Identification of mycobacteria of the MAIS complex and M. tuberculosis by restriction fragment length polymorphism analysis of hsp65 gene

Restriction fragment length polymorphism analysis of hsp65 gene was performed on museum strains of mycobacteria using Hin6I restrictase. Study of restriction profiles allowed us to distinguish mycobacterial species of the MAIS complex and several strains of nontuberculous mycobacteria. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 188–191, August, 2006     

Antibody to mycobacterial 65-kD heat shock protein in commercial antisera.

Inhibition ELISA and immunoblotting were used to examine the antigenic cross-reactivity claimed to exist between mycobacterial 65-kD heat shock protein (hsp65) and human lactoferrin. Commercially available anti-lactoferrin antibodies produced using either Freund's complete (FCA) or Freund's incomplete adjuvant were tested for binding to recombinant mycobacterial hsp65. Both antibody preparations showed reactivity with hsp65, this being greater with the antibody produced using FCA. However, we found no evidence of a cross-reaction. Lactoferrin failed to inhibit anti-hsp65 reactivity, while hsp65 itself did. Affinity purified anti-lactoferrin antibody showed no reaction with hsp65 by ELISA or immunoblotting. These data suggest that commercial anti-lactoferrin preparations are contaminated with antibodies to hsp65. A commercial anti-albumin antibody also bound to hsp65 in ELISA, so this may be a more general phenomenon.     

Monoclonal antibody epitopes of mycobacterial 65-kD heat-shock protein defined by epitope scanning.

The binding sites for MoAbs to the 65-kD heat-shock protein (hsp65) of mycobacteria have been investigated by epitope scanning. Five hundred and twenty-six 8-mer peptides representing the complete sequence of Mycobacterium tuberculosis hsp65 were synthesised in duplicate using the Epitope Scanning Kit (CRB Ltd.). The epitopes of six MoAbs raised to the hsp65 of M. tuberculosis or M. leprae were investigated. We have identified the epitope of a new MoAb (DC16); this epitope is continuous, hydrophilic in nature and 11 amino acids long. We have also confirmed the location of the epitopes of three MoAbs (IIH9, ML30 and IIC8). Thus the epitope scanning technique has proved suitable for the detection of continuous epitopes of hsp65.     

Evaluation of bone marrow and blood cultures for the recovery of mycobacteria in the diagnosis of disseminated mycobacterial infections

This study evaluated the validity of bone marrow (BM) and blood specimens for the diagnosis of disseminated mycobacterial infections (DMIs). From 1990 to February 1997, all specimens were processed with the lysis-centrifugation procedure; thereafter (until December 2001), they were processed with the BACTEC Myco/F Lytic system. Twenty-three paired BM-blood specimens with mycobacteria in at least one specimen were studied from 23 patients. The strains isolated were 14 Mycobacterium avium complex (MAC) and nine M. tuberculosis complex (MTBC). Blood specimens had a statistically significant greater sensitivity for the isolation of MAC than BM (100% vs. 57.1%, respectively), whereas sensitivity for the isolation of MTBC was equal for the two specimen types (66.7%). Although not statistically significant, the times required to detect mycobacteria from blood specimens were lower than those from BM in the MycoF/Lytic system. Overall, blood cultures represented a more sensitive and less invasive alternative to BM cultures for the diagnosis of disseminated mycobacteriosis caused by MAC, especially when the MycoF/Lytic system was used, but provided no advantage for the diagnosis of DMI caused by MTBC.     

Clinical manifestations of nontuberculous mycobacteria infections

The isolation of nontuberculous mycobacteria (NTM) from clinical specimens has become very frequent in the last years. Such organisms are typically environmental and poorly pathogenic for humans; they can, however, be responsible for opportunistic diseases in subjects presenting with various predisposing conditions. Pulmonary infections are responsible for the most frequent disease caused by NTM, although the relevance of mycobacterioses involving other parts of the body is increasing. The risk of disseminated infections characterizing immunocompromised patients is well known, and those numbers are steadily rising. The lymph nodes, cutis and soft tissues, as well as bone and joints, are also important targets of NTM infection. The problems concerning the assessment of the clinical significance of NTM, along with a consideration of the more frequent NTM pathologies, are the major objectives of this review.     

Nitrotyrosine formation after activation of murine macrophages with mycobacteria and mycobacterial lipoarabinomannan.

Murine peritoneal macrophages, elicited with thioglycollate, were stimulated in vitro with lipopolysaccharide (LPS). The production of nitrite, superoxide anion (SOA), and the accumulation of nitrotyrosine in the cells increased after treatment, and all were inhibitable by the NO synthase inhibitor NG-monomethyl-L-arginine monoacetate (L-NMMA). This effect suggests a direct correlation between the accumulation of those metabolites and NO synthase activity. Lipoarabinomannan (LAM) purified from Mycobacterium tuberculosis was added to peritoneal macrophages in the presence of interferon-gamma (IFN-gamma); the cells produced nitrite and SOA, both inhibitable by L-NMMA. There was, as well, accumulation of nitrotyrosine in the macrophage proteins. Strikingly, the amount of nitrotyrosine measured after LAM plus IFN-gamma, or LAM plus the low molecular weight adjuvant glutamylmuramyl dipeptide (GMDP), increased significantly in the presence of L-NMMA. These results suggest that murine macrophages, upon LAM stimulation, might generate reactive nitrogen metabolites by a route other than NO synthase. Nitrotyrosine accumulation after infection of macrophages in vitro, with either live bacille Calmette-Guérin (BCG) or live M. tuberculosis, in the presence or absence of IFN-gamma, showed no correlation with nitrite production, suggesting a low superoxide production.     

结核杆菌HSP65 DNA疫苗的初步研究

目的 构建以结核分枝杆菌热休克蛋白65000u基因为基础的核酸疫苗。方法 采用聚合酶链反应从结核杆菌H37Rv株基因中，扩增出HSP65的编码基因，经限制性核酸内切酶消化后，插入真核表达载体pcDNA3.1(－）的相应酶切位点，并将此重组质粒免疫动物。结果 重组质粒的插入基因经序列测定证实为结核分枝杆菌HSP65。DNA免疫小鼠体内产生特异性抗体。结论 以HSP65编码基因为基础的真核表达载体构建成功，并能引起特异性动物免疫反应，为进一步研究其在结核病防治中的作用奠定了基础。     

Modulation of immunologic responses in nontuberculous mycobacterial infections with indomethacin

We have previously reported that impairedin vitro cellular immunity is a common finding in patients with nontuberculous mycobacterioses and that the subnormal responses may be improved by indomethacin. Subsequently, we have studied thein vivo effects of indomethacin on cell-mediated immune functions of four patients withMycobacterium avium-intracellulare infections. Prior to treatment none of the patients had delayed cutaneous reactions to purified protein derivative (PPD) of the tubercle bacillus, and their lymphocytes had subnormalin vitro proliferation responses to tuberculins fromM. tuberculosis andM. avium-intracellulare and to phytohemagglutinin. The administration of indomethacin reconstituted both thein vitro lymphocyte responses and delayed cutaneous hypersensitivity. We propose that the impairment of T-cell dependent immune functions is mediated by a suppressive factor (or factors) that is a metabolic product(s) of the cyclooxygenase pathway of arachidonic acid metabolism. Preferential inhibition of this pathway with indomethacin allows the expression of cell-mediated responses.     

T cell regulation of the chronic peritoneal neutrophilia during mycobacterial infections.

Intraperitoneal infection of mice with mycobacteria induces the persistent mobilization of neutrophils to the infected peritoneal cavities. The recruitment of the neutrophils was mediated by the immune system since it was enhanced by immunization and reduced in T cell-deficient nude and SCID mice. Anti-mitotic treatments with cyclophosphamide or X-rays led to a reduction in the number of mononuclear cells in the peritoneal cavity of infected mice, followed by a reduction in neutrophil numbers despite the presence of a normal circulating pool of neutrophils. The depletion of T cells with antibodies during mycobacterial i.p. infection led to a reduction in the number of neutrophils. Such a reduction was more extensive if the antibodies were administered early. Our data suggest that T cells are partially involved in the direct recruitment of neutrophils during chronic mycobacteriosis but they also play a role in the priming of other cell types for the mobilization of these phagocytes.     

Mycobacterial cervical lymphadenitis in children: can histological assessment help differentiate infections caused by non-tuberculous mycobacteria from Mycobacterium tuberculosis?

Most mycobacterial lymphadenitis in children in developed countries is caused by non-tuberculous (the so-called 'atypical') mycobacteria. In view of the widely different treatment regimes and the requirement for contact tracing in Mycobacterium tuberculosis infections but not in non-tuberculous mycobacterial infections, it is very important to attempt to define histologically which is the aetiological agent. We have reviewed the histological appearances of mycobacterial cervical lymphadenitis in children and have found that, if any one of several 'atypical' features were seen, the appearances were much more likely to be due to a non-tuberculous mycobacterium. These features include ill-defined (non-palisading) granulomas, irregular or serpiginous granulomas, a predominantly non-specific granulomatous response, predominantly sarcoid-like granulomas or lack of significant caseation. In addition, the non-tuberculous mycobacterial infections showed a different distribution of neutrophil polymorphs, which tended to be seen in the centre of areas of necrosis rather than in Mycobacterium tuberculosis infections where a polymorph infiltrate, if present, was more diffusely scattered. Although no one definitive feature is diagnostic of non-tuberculous mycobacterial infection, some features are helpful in differentiating the two groups of organisms histologically.     

Identification of nontuberculous mycobacteria in clinical samples using molecular methods: a 3-year study

Nontuberculous mycobacteria (NTM) are being increasingly isolated in clinical laboratories and present technical and therapeutic challenges. In the present study, we report our experience with the identification of NTM received from 12 Lisbon hospitals over a 3-year period using GenoType Mycobacterium (CM/AS) assays (HAIN Lifescience GmbH, Nehren, Germany). Together, the two kits identified 96.6% of all NTM isolates tested. Among the 18 NTM species identified, Mycobacterium avium complex was the most frequent, although it accounted for only 34% of all NTM. Introducing these methods for the rapid identification of NTM highlights the importance of NTM as potential pathogens and assisted the selection of adequate therapy.     

Soluble CD4 suppress the antigen driven proliferative response of certain T cell clones specific for mycobacteria and for peptides by mycobacterial heat shock proteins.

We have investigated the effect of soluble recombinant CD4 (sCD4) on the antigen specific (BCG, peptides of mycobacterial 65 kDa hsp) responses of T cell lines of T cell clones. The majority of the antigen specific clones could be suppressed in their antigen driven response by the addition of sCD4, while others, including the parental polyclonal T cell line, were not. The suppression of the specific T cell response was reversed by the addition of anti-CD3, did not affect the proliferative response to IL-2, and was independent of the amount of antigen. A decreased capacity to produce IFN-gamma in response to the antigen by the addition of sCD4 was seen only with those clones that were also inhibited in their specific proliferative response. This model may be used to delineate further the interaction between T cells and the antigen presenting cell, and the finding may limit the possible in vivo use of sCD4 in the therapy of human immunodeficiency virus (HIV) infections.     

The identification of mycobacteria from solid media and directly from VersaTREK Myco bottles using the Sherlock Mycobacteria Identification HPLC system

The Sherlock Mycobacteria Identification HPLC system correctly identified to the species level 61 (67.8%) of 90 isolates growing on solid media, and 73 (45.3%) of 161 isolates directly from positive VersaTREK Myco bottles. When these data were re-analysed with a revised database, correct identifications increased to 91.1% and 83.2%, respectively. All Mycobacterium tuberculosis isolates were identified correctly, regardless of the inoculum source or database used. The use of the revised database with isolates obtained directly from positive VersaTREK Myco bottles allows the identification of most isolates within clinically relevant time-frames.     

Specificity of antibodies induced after immunization of mice with the mycobacterial heat shock protein of 65 kD.

We have previously shown in mice and monkeys that mycobacterial heat shock proteins (hsp) of 65 and 70 kD exert a strong in vivo helper effect when conjugated to synthetic peptides or bacterial oligosaccharides and given in the absence of any adjuvants. Considering the degree of homology existing in the phylogeny among hsp belonging to the same family, we studied whether antibodies induced in mice with this protocol of immunization with the mycobacterial 65-kD hsp (hsp65) would cross-react, and to what extent, with hsp homologues from other origins, notably with the Escherichia coli GroEL protein and with the human homologue (hsp60). The results obtained show that antibodies to the mycobacterial hsp65 cross-reacted with the E. coli GroEL protein, both in ELISA and Western blot experiments, but not with the human hsp60. In competitive ELISA experiments, the binding of these antibodies to solid-phase hsp65 was very effectively inhibited by low concentrations of the mycobacterial hsp65; however, for human hsp60, 100 times higher concentrations were required in order to obtain similar patterns of inhibition. Finally, murine antibodies to the mycobacterial hsp65 always failed to give positive results in Western blot experiments using extracts of murine cells. Taken together, these data suggest that, after immunization of mice with the mycobacterial hsp65 conjugated to peptides or oligosaccharides in the absence of adjuvants, anti-hsp65 antibodies are induced which cross-react well with hsp homologues from other prokaryotes (e.g. E. coli GroEL), but which weakly bind the human hsp homologue. These results may have implications for the potential use of microbial hsp molecules in the design of conjugated vaccine constructs.     

Isolation of nontuberculous mycobacteria from hospital waters in Turkey

Nontuberculous mycobacteria (NTM) are ubiquitous in hot and cold water distribution systems. With molecular typing methods it was shown that water can be the source of colonization and infection with NTM. The aim of our study was the investigation of NTM in hot and cold water samples taken from various departments of two hospitals in Istanbul, Turkey. Totally, 160 water samples were examined. The temperature, pH, and free chlorine levels of water samples were measured between 10–41 °C, 6.78–7.98 and <0.3–0.5 mg/L, respectively. NTM were detected in 33 (20.6%) samples. Totally 20 (60.6%), 10 (30.3%) and 3 (9.1%) isolates were identified as Mycobacterium lentiflavum, Mycobacterium gordonae, and Mycobacterium peregrinum, respectively. M. lentiflavum, which was the most frequently isolated NTM, is characterized by multiple resistance to antimycobacterial drugs. Although no infections with this mycobacterium were reported from our country so far, preventive measures may be considered in patients under immunosuppression. Because no significant correlations were found among the presence of NTM or species distribution and water temperature, pH or free chlorine levels, other factors need to be investigated.     

Multiprimer PCR system for differential identification of mycobacteria in clinical samples.

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay.     

结核杆菌热休克蛋白—65重组质粒的构建及其在真核细胞中的初步表达

目的：构建结核杆菌热休克蛋白－65（HSP65）真核表达质粒,并探讨其在人肺癌细胞系A549中的表达,方法：用PCR的方法从结核杆菌H37Rv基因组中扩增出HSP65基因,并定向克隆到真核表达质粒pcDNA3.1( ）,构建成重组质粒pcDNA3.1-HSP65;然后用电穿孔的方法将质粒pcDNA3.1－65转染到A549细胞,经G418筛选后获得抗性细胞克隆;用RT－PCR的方法检抗性细胞中HSP65mRNA的表达,结果：经酶切鉴定和DNA序列测定证实重组质粒pcDNA3.1－HSP65构建正确;RT－PCR检测结果发现,重组质粒pcDNA3.1－HSP65转染的A549细胞总RNA中结核杆菌HSP65mRNA为阳性,结论：真核表达质粒pcDNA3.1－HSP65构建成功,并可在真核细胞A549细胞中稳定表达。     

GenoType mycobacterium assay for identification of mycobacterial species isolated from human clinical samples by using liquid medium

The GenoType Mycobacterium assay was used to identify 98 mycobacteria isolates by using liquid cultures from positive BACTEC, MGIT, and ESP bottles. This system identifies 16 mycobacteria. There was complete agreement between the GenoType results and the laboratory identifications for Mycobacterium tuberculosis complex and other Mycobacterium spp. GenoType also identified mixed mycobacterial infections.     

Bacille Calmette–Guérin/DNAhsp65 prime‐boost is protective against diabetes in non‐obese diabetic mice but not in the streptozotocin model of type 1 diabetes

Type I diabetes is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this study was to evaluate the prophylactic potential of a prime‐boost strategy involving bacille Calmette–Guérin (BCG) and the pVAXhsp65 vaccine (BCG/DNAhsp65) in diabetes induced by streptozotocin (STZ) in C57BL/6 mice and also in spontaneous type 1 diabetes in non‐obese diabetic (NOD) mice. BCG/DNAhsp65 vaccination in NOD mice determined weight gain, protection against hyperglycaemia, decreased islet inflammation, higher levels of cytokine production by the spleen and a reduced number of regulatory T cells in the spleen compared with non‐immunized NOD mice. In the STZ model, however, there was no significant difference in the clinical parameters. Although this vaccination strategy did not protect mice in the STZ model, it was very effective in NOD mice. This is the first report demonstrating that a prime‐boost strategy could be explored as an immunomodulatory procedure in autoimmune diseases.     

Molecular detection and identification of mycobacterium tuberculosis complex and four clinically important nontuberculous mycobacterial species in smear-negative clinical samples by the genotype mycobacteria direct test

Although the sensitivity and specificity of nucleic acid amplification assays are high with smear-positive samples, the sensitivity with smear-negative and extrapulmonary samples for the diagnosis of tuberculosis in suspicious tuberculosis cases still remains to be investigated. This study evaluates the performance of the GenoType Mycobacteria Direct (GTMD) test for rapid molecular detection and identification of the Mycobacterium tuberculosis complex and four clinically important nontuberculous mycobacteria (M. avium, M. intracellulare, M. kansasii, and M. malmoense) in smear-negative samples. A total of 1,570 samples (1,103 bronchial aspiration, 127 sputum, and 340 extrapulmonary samples) were analyzed. When we evaluated the performance criteria in combination with a positive culture result and/or the clinical outcome of the patients, the overall sensitivity, specificity, and positive and negative predictive values were found to be 62.4, 99.5, 95.9, and 93.9%, respectively, whereas they were 63.2, 99.4, 95.7, and 92.8%, respectively, for pulmonary samples and 52.9, 100, 100, and 97.6%, respectively, for extrapulmonary samples. Among the culture-positive samples which had Mycobacterium species detectable by the GTMD test, three samples were identified to be M. intracellulare and one sample was identified to be M. avium. However, five M. intracellulare samples and an M. kansasii sample could not be identified by the molecular test and were found to be negative. The GTMD test has been a reliable, practical, and easy tool for rapid diagnosis of smear-negative pulmonary and extrapulmonary tuberculosis so that effective precautions may be taken and appropriate treatment may be initiated. However, the low sensitivity level should be considered in the differentiation of suspected tuberculosis and some other clinical condition until the culture result is found to be negative and a true picture of the clinical outcome is obtained.