were evaluated. Sequence comparison revealed that the 22 Korean CCV strains had an 87.2 to 100% nucleotide homology. Comparing to the typical reference CCV strains (type II), the nucleotide sequence of Korean strains had homology ranged from 86.3% to 98.3% (89.1% to 99.2% for the amino acid sequence) and 87.7% to 97.8% (92.4% to 100% for the amino acid sequence) when compared to FCoV-like CCV strains (type I). Three amino acid variations in the M gene were characteristic for the Korean CCV strains. Phylogenetic analysis demonstrated that the 22 Korean CCV strains belonged to four typical CCV clusters (i.e., a unique Korean CCV cluster, a type II and transmissible gastroenteritis virus cluster, an intermediate cluster between type I and II, and a type I cluster). This study was the first to identify genetic differences of the M gene from Korean CCV strains and provided a platform for molecular identification of different Korean CCV strains.

A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples / 中国病毒学·英文版

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit(R) for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR.The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50.The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg.Based on the serial dilution of the live-attenuated PPR vaccine virus,the detection limit was ～0.0001 cell culture infectious dose 50％ units (TCID50).Additionally,swab materials spiked with known titre of vaccine virus were equally well detected in the assay.The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples.The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples.The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats.Therefore,the established,simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

Molecular evolutionary analysis of matrix protein and nucleoprotein genes of novel influenza virus A/H1N1 in 2009 pandemic / 第二军医大学学报

Objective: To analyze evolutionary characteristics of the matrix protein (M) and nucleoprotein (NP) genes of influenza virus A/H1N1 in 2009 pandemic. Methods: The M and NP genes of A/H1N1 viruses were downloaded from NCBI database. MEGA4.0 software and NJ method were used for sequence alignment, protein sequence alignment, and the phylogenetic tree construction. Meanwhile, Epi Info software was used to analyze the linear trend of evolutionary distance of the M and NP genes of human H1N1 strains isolated during 1918 to 2009. Results: The M and NP gene sequences were similar among the novel A/H1N1 viruses, but different from those of the previous influenza H1N1 viruses. Using reference sequences of human H1N1 strains isolated during 1918 to 2008, we found that changes in evolutionary distances of the M genes between novel A/H1N1 strains and each of the reference A/H1N1 strains increased with increasing year intervals (Ptrend = 0.001). Compared with the amino acid sequence of M2 protein of reference human A/H1N1 virus strains isolated during 1918 to 2008, the novel A/H1N1 viruses had the amino acid substitutions at 6 sites: 11, 43, 54, 57, 77, and 78. Compared with swine and avian A/H1N1, the novel A/H1N1 virus only had the amino acid substitutions at 43 and 77. Conclusion: The NP gene of novel A/H1N1 virus, which is routinely considered as a conserved sequence, is different from those of the previously isolated human H1N1 influenza viruses; the related mechanisms and consequences on viral activity remain to be elucidated. The substitution to threonine at 11 and 43 amino acids of M2 protein might contribute to amantadine resistance of the novel H1N1 virus pandemic in 2009.

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines / 中国病毒学

The M and NP genes of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA,and cloned into pMD 18-T vector respectively.The expression plasmid containing the M gene (pHM6-m) or the NP gene (pHM6-np) was then constructed by inserting the M or NP gene into the pHM6 eukaryote expression vector; the constructed plasmid was then sequenced.32 BALB/c mice (6-week-old) were divided into four groups at random.Three groups of BALB/c mice were inoculated one time the intramuscular route with either 30 μg of plasmid pHM6-m,30 μg of plasmid pHM6-np or the mixture of plasmid pHM6-m (15 μg ) and pHM6-np(15 μg) respectively.A additional group of mice were injected with 100 μ1 PBS as controls.Two weeks later,all mice were challenged with homologous H5N1 avian influenza virus,and observed in the following 12 days.The survival rates of mice in the pHM6-m group,the pHM6-np group and mixed plasmids group were 62.5% ,25.0% and 50.0%,respectively.Results showed that effective protection could be provided by either pHM6-m or pHM6-np,but pHM6-m provided a better protective effect than pHM6-np.