Characteristic of KPC-12, a KPC Variant Conferring Resistance to Ceftazidime-Avibactam in the Carbapenem-Resistant Klebsiella pneumoniae ST11-KL47 Clone Background.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a great threat to public health worldwide. Ceftazidime-avibactam (CZA) is an effective ß-lactam/ß-lactamase inhibitors against CRKP. However, reports of resistance to CZA, mainly caused by Klebsiella pneumoniae carbapenemase (KPC) variants, have increased in recent years. In this study, we aimed to describe the resistance characteristics of KPC-12, a novel KPC variant identified from a CZA resistant K. pneumoniae. Methods: The K. pneumoniae YFKP-97 collected from a patient with respiratory tract infection was performed whole-genome sequencing (WGS) on the Illumina NovaSeq 6000 platform. Genomic characteristics were analyzed using bioinformatics methods. Antimicrobial susceptibility testing was conducted by the broth microdilution method. Induction of resistant strain was carried out in vitro as previously described. The G. mellonella killing assay was used to evaluate the pathogenicity of strains, and the conjugation experiment was performed to evaluate plasmid transfer ability. Results: Strain YFKP-97 was a multidrug-resistant clinical ST11-KL47 K. pneumoniae confers high-level resistance to CZA (16/4 µg/mL). WGS revealed that a KPC variant, KPC-12, was carried by the IncFII (pHN7A8) plasmids (pYFKP-97_a and pYFKP-97_b) and showed significantly decreased activity against carbapenems. In addition, there was a dose-dependent effect of bla KPC-12 on its activity against ceftazidime. In vitro inducible resistance assay results demonstrated that the KPC-12 variant was more likely to confer resistance to CZA than the KPC-2 and KPC-3 variants. Discussion: Our study revealed that patients who was not treated with CZA are also possible to be infected with CZA-resistant strains harbored a novel KPC variant. Given that the transformant carrying bla KPC-12 was more likely to exhibit a CZA-resistance phenotype. Therefore, it is important to accurately identify the KPC variants as early as possible.

CH4 control and nitrogen removal from constructed wetlands by plant combination.

Global warming trend is accelerating. This study proposes a green and economical methane (CH4) control strategy by plant combination in constructed wetlands (CWs). In this study, a single planting of Acorus calamus L. hybrid constructed wetland (HCW-A) and a mixed planting of Acorus calamus L. and Eichhornia crassipes (Mart.) Solms hybrid constructed wetland (HCW-EA) were constructed. The differences in nitrogen removal performance and CH4 emissions between HCW-A and HCW-EA were compared and analyzed. The findings indicated that HCW-EA demonstrated significant improvements over HCW-A, with NH4+-N and TN removal rates increasing by 21.61% and 16.38% respectively, and CH4 emissions decreased by 43.36%. The microbiological analysis results showed that plant combination promoted the enrichment of Proteobacteria, Alphaproteobacteria and Bacillus. More nitrifying bacteria carrying nxrA genes and denitrifying bacteria carrying nirK genes accelerated the nitrogen transformation process. In addition, the absolute abundance ratio of pmoA/mcrA increased, reducing the release of CH4.

Bedaquiline, Delamanid, Linezolid, Clofazimine, and Capreomycin MIC Distributions for Drug Resistance Mycobacterium tuberculosis in Shanghai, China.

Background: New antituberculosis drugs have recently been approved for the treatment of multidrug-resistant tuberculosis TB (MDR-TB). We aimed to describe the distributions of bedaquiline, delamanid, linezolid, clofazimine, and capreomycin MIC values for M. tuberculosis. Methods: M. tuberculosis clinical isolates were originally isolated from 2020 to 2021 from 1452 different pulmonary tuberculosis patients of the Shanghai Pulmonary Hospital in China. The drug susceptibility testing was performed using the Sensititre custom plates (SHTBMY) (TREK Diagnostic Systems, Thermo Fisher Scientific In., USA) consisting of a 96-well microtitre plate containing 4 (bedaquiline, delamanid, clofazimine, capreomycin) antimicrobial agents. MICs were determined for linezolid using a microdilution method. Results: Based on the latest definitions, 156 (10.74%) were MDR-TB, 93 (6.40%) were pre-XDR-TB, and 27 (1.86%) were XDR-TB. The rate of BDQ resistance in cases of MDR-TB was 7.69%, while it was observed to be 10.75% in cases of pre-XDR-TB, and significantly higher at 37.04% in cases of XDR-TB. The lowest rate of drug resistance against M. tuberculosis was DLM (0.14%). For LZD, 11 (0.76%) clinical isolates were resistant, based on the CLSI breakpoint of 1µg/mL. The five strains with a MIC value of >32 for LZD resistance were XDR-TB isolates. Among all MDR, pre-XDR, and XDR isolates tested, LZD' MIC50 increased from 0.25 and 0.5 to 1µg/mL. The MIC90 value of LZD against XDR-TB isolates was 32µg/mL. For CFZ, six isolates with elevated MICs of ≥2µg/mL. CFZ's MIC50 and MIC90 values in all isolates were 0.12µg/mL and 0.25µg/mL, respectively. Conclusion: The study findings indicate that BDQ, DLM, CFZ, and LZD may exhibited excellent in vitro activity against MDR-TB isolates. Detection of resistance to BDQ and LZD was alarming for XDR-TB isolates. It is necessary to perform universal drug sensitivity testing for M. tuberculosis, especially MDR-TB and XDR-TB patients.

Isolation and Identification of Culturable Bacteria from South China Seawater and Preliminary Screening of Marine Biocontrol Bacteria.

Marine microorganisms have evolved special metabolic pathways to produce numerous bioactive substances with novel structures and unique functions. This study analyzed the diversity of culturable bacteria in marine water samples from the South China Sea and screened the isolated bacteria with pathogenic fungi. A total of 200 culturable strains of 72 different bacteria were obtained from 56 water samples from the South China Sea. They belonged to three phyla and four classes, namely Gammaproteobacteria, Alphaproteobacteria, Bacilli and Actinomycetia. Bacilli was the dominant class, comprising up to 59.72%, followed by Gammaproteobacteria (20.83%). Bacillus, Pseudomonas, Paenibacillus and Rhizobium were the most dominant genera. Among these strains, HY-88 and HY-91 encoding BamC, FenB and PKSI genes were selected and identified as Bacillus subtilis. The respective inhibition rates of the HY-88 caused by plate confrontation against Magnaporthe grisea, Fusarium oxysporum, Botrytis cinerea, anthrax and Botrytis cinerea were 90.91%, 54.29%, 52.17% and 51.72%, in comparison with HY-91 86.36%, 48.57%, 47.83% and 34.48%. In addition, the supernatant of HY-88 showed a lesion inhibition rate of 74.5%, which was significantly higher than HY-91 (60.55%). In addition, HY-88 and HY-91 showed strong antifungal activity to Colletotrichum viniferum on detached Shine Muscat grapes. Tolerance tests showed that the HY-88 and HY-91 grew at 10-40 °C, 7-10% NaCl and pH 3-11. HY-88 and HY-91 could inhibit various fungal plant diseases, which lays a foundation for the development of new biopesticides.

Carbon monoxide ameliorates lipopolysaccharide-induced acute lung injury via inhibition of alveolar macrophage pyroptosis.

Carbon monoxide (CO) has been reported to exhibit a therapeutic effect in lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the precise mechanism by which CO confers protection against ALI remains unclear. Pyroptosis has been recently proposed to play an essential role in the initiation and progression of ALI. Thus, we investigated whether pyroptosis is involved in the protection of CO against ALI and its underlying mechanism. First, an LPS-induced ALI mouse model was established. To determine the role of pyroptosis, we evaluated histological changes and the expression levels of cleaved caspase-11, N-gasdermin D (GSDMD), and IL-1ß in lung tissues, which are the indicators of pyroptosis. Inhalation of CO exhibited protective effects on LPS-induced ALI by decreasing TNF-α and IL-10 expression and ameliorating pathological changes in lung tissue. In vitro, CO significantly reduced the expression of cleaved caspase-11, N-GSDMD, IL-1ß, and IL-18. In addition, it increased nuclear factor E2-related factor 2 (NRF-2) expression in a time-dependent manner in RAW 264.7 cells and decreased N-GSDMD expression. The expression of cleaved GSDMD and release of LDH were increased after treatment with a specific NRF-2 inhibitor, ML385, indicating that NRF-2 mediates the inhibition of pyroptosis by CO. Taken together, these results demonstrated that CO upregulated NRF-2 to inhibit pyroptosis and subsequently ameliorated LPS-induced ALI.

The frequency and dynamics of CD4+ mucosal-associated invariant T (MAIT) cells in active pulmonary tuberculosis.

MAIT cells are unconventional innate-like T lymphocytes contributing to host immune protection against Mycobacteria tuberculosis (Mtb) infection. CD4- MAIT cells play a major role in immune protection against tuberculosis (TB), however, the role of CD4+ MAIT cells was elusive due to their low abundance. We firstly investigated the frequency and functions of CD4+ MAIT cells in pulmonary tuberculosis (PTB) patients before and after anti-TB treatment. We found that the frequency of Mtb-reactive CD4+ MAIT cells and IFN-γ, granzyme B (GrzB), CD69 expression on them were increased while LAG-3+ cells of them were decreased in PTB patients. After the treatment, the frequency of Mtb-reactive CD4+ MAIT cells and CD69, IFN-γ, GrzB expression on them were decreased while LAG-3 increased. The results indicated the expression profile is distinct between CD4+ MAIT cells and CD4- MAIT cells in PTB patients, the increased IFN-γ and GrzB expression of CD4+ MAIT cells play a role in anti-TB immunity.

Difference in drug susceptibility distribution and clinical characteristics between Mycobacterium avium and Mycobacterium intracellulare lung diseases in Shanghai, China.

Introduction. Mycobacterium avium complex (MAC) has been reported as the most common aetiology of lung disease involving nontuberculous mycobacteria.Hypothesis. Antimicrobial susceptibility and clinical characteristics may differ between Mycobacterium avium and Mycobacterium intracellulare.Aim. We aimed to evaluate the differences in antimicrobial susceptibility profiles between two major MAC species (Mycobacterium avium and Mycobacterium intracellulare) from patients with pulmonary infections and to provide epidemiologic data with minimum inhibitory concentration (MIC) distributions.Methodology. Between January 2019 and May 2020, 45 M. avium and 242 M. intracellulare isolates were obtained from Shanghai Pulmonary Hospital. The demographic and clinical characteristics of patients were obtained from their medical records. The MICs of 13 antimicrobials were determined for the MAC isolates using commercial Sensititre SLOWMYCO MIC plates and the broth microdilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI; Standards M24-A2). MIC50 and MIC90 values were derived from the MIC distributions.Results. M. intracellulare had higher resistance rates than M. avium for most tested antimicrobials except clarithromycin, ethambutol, and ciprofloxacin. Clarithromycin was the most effective antimicrobial against both the M. avium (88.89 %) and M. intracellulare (91.32 %) isolates, with no significant difference between the species (P=0.601). The MIC90 of clarithromycin was higher for M. avium (32 µg ml-1) than M. intracellulare (8 µg ml-1). The MIC50 of rifabutin was more than four times higher for M. intracellulare (1 µg ml-1) than M. avium (≤0.25 µg ml-1). The percentages of patients aged >60 years and patients with sputum, cough, and cavitary lesions were significantly higher than among patients with M. intracellulare infection than M. avium infections.Conclusions. The pulmonary disease caused by distinct MAC species had different antimicrobial susceptibility, symptoms, and radiographic findings.

Rifampin-resistance-associated mutations in the rifampin-resistance-determining region of the rpoB gene of Mycobacterium tuberculosis clinical isolates in Shanghai, PR China.

Introduction. Resistance to rifampin (RIF) in Mycobacterium tuberculosis infection is associated with mutations in the rpoB gene coding for the ß-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive.Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance.Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against M. tuberculosis.Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for M. tuberculosis isolates were determined using commercial Sensititre M. tuberculosis MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis.Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01).Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by M. tuberculosis strains harbouring these mutations.

Use of whole-genome sequencing to predict Mycobacterium tuberculosis drug resistance in Shanghai, China.

OBJECTIVE: To evaluate the performance of whole-genome sequencing (WGS) for predicting Mycobacterium tuberculosis (MTB) drug resistance. METHODS: 276 rifampin-resistance tuberculosis (RR-TB) and 30 rifampicin-sensitive clinical isolates were randomly selected from patients with tuberculosis in Shanghai Pulmonary Hospital (SPH). Phenotypic drug susceptibility testing (DST) against six anti-TB drugs was performed, and WGS was used to predict the drug resistance using an online 'TB-Profiler' tool. RESULTS: Using phenotypic susceptibility as the gold standard, the overall sensitivities and specificities for WGS were 94.53% and 92.00% for isoniazid, 97.10% and 100.00% for rifampicin, 97.46% and 64.36% for ethambutol, 97.14% and 95.83% or streptomycin, 93.02% and 98.87% for ofloxacin, and 75.00% and 100.00% for amikacin, respectively. The concordances of WGS-based DST and phenotypic DST were: isoniazid (94.12%), rifampicin (97.39%), ethambutol (77.12%), streptomycin (96.73%), ofloxacin (96.41%) and amikacin (97.06%). CONCLUSIONS: WGS could be a promising approach to predict resistance to isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and amikacin.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.

[Isolation and Identification of a Novel Cypovirus from Daphnis nerii].

In order to develop a novel effective biological insecticide for controlling oleander hawk moth, a new pathogen was isolated from naturally diseased Daphnis nerii. Based on scanning electron microscopy, full-length amplification of cDNAs (FLAC), and phylogenetic analysis of genome segments 2and 10,the virus was identified as a new type of cypovirus (Da phnis nerii cypovirus [DnCPV]). Electrophoresis analysis showed that DnCPV had a genome comprising 10double-stranded RNA (dsRNA) segments, ranging from 892 to 4160bp.Using FLAC, the cDNAs from the 10 dsRNA segments of the new CPV were cloned and genome segments 2and 10 were sequenced. Sequencing results showed that segment 2 encoded RNA-dependent-RNA-polymerases (RdRps) and segment 10 encoded polyhedrin. These two segments shared conserved terminal sequences of AGUCAAA and AGC at the 5'and 3'ends,respectively.These conserved terminal sequences were not consistent with any of the known CPV types.Phylogenetic analysis of the RdRp and polyhedrin indicated that this CPV was more closely related to CPV type 19 and type 5than other CPV types. Based on the unique conserved terminal sequences and the electrophoresis pattern of the new virus, we tentatively named it DnCPV Nanchang isolate: DnCPV-NC.

[Improvement of River Water Quality by Aeration: WASP Model Study].

Aeration is one of the main technical methods to remediate polluted rivers, and mathematical models are the main ways to predict and assess the environmental pollution. WASP model was used to study the effect of aeration on improving the water quality of a small river over a time span. The preliminary results showed that the simulation values were consistent with practical monitoring data, which could be useful for the management and control of polluted water. Furthermore, simulations under different aeration conditions suggested that aeration technology could significantly lower the levels of chemical oxygen demand (COD) and ammonia-nitrogen (NH4(+)-N) of river water, thus improving the water quality. With the improvement of dissolved oxygen (DO), the water quality could be further improved. However, the increase in the removal rate of pollutants would slow down. Also, there were remarkable differences among various months throughout the whole year, and aeration from May to September had better results. Considering economic costs and environmental benefits, river aeration with a 4 mg x L(1) DO standard in summer was determined as the best condition.

Relationship between human LTA4H polymorphisms and extra-pulmonary tuberculosis in an ethnic Han Chinese population in Eastern China.

Two single nucleotide polymorphisms in Leukotriene A4 hydrolase (LTA4H) gene were reported to be associated with protection from pulmonary tuberculosis in Vietnamese population. But these associations were not found in the Russians. To investigate the association of LTA4H polymorphisms with tuberculosis in a Han Chinese population in Eastern China, we genotyped 5 SNPs of LTA4H gene in 743 of pulmonary tuberculosis patients, 372 of extra-pulmonary tuberculosis patients and 888 of healthy controls individuals. The CC and TT homozygotes of rs1978331 and rs2540474 were identified to have higher rates (P < 0.01) and be risk factors in the patients with extra-pulmonary tuberculosis (OR = 1.412; 95% CI = 1.104-1.804 and(OR = 1.380; 95% CI = 1.080-1.764). However, no significant association was found between any of the SNPs and pulmonary tuberculosis. In the extra-pulmonary tuberculosis subgroups. LTA4H gene were significantly associated with tuberculous meningitis, lymph node tuberculosis, bone tuberculosis and other extra-pulmonary tuberculosis except for pleural tuberculosis. The present findings suggest that polymorphisms in the LTA4H gene may affect susceptibility to extra-pulmonary tuberculosis and change the risk of developing the disease in the Han nationality in the East China.

Identification of cultivable bacteria in the intestinal tract of Bactrocera dorsalis from three different populations and determination of their attractive potential.

BACKGROUND: This study aimed to identify the cultivable bacteria inhabiting the intestinal tract of adult oriental fruit flies (Bactrocera dorsalis) from laboratory-reared, laboratory sterile sugar-reared, and field-collected populations, and to evaluate the attractiveness of the metabolites produced by the above bacteria to their hosts. RESULTS: Fifteen bacterial isolates chosen from the three populations were determined at species level. These 15 strains were cultured and the attractiveness of the whole Luria-Bertani broth, filtered and autoclaved supernatants to B. dorsalis adults was determined using bioassays. The bioassays showed that all bacterial strains were significantly more attractive to B. dorsalis adults than the media-only control. Among them, Bacillus cereus, Enterococcus faecalis, Enterobacter cloacae and Citrobacter freundii were the most attractive bacteria. Furthermore, results of a subsequent field test showed that the six bacterial strains were significantly more attractive than the control, with B. cereus and E. faecalis attracting significantly more flies. CONCLUSIONS: A cultivable bacterial community composed of Enterobacteriaceae, Enterococcaceae, and Bacillaceae was identified in the intestinal tract of B. dorsalis. Metabolites from B. cereus attracted the greatest number of B. dorsalis adults in the laboratory and field. These results provide useful information for the development of bacterial biocontrol agents or implementation as an insecticide.

Clinical diagnostic utility of adenosine deaminase, interferon-γ, interferon-γ-induced protein of 10 kDa, and dipeptidyl peptidase 4 levels in tuberculous pleural effusions.

OBJECTIVE: Current tools for the diagnosis of tuberculous pleural effusions are suboptimal. The study was undertaken to evaluate the accuracy of pleural fluid adenosine deaminase (ADA), interferon (IFN)-γ, interferon-γ-induced protein of 10 kDa (IP-10), and dipeptidyl peptidase (DPP) 4 levels in differentiating tuberculous pleural effusion (TPE) and non-TPE. METHODS: A total of 122 samples of pleural effusion were studied. Pleural fluid ADA activity was measured with the colorimetric method, and IP-10, IFN-γ, and DPP4 levels were measured with enzyme-linked immunosorbent assay. RESULTS: ADA activity and IP-10, IFN-γ, and DPP4 levels were significantly higher in TPE than in non-TPE (88.9 ± 62.7 U/L vs 18.1 ± 16.2 U/L, P < .05; 147.5 ± 117.3 ng/L vs 24.9 ± 19.7 ng/L, P < .05; 627.2 ± 345.3 ng/L vs 152.6 ± 71.4 ng/L, P < .05; and 560.6 ± 451.2 vs 56.8 ± 57.7, P < .05, respectively). The diagnostic sensitivity and specificity of ADA activity (cutoff value of 40 U/L) were 93.6% and 90.9%, respectively, and higher than those of IFN-γ (91.0% and 88.6% at the cutoff value of 225 ng/L, respectively), DPP4 (88.5% and 81.8% at the cutoff value of 75 ng/L, respectively), and IP-10 (83.3% and 86.4% at the cutoff value of 44 ng/L, respectively). CONCLUSION: The roles of ADA and IFN-γ in the differential diagnosis of tuberculous pleurisy are pivotal. ADA or IFN-γ in combination with DPP4 or IP-10 can aid in differentiation between TPE and non-TPE with improved specificity and diagnostic efficiency.

Association of SP110 gene polymorphisms with susceptibility to tuberculosis in a Chinese population.

Development of tuberculosis is mediated by both environmental and genetic factors. The Ipr1 (intracellular pathogen resistance-1) gene has been identified at the sst1 (super-susceptibility to tuberculosis 1) locus on mouse chromosome 1. As Ipr1 plays a major role in mediating innate immunity in a mouse model of Mycobacterium tuberculosis infection, the human Ipr1 homologue, SP110 is a recognised candidate gene for control M. tuberculosis infection. This study was designed to investigate sequence variants of the SP110 gene in Chinese and test whether the SP110 gene is a susceptibility factor for tuberculosis. In a sample of 308 smear-positive patients with pulmonary tuberculosis and 628 exposed, apparently healthy controls, we have genotyped 6 SP110 gene variants that were either available from public databases, including HapMap data, or identified by DNA re-sequencing. DNA re-sequencing revealed 7 novel SP110 variants in the 5'-UTR and intronic regions. Two of SP110 SNPs, rs11556887 and rs1135971 were significantly associated with disease. Analysis of the haplotypes revealed two haplotypes are significantly associated with TB. Other variants of SP110 in this case-control approach could not find any significant differences. Our study demonstrates that genotypes and haplotypes of SP110 might be associated with susceptibility to tuberculosis in Chinese population.

Relationship between single nucleotide polymorphism of interleukin-18 and susceptibility to pulmonary tuberculosis in the Chinese Han population.

Interleukin-18 (IL-18) is a multi-functional cytokine capable of inducing either Th1 or Th2 polarization depending on the immunologic milieu. IL-18 may influence the host response to Mycobacterium tuberculosis (M.tb) infection. To investigate the relationship between single nucleotide polymorphisms of the IL-18 and susceptibility to pulmonary tuberculosis in the Chinese Han population, the IL-18 gene was sequenced to detect polymorphisms and to examine the genotype frequencies in 300 patients and 702 healthy controls. DNA sequencing revealed three IL-18 variants: rs1946518, rs5744247, and rs549908. It also revealed that allele A of rs1946518 confers a 1.47-fold increased risk of developing tuberculosis (TB) (P = 0.0001, OR [95%CI] = 1.47 [1.21-1.78]), and that the C allele of rs5744247 confers a 0.77-fold decreased risk of disease (P = 0.01, R [95%CI] = 0.77 [0.632-0.937]). The genotypes rs1946518, rs5744247 and rs549908 were found to be significantly associated with TB. Estimation of the frequencies of haplotypes revealed a potential risk haplotype AGA (P = 0.01, OR [95%CI] = 1.41 [1.15-1.72]) and a protective haplotype CCA (P = 0.01, OR [95%CI] = 0.70 [0.57-0.85]) for TB. The present findings suggest that polymorphisms in the IL-18 gene may affect susceptibility to TB and increase the risk of developing the disease in the Chinese Han population.

Interleukin-10 gene promoter polymorphisms and their protein production in pleural fluid in patients with tuberculosis.

Associations of interleukin-10 (IL-10) gene promoter polymorphisms and pleural tuberculosis risk remain unclear. The objective of this study was to determine IL-10 gene promoter polymorphisms at -1082, -819 and -592 sites and their protein production in pleural fluid (PF) in patients with and without pleural tuberculosis. IL-10 gene promoter polymorphisms at the -1082, -819 and -592 sites were genotyped using a SNaPshot assay. Protein levels of IL-10 in PF were measured using an enzyme-linked immunosorbent assay. There were no significant differences in the genotype and allele frequencies of IL-10 gene promoter polymorphisms at position -1082 between the pleural tuberculosis and the control groups. However, the frequency of -819 T or -592 A alleles was significantly more common in patients with pleural tuberculosis than controls. The protein levels of IL-10 in PF were statistically higher in the pleural tuberculosis group than in the control group. Moreover, the polymorphisms at the -1082, -819 and -592 sites were associated with protein levels of IL-10 in PF in the pleural tuberculosis group, while in the control group, only the polymorphism at position -1082 correlated with the protein levels. These findings support the association between IL-10 promoter polymorphisms at -819 and -592 sites and their protein production with pleural tuberculosis risk.

Nontuberculous mycobacteria: susceptibility pattern and prevalence rate in Shanghai from 2005 to 2008.

BACKGROUND: An increasing incidence of disease caused by nontuberculous mycobacteria (NTM) is being reported. The purpose of this study was to determine the isolation rates of NTM from various clinical specimens, and their antimicrobial susceptibility patterns, over a 4-year period in Shanghai. METHODS: All NTM isolated between 2005 and 2008 at Shanghai Pulmonary Hospital, a key laboratory of mycobacteria tuberculosis in Shanghai, China, were identified with conventional biochemical tests and 16S rRNA gene sequencing. Antimicrobial susceptibility for all NTM was determined using the BACTEC MGIT 960 system. RESULTS: A total of 21,221 specimens were cultured, of which 4868 (22.94%) grew acid fast bacilli (AFB), and 248 (5.09%) of the AFB were NTM. The prevalence rate of NTM was determined as 4.26%, 4.70%, 4.96% and 6.38% among mycobacteria culture positive samples in years 2005, 2006, 2007 and 2008 respectively. These data indicated that the prevalence rate has continuously increased. Sixteen different species of NTM were identified, the most commonly encountered NTM in Shanghai were M. chelonae (26.7%), followed by M. fortuitum (15.4%), M. kansasii (14.2%), M. avium-intracellulare complex (13.1%) and M. terrae (6.9%). The rare species identified were M. marinum, M. gastri, M. triviale, M. ulcerans, M. smegmatis, M. phlci, M. gordonae, M. szulgai, M. simiae, M. scrofulaceum and M. xenopi. The five most commonly identified NTM species showed high drug resistance to general anti-tuberculosis drugs, particularly, M. chelonae and M. fortuitum appear to be multi-drug resistance. CONCLUSIONS: The prevalence of NTM in Shanghai showed a tendency to increase over the course of the study. The five most commonly isolated NTM species showed high drug resistance to first line anti-tuberculosis drugs.

Rapid method for identification of six common species of mycobacteria based on multiplex SNP analysis.

A multiplex method using the SNaPshot technique was developed to screen for six common mycobacterial species: Mycobacteria tuberculosis, M. avium, M. intracellulare, M. chelonae, M. kansasii, and M. gordonae. A total of 468 mycobacterial clinical isolates were subjected to analysis for the presence of the six mycobacterial species by the multiplex SNaPshot method. Of the 468 mycobacterial isolates, 464 (99.15%) could be correctly identified by this assay. The multiplex SNaPshot technique is a promising discriminatory tool for rapid and accurate identification of frequently encountered clinical mycobacterial species.