Prevalence and evolution of drug resistance HIV-1 variants in Henan, China

To understand the prevalence and evolution of drug resistant HIV strains in Henan China after the implementation of free antiretroviral therapy for AIDS patients. 45 drug naive AIDS patients, 118 AIDS patients who received three months antiretroviral therapy and 124 AIDS patients who received six months antiretroviral treatment were recruited in the southern part of Henan province. Information on general condition, antiretroviral medicines, adherence and clinical syndromes were collected by face to face interview. Meanwhile, 14ml EDTA anticoagulant blood was drawn. CD4/CD8 T cell count, viral load and genotypic drug resistance were tested. The rates of clinical improvement were 55.1% and 50.8% respectively three months and six months after antiretroviral therapy. The mean CD4 cell count after antiretroviral therapy was significantly higher than in drug naive patients. The prevalence rate of drug resistant HIV strains were 13.9%, 45.4% and 62.7% in drug naive patients, three month treatment patients and six month treatment patients, respectively. The number of resistance mutation codons and the frequency of mutations increased significantly with continued antiretroviral therapy. The mutation sites were primarily at the 103, 106 and 215 codons in the three-month treatment group and they increased to 15 codon mutations in the six-month treatment group. From this result, the evolution of drug resistant strains was inferred to begin with the high level NNRTI resistant strain, and then develop low level resistant strains to NRTIs. The HIV strains with high level resistance to NVP and low level resistance to AZT and DDI were highly prevalent because of the AZT＋DDI＋NVP combination therapy. These HIV strains were also cross resistant to DLV, EFV, DDC and D4T. Poor adherence to therapy was believed to be the main reason for the emergence and prevalence of drug resistant HIV strains. The prevalence of drug resistant HIV strains was increased with the continuation of antiretroviral therapy in the southern part of Henan province. Measures, that could promote high level adherence, provide new drugs and change ART regimens in failing patients, should be implemented as soon as possible.     

Diversity and functional analysis of Chinese bumblebee gut microbiota reveal the metabolic niche and antibiotic resistance variation of Gilliamella

Bumblebees play an important role in maintaining the balance of natural and agricultural ecosystems,and the characteristic gut microbiota of bumblebees exhibit significant mutualistic functions.China has the highest diversity of bumblebees;however,gut microbiota of Chinese bumblebees have mostly been investigated through cultureindependent studies.Here,we analyzed the gut communities of bumblebees from Sichuan,Yunnan,and Shaanxi provinces in China through 16S ribosomal RNA amplicon sequencing and bacterial isolation.It revealed that the bumblebees examined in this study harbored two gut enterotypes as previously reported:one is dominated by Gilliamella and Snodgrassella,and the other is distinguished by prevalent environmental species.The gut compositions obviously varied among different individual bees.We then isolated 325 bacterial strains and the comparative genomic analysis of Gillianiella strains revealed that galactose and pectin digestion pathways were conserved in strains from bumblebees,while genes for the utilization of arabinose,mannose,xylose,and rhamnose were mostly lost.Only two strains from the Chinese bumblebees possess the multidrug-resistant gene emrB,which is phylogenetically closely related to that from the symbionts of soil entomopathogenic nematode.In contrast,tetracycline-resistant genes were uniquely present in three strains from the USA.Our results illustrate the prevalence of strain-level variations in the metabolic potentials and the distributions of antibiotic-resistant genes in Chinese bumblebee gut bacteria.     

Immune Responses and Protective Efficacy Induced by 85B Antigen and Early Secreted Antigenic Target-6 kDa Antigen Fusion Protein Secreted by Recombinant Bacille Calmette-Guérin

In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin （rBCG） strains expressing an 85B antigen （Ag85B） and early secreted antigenic target-6 kDa antigen （ESAT6） of Mycobacterium tuberculosis （MTB） fusion protein. Both rBCG strains have the same protein insertion but in a different order （Ag85B-ESAT6 and ESAT6-Ag85B）. The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B- ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The T-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group （P〈0.05）. Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups. Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups （p〉0.05）.     

Three metaltolerance Halobacillus sp. isolated from salt lakes in Shaanxi, China

Three Gram positive, spore forming, halophilic bacterial strains were isolated from a salt lake in shanxi China and subjected to a polyphasic taxonomic study. Strains A375, A381 and A389 grew in the presence of 0~24% (w/v) NaCl in complex medium. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains A375, A381 and A389 were located in the genus Halobacillus. Levels of 16S rRNA gene sequence similarity between the isolated strains and the type trains of Halobacillus species were in range 91.6%~98.9%. On the basis of phenotypic and chemotaxonomic properties and phylogenetic analysis, strains A375, A381 and A389 should be placed in the Halobacillus species. Metaltolerance test verify that all strains can tolerate different levels of metal salts.     

Loss of heterozygosity by SCRaMbLEing

Genetic variation drives phenotypic evolution within populations. Genetic variation can be divided into different forms according to the size of genomic changes. However, study of large-scale genomic variation such as structural variation and aneuploidy is still limited and mainly based on the static, predetermined feature of individual genomes. Here, using SCRaMbLE,different levels of loss of heterozygosity(LOH) events including short-range LOH, long-range LOH and whole chromosome LOH were detected in evolved strains. By contrast, using rapid adaptive evolution, aneuploidy was detected in the adaptive strains. It was further found that deletion of gene GLN3, long-range LOH in the left arm of synthetic chromosome Ⅹ, whole chromosome LOH of synthetic chromosome Ⅹ, and duplication of chromosome Ⅷ(trisomy) lead to increased rapamycin resistance in synthetic yeast. Comparative analysis of genome stability of evolved strains indicates that the aneuploid strain has a higher frequency of degeneration than the SCRaMbLEd strain. These findings enrich our understanding of genetic mechanism of rapamycin resistance in yeast, and provide valuable insights into yeast genome architecture and function.     

Mining Genomic Patterns in Mycobacterium tuberculosis H37Rv Using a Web Server Tuber-Gene

Mycobacterium tuberculosis (MTB), causative agent of tuberculosis, is one of the most dreaded diseases of the century. It has long been studied by researchers throughout the world using various wet-lab and dry-lab techniques. In this study, we focus on mining useful patterns at genomic level that can be applied for in silico functional characterization of genes from the MTB complex. The model developed on the basis of the patterns found in this study can correctly identify 99.77% of the input genes from the genome of MTB strain H37Rv. The model was tested against four other MTB strains and the homologue M. bovis to further evaluate its generalization capability. The mean prediction accuracy was 85.76%. It was also observed that the GC content remained fairly constant throughout the genome, implicating the absence of any pathogenicity island transferred from other organisms. This study reveals that dinucleotide composition is an efficient functional class discriminator for MTB complex. To facilitate the application of this model, a web server Tuber-Gene has been developed, which can be freely ac- cessed at http://www.bifmanit.org/tb2/.     

Exploring Mycobacterium tuberculosis infection-induced alterations in gene expression in macrophage by microarray hybridization

Tuberculosis remains a major global threat to human health, with 8 million cases of clinical tuberculosis and 3 million deaths annually[1] (www.stoptb.org/tuberculosis/#facts.html). Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. The emergence of multi-drug resistant strains of Mtb and co-infection with the human immunodeficiency virus (HIV) have further emphasized the need for effective prevention and treatment of tuberculosis. Mtb is a facultative intracellular pat…     

The Distribution of Different Clades of Seneca Valley Viruses in Guangdong Province,China

Seneca Valley virus (SVV), a newly determined etiological agent of vesicular disease in swine, causes porcine idiopathic disease and occasional acute death in piglets. Recently, an increased number of SVV infection cases have been reported in the United States (US) and China, resulting in significant economic losses to the swine industry. The first identification of SVV in China was reported in Guangdong Province, a major swine producing province. The cases of SVV were continuously reported in Guangdong in 2015 and 2016. However, the spread of SVV in Guangdong in 2017 remains unknown.In this study, we determined two new SVV strains, CH-GD-2017-1 and CH-GD-2017-2, from Guangdong. The genetic analysis suggested that the two Guangdong strains showed different characteristics to previous Guangdong strains. They showed lower nucleotide similarity with strains isolated in 2015 and 2016, and were more similar to the US strains.Phylogenetic analyses indicated that the new strains were clustered in a different clade with previous Guangdong strains.We found 28 mutated amino acids in the new strains, compared with the first Guangdong strain, SVV CH-01-2015. In the geographic analysis, we found that the US and China reported more SVV cases than other countries, and most of the SVV cases were reported in east and central China—of which, Guangdong Province is one of the major epidemic regions. In conclusion, our findings indicate that SVV continued to spread in Guangdong Province in 2017, and two different clades of SVVs have emerged in this region.     

Effect of mixed infection on TB dynamics

Poor living conditions,overcrowding and strain diversity are some of the factors that influence mixed infection in Tuberculosis (TB) at the population level.We formulate a mathematical model for mixed infection in TB using nonlinear ordinary differential equa-tions where such factors were represented as probabilities of acquiring mixed infection.A qualitative analysis of the model shows that it exhibits multiple endemic equilibria and backward bifurcation for certain parameter values.The reactivation rate and trans-mission rate of individuals with mixed infection were of importance as well as the prob-abilities for latent individuals to acquire mixed infection.We calculate the prevalence of mixed infection from the model and the effect of mixed infection on TB incidence,TB prevalence and Mycobacterium tuberculosis (MTB) infection rate.Numerical simu-lations show that mixed infection may explain high TB incidences in areas which have a high strain diversity,poor living conditions and are overcrowded even without HIV.     

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c（Hsp70359–610） fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70（mHsp70） is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte（CTL） response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2（M2e）, was fused to the C-terminus of Mycobacterium tuberculosis Hsp70（Hsp70c）, to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c（Hsp70359–610）. And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.     

痰涂片标本用于结核分枝杆菌耐药性检测的研究

目的 研究抗酸染色结核分枝杆菌(简称结核杆菌)阳性痰涂片标本直接用于耐药性检测的方法。方法 对18株临床分离培养的结核杆菌用利福平进行药敏试验。分别提取菌株DNA和与之对应的痰涂片标本的菌体DNA,用聚合酶链反应(PcR)扩增ropB基因后进行固相杂交和核酸测序检测结核杆菌的耐药性。结果 18株结核杆菌中有12株对利福平耐药。经PCR扩增的ropB片段与探针杂交后,敏感菌株未发现rpoB基因的突变,自耐药菌株提取的DNA中rpoB突变体的检出率为100％(12／12),痰涂片提取DNA的检出率为91．7％(11／12)。所有耐药菌株DNA与痰涂片DNA核酸测序结果相吻合,都有rpoB基因核心区域碱基突变。结论 抗酸染色痰涂片阳性标本可直接用于检测结核杆菌利福平耐药基因rpoB突变体,是一种值得临床实验室推广使用的耐药菌诊断方法。     

结核分枝杆菌rpoB基因突变的检测(简报)

结核病主要是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种慢性传染性疾病。利福平是结核病化疗方案中一个关键性的药物,它在结核病的短程化疗中起着重要的作用。但是,在我国结核菌对利福平的耐药发生率呈上升局势,而通过传统的依赖生物生长的药敏试验方法进行结核菌对利福平耐药性检测所需时间较长(4-8周),不能满足临床早期开展有效化疗的需要,所以迫切需要建     

应用专一性寡核苷酸微阵列可靠地检测结核分枝杆菌利福平耐药性

DNA微阵列代表聚合酶链反应产物诊断测序的发展方向 .根据结核分枝杆菌rpoB基因利福平抗药性决定区域内点突变及其它重排的特征 .研制一种快速地鉴定结核分枝杆菌利福平耐药菌株的中等密度微阵列方法 .利福平抗药性通过使荧光标记扩增遗传物质与微阵列杂交测定 .检测5 3株利福平耐药结核分枝杆菌和 15株利福平敏感结核分枝杆菌 .微阵列方法的检测结果与药物敏感性试验和DNA测序结果完全一致 .临床标本PCR扩增后仅 1 5h可检出利福平耐药临床分离株 .表明寡核苷酸微阵列是高效的、专一性的方法 ,可作为检测利福平抗药性的快速方法以弥补传统培养方法的不足     

中国耐多药结核分枝杆菌临床分离株rpoB基因突变特点

为阐明中国耐多药结核分枝杆菌临床分离株rpoB基因的突变特征,对86株结核分枝杆菌临床分离株rpoB基因两个区域,包括81个碱基利福平抗药性决定区(rifampin resistance determining region,RRDR)和V176F区进行序列测定。其中72株耐多药分离株中的65株rpoB基因的RRDR区存在22种不同类型突变、21种点突变和一个插入突变。最常见的突变部位分别位于密码子531(41％)、526(40％)和516(4％),10％耐药分离株未检测到突变。鉴定了RRDR内6个新的等位基因,以及RRDR外部区域5个新的突变。所有分离菌株V176均无突变。     

Use of multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis in Panama

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.     

结核分枝杆菌乙胺丁醇耐药机制的研究进展

耐多药结核病的出现和流行对结核病的防控造成了严重威胁。乙胺丁醇(Ethambutol, EMB)是一线抗结核药物,常与异烟肼、利福平等联合应用,还可用于耐药结核病的治疗。但近年来EMB耐药形势严峻,我国复治结核病患者中EMB耐药率已达17.2%,并呈上升趋势;耐多药结核病患者中,EMB耐药率约为51.3%~66.7%,情况不容乐观。明确EMB耐药的产生机制对于有效防控EMB耐药率的上升、充分发挥EMB的作用十分重要,因此本文对结核分枝杆菌EMB的耐药现状、EMB的作用机制及其耐药产生机制方面的研究进展进行了综述。     

A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis

Prompt detection of drug resistance in Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a Multi-PCR-SSCP method that detects more than 80% commonly observed isoniazid (INH) and rifampin (RIF) resistance M. tuberculosis in a single assay. The usefulness of the newly developed method was evaluated with 116 clinical isolates of M. tuberculosis. Distinct SSCP patterns were observed for different mutations and the correlation between Multi-PCR-SSCP results and DNA sequencing data was strong. Using the culture-based phenotypic drug susceptibility testing as a reference, the sensitivity of the newly developed Multi-PCR-SSCP assay was determined to be 80% and 81.8% for INH and RIF, respectively. The specificity of the assay was 100% and 92%, for INH and RIF, respectively. Multi-PCR-SSCP provides a rapid and potentially more cost-effective method of detecting multidrug-resistant TB.     

At Baltic crossroads: a molecular snapshot of Mycobacterium tuberculosis population diversity in Kaliningrad, Russia

The Kaliningrad region is the westernmost part of the Russian Federation; it includes an enclave on the Baltic Sea inside the European Union separated from mainland Russia by Lithuania and Poland. The incidence of tuberculosis in Kaliningrad has shown a steady and dramatic increase from 83/100 000 in 2000 to 134/100 000 in 2006; the rate of multidrug-resistant tuberculosis (MDR-tuberculosis) in the Kaliningrad region was reported to be 30.5% among newly diagnosed tuberculosis patients. This study presents a first molecular snapshot of the population diversity of Mycobacterium tuberculosis in this region. A total of 90 drug-resistant and susceptible M. tuberculosis strains from Kaliningrad were subjected to spoligotyping, 12-locus MIRU typing and mutation analysis of the drug resistance genes rpoB and katG . A comparison with international databases showed that the M. tuberculosis population in this region shares a joint pool of strains with the European part of Russia, and also exhibits a certain affinity with those of its northern European neighbours, such as Poland and Germany. Comparison of the genotyping and drug resistance data emphasized that the high prevalence of the MDR Beijing genotype strains is a major cause of the adverse epidemiological situation of MDR-tuberculosis in the Kaliningrad region.     

Simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by polymerase chain reaction-single strand conformation polymorphism and sequence analysis of the RNA polymerase gene (rpoB)

Interspecies variations and mutations associated with rifampin resistance in rpoB of Mycobacterium allow for the simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by PCR-SSCP analysis and PCR- sequencing. One hundred and ten strains of rifampin-susceptible M. tuberculosis, 14 strains of rifampin-resistant M. tuberculosis, and four strains of the M. avium complex were easily identified by PCR-SSCP. Of another seven strains, which showed unique SSCP patterns, three were identified as rifampin-resistant M. tuberculosis and four as M. terrae complex by subsequent sequence analysis of their rpoB DNAs (306 bp). These results were concordant with those obtained by susceptibility testing, biochemical identification, and 16S rDNA sequencing.     

广泛耐药结核分枝杆菌耐药机制及其疾病诊断的研究进展

自20世纪90年代以来,全球结核病疫情回升,结核分枝杆菌耐药是其中的一个重要原因.广泛耐药结核病是指在耐多药结核病(即同时对异烟肼和利福平耐药的结核分枝杆菌引起的结核病)的基础上,还对氟喹诺酮类药物和至少3种二线静脉用抗结核药物(卷曲霉素、卡那霉素、阿米卡星)中的1种耐药的结核分枝杆菌引起的结核病.我国是结核病高流行国...