Comparing the Genotype and Drug Susceptibilities between Mycobacterium avium and Mycobacterium intracellulare in China.

OBJECTIVE: Mycobacterium avium (M. avium) and Mycobacterium intracellulare (M. intracellulare) are the major causative agents of nontuberculous mycobacteria (NTM)-related pulmonary infections. However, little is known about the differences in drug susceptibility profiles between these two species. METHODS: A total of 393 NTM isolates were collected from Shanghai Pulmonary Disease Hospital. Sequencing of partial genes was performed to identify the strains at species level. The minimum inhibitory concentration (MIC) was used to evaluate the drug susceptibility against 20 antimicrobial agents. Variable number of tandem repeat (VNTR) typing was conducted to genotype these two species. RESULTS: A total of 173 (44.0%) M. avium complex (MAC) isolates were identified, including 41 (10.4%) M. avium isolates and 132 (33.6%) M. intracellulare isolates. Clarithromycin and amikacin were the two most effective agents against MAC isolates. The Hunter-Gaston Discriminatory Index (HGDI) values for VNTR typing of M. avium and M. intracellulare isolates were 0.993 and 0.995, respectively. Levofloxacin resistance was more common among the unclustered strains than among the clustered strains of M. intracellulare. CONCLUSION: M. intracellulare was the most common NTM species in China. Clarithromycin and amikacin had high antimicrobial activities against MAC. VNTR typing of MAC isolates revealed a high discriminatory power. Levofloxacin resistance was associated with unclustered strains of M. intracellulare.

Antimicrobial Susceptibility Testing and Molecular Characterization of Mycobacterium fortuitum Isolates in China.

We performed molecular identification of clinical isolates of Mycobacterium fortuitum (M. fortuitum) and conducted drug susceptibility testing to analyze the in vitro susceptibility of clinical M. fortuitum isolates and potential molecular mechanism conferring resistance to fluoroquinolone and macrolide drugs. The results showed that moxifloxacin had the highest in vitro activity against M. fortuitum, and most M. fortuitum isolates were resistant to clarithromycin and linezolid in China. The loss of genetic mutation in clarithromycin- and amikacin-resistant isolates indicates that some other intrinsic mechanism conferring clarithromycin and amikacin resistance plays an essential role in M. fortuitum infection.

Prevalence and Risk Factors of Primary Drug-Resistant Tuberculosis in China.

OBJECTIVE: To investigate the prevalence of primary drug-resistant tuberculosis (TB) and associated risk factors in China. We also explored factors contributing to the transmission of multidrug-resistant tuberculosis (MDR-TB). METHODS: A total of 2794 representative, Mycobacterium tuberculosis isolates from treatment-naive patients were subjected to drug susceptibility testing, and risk factors for drug-resistant TB were analyzed. We also analyzed MDR-TB strain sublineages, drug-resistance-conferring mutations, and risk factors associated with clustered primary MDR strains. RESULTS: Among 2794 Mycobacterium tuberculosis isolates from treatment-naive patients, the prevalence of any resistance to first-line drugs was 33.2% and the prevalence of MDR-TB was 5.7%. We did not find any risk factors significantly associated with resistance to first-line drugs. The 93 primary MDR-TB isolates were classified into six sublineages, of which, 75 (80.6%) isolates were the RD105-deleted Beijing lineage. The largest sublineage included 65 (69.9%) isolates with concurrent deletions of RD105, RD207, and RD181. Twenty-nine (31.2%) primary MDR strains grouped in clusters; MDR isolates in clusters were more likely to have S531L rpoB mutation. CONCLUSION: This study indicates that primary drug-resistant TB and MDR-TB strains are prevalent in China, and multiple measures should be taken to address drug-resistant TB.

Isolation and full-genome sequence of two reticuloendotheliosis virus strains from mixed infections with Marek's disease virus in China.

Reticuloendotheliosis virus (REV), classified as a gammaretrovirus, has a variety of hosts, including chickens, ducks, geese, turkeys, and wild birds. REV causes a series of pathological syndromes, especially the immunosuppression of the host, which may lead to an increased susceptibility to other pathogens, thus greatly damaging the poultry industry. Mixed infections of REV and Marek's disease virus (MDV) have been reported in many countries, including China. Previous reports revealed that MDV vaccines were not efficacious, and even less-virulent MDV strains would cause some losses due to mixed infections with REV. Additionally, contaminants in the MDV vaccine might be the main source of REV. In this study, two clinical samples were collected from two flocks of chickens that were diagnosed with MDV. Subsequently, two REV isolates were obtained from the clinical samples. The isolates, named CY1111 and SY1209, were further confirmed through an indirect immunofluorescence assay and electron microscopy. Complete genome sequences of the two REV strains were determined to test the relationship between them and other REV strains. Phylogenetic trees showed that the two REV strains were closely related to most REV strains that were isolated from a variety of hosts. Therefore, REVs might spread freely among these hosts under natural conditions. Additionally, most REV strains in China were in the same clade. The present work offers some information regarding REV in China.

Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model.

Understanding the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clarifying antiviral immunity in hosts are critical aspects for the development of vaccines and antivirals. Mice are frequently used to generate animal models of infectious diseases due to their convenience and ability to undergo genetic manipulation. However, normal adult mice are not susceptible to SARS-CoV-2. Here, we developed a viral receptor (human angiotensin-converting enzyme 2, hACE2) pulmonary transfection mouse model to establish SARS-CoV-2 infection rapidly in the mouse lung. Based on the model, the virus successfully infected the mouse lung 2 days after transfection. Viral RNA/protein, innate immune cell infiltration, inflammatory cytokine expression, and pathological changes in the infected lungs were observed after infection. Further studies indicated that neutrophils were the first and most abundant leukocytes to infiltrate the infected lungs after viral infection. In addition, using infected CXCL5-knockout mice, chemokine CXCL5 was responsible for neutrophil recruitment. CXCL5 knockout decreased lung inflammation without diminishing viral clearance, suggesting a potential target for controlling pneumonia.

Dysbiosis of gut microbiome affecting small intestine morphology and immune balance: a rhesus macaque model.

There is a growing appreciation for the specific health benefits conferred by commensal microbiota on their hosts. Clinical microbiota analysis and animal studies in germ-free or antibiotic-treated mice have been crucial for improving our understanding of the role of the microbiome on the host mucosal surface; however, studies on the mechanisms involved in microbiome-host interactions remain limited to small animal models. Here, we demonstrated that rhesus monkeys under short-term broad-spectrum antibiotic treatment could be used as a model to study the gut mucosal host-microbiome niche and immune balance with steady health status. Results showed that the diversity and community structure of the gut commensal bacteria in rhesus monkeys were both disrupted after antibiotic treatment. Furthermore, the 16S rDNA amplicon sequencing results indicated that Escherichia-Shigella were predominant in stool samples 9 d of treatment, and the abundances of bacterial functional genes and predicted KEGG pathways were significantly changed. In addition to inducing aberrant morphology of small intestinal villi, the depletion of gut commensal bacteria led to increased proportions of CD3 + T, CD4 + T, and CD16 + NK cells in peripheral blood mononuclear cells (PBMCs), but decreased numbers of Treg and CD20 + B cells. The transcriptome of PBMCs from antibiotic-treated monkeys showed that the immune balance was affected by modulation of the expression of many functional genes, including IL-13, VCAM1, and LGR4.

Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1 -/-mice infected with influenza virus (H1N1).

Respirovirus infection can cause viral pneumonia and acute lung injury (ALI). The interleukin-1 (IL-1) family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo. IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation. We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 (IL-1R1) deficient mice. Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6, TNF-α, G-CSF, KC, and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1 -/- mice in comparison with that of wild type infected mice. The adaptive immune response against the H1N1 virus in IL-1R1 -/- mice was impaired with downregulated anti-viral Th1 cell, CD8+ cell, and antibody functions, which contributes to attenuated viral clearance. Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1 -/- mice compared with that in WT infected mice. Moreover, the infected IL-1R1 -/- mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung. Together, these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury, particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.

Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo).

Data from EV-D68-infected patients demonstrate that pathological changes in the lower respiratory tract are principally characterized by severe respiratory illness in children and acute flaccid myelitis. However, lack of a suitable animal model for EV-D68 infection has limited the study on the pathogenesis of this critical pathogen, and the development of a vaccine. Ferrets have been widely used to evaluate respiratory virus infections. In the current study, we used EV-D68-infected ferrets as a potential animal to identify impersonal indices, involving clinical features and histopathological changes in the upper and lower respiratory tract (URT and LRT). The research results demonstrate that the EV-D68 virus leads to minimal clinical symptoms in ferrets. According to the viral load detection in the feces, nasal, and respiratory tracts, the infection and shedding of EV-D68 in the ferret model was confirmed, and these results were supported by the EV-D68 VP1 immunofluorescence confocal imaging with α2,6-linked sialic acid (SA) in lung tissues. Furthermore, we detected the inflammatory cytokine/chemokine expression level, which implied high expression levels of interleukin (IL)-1a, IL-8, IL-5, IL-12, IL-13, and IL-17a in the lungs. These data indicate that systemic observation of responses following infection with EV-D68 in ferrets could be used as a model for EV-D68 infection and pathogenesis.

The Abbott RealTime MTB assay and the Cepheid GeneXpert assay show comparable performance for the detection of Mycobacterium tuberculosis in sputum specimens.

OBJECTIVES: To evaluate the performance of the newly developed technology Abbott RealTime MTB assay (RealTime MTB assay) for the detection of Mycobacterium tuberculosis in sputum specimens and to compare its performance with that of the Cepheid GeneXpert assay. METHODS: Sputum specimens were collected from 270 subjects suspected to have tuberculosis (TB). Smear microscopy, culture, identification, RealTime MTB, and GeneXpert assays were performed according to standard protocols. Accuracy measures of the method evaluated were determined using solid culture as the reference standard. RESULTS: The RealTime MTB assay showed similar positive detection rates as the GeneXpert assay in smear-positive, culture-positive, and smear/culture-negative groups; no significant differences were found in these groups between the two assays. The RealTime MTB assay demonstrated a sensitivity of 100% and a specificity of 84.4%; the GeneXpert assay had a sensitivity of 96.9% and specificity of 89.6%. After the resolution of discordant results by PCR-based molecular method, the sensitivities and specificities of the RealTime MTB and GeneXpert assays were 100% vs. 97% and 90.0% vs. 95.6%, respectively; no significant difference in sensitivity or specificity was found between the RealTime MTB and GeneXpert assays. CONCLUSIONS: This study demonstrated that the Abbott RealTime MTB and Cepheid GeneXpert assays have similar sensitivity and specificity. The Abbott RealTime MTB assay is a highly promising method for the diagnosis of TB.

Distinct expression pattern of miRNAs in Marek's disease virus infected-chicken splenic tumors and non-tumorous spleen tissues.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression. Emerging evidence suggests that differential miRNA expression is associated with viral infection and tumorigenesis. Recently discovered microRNAs in the Marek's disease virus (MDV) genome have been suggested to have regulatory roles during MDV oncogenesis. To gain more insight into the molecular mechanisms of the tumorigenesis of MDV, we used microarrays to screen host and viral miRNAs that were sensitive to infection by MDV. Microarray analysis showed significant differential expression of 79 miRNAs, which was confirmed by qRT-PCR analysis. These data suggest that differentially expressed miRNAs may have major roles in MDV-induced tumorigenesis. In addition, we found two clades of chicken miRNAs had increased expression in splenic tumors and non-tumorous spleen tissues from GA-infected chickens. Thus, the expression of these miRNAs can be considered signatures for MDV infection and tumorigenesis.