Effect of methodology, dilution, and exposure time on the tuberculocidal activity of glutaraldehyde-based disinfectants

The Association of Official Analytical Chemists (AOAC) test for assessing the tuberculocidal activity of disinfectants has been shown to be variable. A modified AOAC test, which substituted Middlebrook 7H9 broth as the primary subculture medium and used neutralization by dilution, was compared with the standard AOAC method to assess the mycobactericidal activity of three glutaraldehyde-based disinfectants at 20 degrees C and various exposure times. These changes had a marked effect on results, with the modified AOAC test providing more positive penicylinders per 10 replicates in 12 of the 13 comparisons that provided positive results. These differences were observed with both Mycobacterium bovis (ATCC 35743) and a clinical isolate of Mycobacterium tuberculosis. The effects of various exposure times to and dilutions of the glutaraldehyde-based disinfectants were also examined. The minimum exposure time needed to inactivate reliably M. bovis or M. tuberculosis with 2% glutaraldehyde was 20 min at 20 degrees C. Diluting 2% glutaraldehyde caused a significant decline in mycobactericidal activity. Modification of the standard AOAC test to improve its sensitivity in detecting the failure of disinfectants to inactivate mycobacteria is indicated.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Rapid screening method for Mycobactericidal activity of chemical germicides that uses Mycobacterium terrae expressing a green fluorescent protein gene

The slow growth of mycobacteria in conventional culture methods impedes the testing of chemicals for mycobactericidal activity. An assay based on expression of the green fluorescent protein (GFP) by mycobacteria was developed as a rapid alternative. Plasmid pBEN, containing the gene encoding a red-shifted, high-intensity GFP mutant, was incorporated into Mycobacterium terrae (ATCC 15755), and GFP expression was observed by epifluorescence microscopy. Mycobactericidal activity was assessed by separately exposing a suspension of M. terrae(pBEN) to several dilutions of test germicides based on 7.5% hydrogen peroxide, 2.4% alkaline glutaraldehyde, 10% acid glutaraldehyde, and 15.5% of a phenolic agent for contact times ranging from 10 to 20 min (22 degrees C), followed by culture of the exposed cells in broth (Middlebrook 7H9) and measurement of fluorescence every 24 h. When the fluorescence was to be compared with CFU, the samples were plated on Middlebrook 7H11 agar and incubated for 4 weeks. No increase in fluorescence or CFU occurred in cultures in which the cells had been inactivated by the germicide concentrations tested. Where the test bacterium was exposed to ineffective levels of the germicides, fluorescence increased after a lag period of 1 to 7 days, corresponding to the level of bacterial inactivation. In untreated controls, fluorescence increased rapidly to reach a peak in 2 to 4 days. A good Pearson correlation coefficient (r > or =0.85) was observed between the intensity of fluorescence and the number of CFU. The GFP-based fluorescence assay reduced the turnaround time in the screening of chemical germicides for mycobactericidal activity to < or =7 days.     

Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis

An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.     

Inactivation of Mycobacterium bovis in cattle slurry by five volatile chemicals

This research was undertaken to evaluate volatile chemicals which retained mycobactericidal activity in cattle slurry. Mycobacterium bovis, suspended in sterilized cattle slurry, was treated with different concentrations of five volatile chemicals with mycobactericidal activity. Following treatment of the slurry for specified time intervals, the reaction mixture was lyophilized to remove the volatile chemicals, and samples of the reconstituted slurry were used to inoculate flasks of Lowenstein-Jensen medium to determine survival or inactivation of the mycobacteria. Acetone, at a concentration of 22.5%, inactivated M. bovis in less than 24 h. Ammonium hydroxide, at a concentration of 1%, was mycobactericidal after 36 h. Chloroform at a concentration of 0.5%, ethyl alcohol at a concentration of 17.5% and xylene at a concentration of 3% inactivated the mycobacteria within 48 h. Some of the volatile chemicals with mycobactericidal activity are potentially useful at farm level.     

Survival of Mycobacterium bovis on feedstuffs commonly used as supplemental feed for white-tailed deer (Odocoileus virginianus)

Mycobacterium bovis, the causative agent of bovine tuberculosis, has become established in free-ranging white-tailed deer Odocoileus virginianus in northeastern Michigan. The practice of supplemental feeding of white-tailed deer during the winter is believed to contribute to transmission of M. bovis between deer. The current study was conducted to determine the ability of M. bovis to survive on various feedstuffs commonly used as supplemental feed for deer in northeast Michigan (i.e., apples, corn, carrots, sugar beets, potatoes, and hay) and the effect of maintenance at -20 C, 8 C, and 23 C on survival. Mycobacterium bovis survived on all feedstuffs at all temperatures tested for at least 7 days. At 23 C, M. bovis could still be isolated from samples of apples, corn and potatoes at 112 days. This study suggests that contamination of feedstuffs by M. bovis-infected deer could act as a source of indirect transmission between deer because M. bovis is able to survive in temperatures similar to those recorded during winter months in northeastern Michigan. Current efforts to ban or control supplemental feeding of deer should have a positive effect on decreasing transmission of M. bovis among deer.     

Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 x 10(3) to 3.6 x 10(3) gene copies g of soil(-1), depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 degrees C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

An Examination of the Effect of Three Phenolic Disinfectants on Mycobacterium tuberculosis

The effect of a phenolic disinfectant ( o -phenylphenol 45% w/w) with linseed oil soap or with soya oil soap on Mycobacterium tuberculosis was determined by three methods. Neither the geometrical dilution test nor the modified capacity use-dilution test revealed any differences between the two disinfectants. However, paradoxically both methods proved that the highest concentration of the disinfectants tested (3·5% v/v) exhibited a very low germicidal effect on M. tuberculosis , whereas lower concentrations showed a much better effect. When determining the tuberculocidal effects of various concentrations of the two disinfectants at different exposure times, the higher concentrations showed very low effects, even after the longest exposure time. At concentrations of 2·0 and 1·0% (v/v), the disinfectants displayed the most rapid effects. In the present investigation the disinfectant with the linseed oil soap seemed to destroy the cells more quickly than that with the soya oil soap. The third disinfectant containing p -chloro- m -cresol and o -benzyl- p -chlorophenol with a total of 9·2% (w/w) phenols in a detergent system, did not display, when employing the capacity use-dilution test, the same phenomenon in the concentrations used, but the experiment showed that the recommended use-dilution concentration ought to be doubled.     

Evaluation of a novel commercial quaternary ammonium compound for eradication of Mycobacteria,HCV and HBV in Egypt

Endoscopes are a common source of outbreaks of healthcare-associated infections. It is therefore important to identify high-level disinfectants capable of eliminating or killing all vegetative bacteria, mycobacteria, and viruses. Aldehydebased disinfectants are most commonly used in clinical practice but resistance has recently been detected and side effects associated with these disinfectants are well documented. In this study, we evaluated Virusolve+® EDS, a novel quaternary ammonium compound formulation supplied by Amity international, against Mycobacterium bovis (ATCC-27289), hepatitis C virus (HCV)-positive serum and hepatitis B surface antigen-positive serum. We also compared its efficacy against Cidex® (glutaraldehyde 2%), an aldehyde-based disinfectant. M. bovis showed no growth after 10 weeks with either Virusolve+® or Cidex®. Virusolve+® achieved a 10⁴- fold reduction in the initial 10⁶ HCV load under clean conditions (without red blood cells) for 20 min, whereas Cidex® achieved this reduction under clean and dirty conditions (without and with red blood cells, respectively) after both 10 and 20 min. Both Virusolve+® and Cidex® were able to eradicate hepatitis B virus (HBV) infectivity under clean conditions after 10 and 20 min, whereas under dirty conditions they were only able to eradicate virus infectivity after 20 min. Virusolve+® EDS when compared with Cidex® showed equal mycobactericidal activity completely eradicating M. bovis. However, both showed comparable virucidal activity against HBV, which was more effective under clean conditions, emphasizing the importance of the cleaning step in endoscope reprocessing. Cidex® was more effective at eradicating HCV under dirty conditions after a short contact time.     

Methyl chloride utilising bacteria are ubiquitous in the natural environment

A 17-kDa protein (CadI) was induced by cadmium in Mycobacterium bovis and Mycobacterium tuberculosis. Comparison of the N-terminal sequence from M. bovis CadI with the annotated M. tuberculosis genome database identified Rv2641 as the encoding gene. Long and short promoter fragments from M. bovis cadI were fused to the lacZ reporter gene in pYUB76. Only the long fragment directed cadmium-inducible activity when electroporated into M. bovis. The cadI promoter has potential for both constitutive and inducible expression studies in M. bovis and M. tuberculosis.     

Effect of proton pump inhibitor alone or in combination with clarithromycin on mycobacterial growth in human alveolar macrophages

The effect of omeprazole, a clinically used proton pump inhibitor, alone or in combination with clarithromycin was evaluated against Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium tuberculosis, using a human alveolar macrophage model of infection. Omeprazole exhibited no significant effect on the growth of the two M. avium complex strains or on the mycobactericidal activity of clarithromycin against them. In contrast, omeprazole significantly promoted the growth of Mycobacterium tuberculosis and the anti-mycobacterial activity of clarithromycin against it in human alveolar macrophages. It was speculated that intracellular acidic milieu around M. tuberculosis might be one reason for the lower activity of clarithromycin in the treatment of human tuberculosis.     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.     

应用多重PCR方法检测石蜡包埋组织标本中的分枝杆菌DNA

应用多重PCR方法检测并鉴别石蜡包埋组织中的结核分枝杆菌复合体与非结核分枝杆菌DNA扩增片段类型 ,为结核分枝杆菌复合体感染与非结核分枝杆菌感染的病理学诊断提供一种补充的鉴别诊断方法。应用三对具有特异性的寡核苷酸引物 ,进行多重PCR扩增。这三对引物分别对应于分枝杆菌 6 5kD表面抗原、结核分枝杆菌插入序列IS6 1 1 0及人类β 珠蛋白基因的部分序列 ,其扩增产物分别为 3 83bp、1 2 3bp和 2 6 8bp。此种多重PCR方法检测的灵敏度为 0 6pg。经多重PCR扩增后进行凝胶电泳 ,结核分枝杆菌复合体 (结核分枝杆菌、牛型结核分枝杆菌、BCG)均可见 3 83bp、1 2 3bp片段 ,而非结核分枝杆菌 (鸟、龟、瘰疬、蟾蜍、堪萨斯、胞内、耻垢分枝杆菌 )仅见 3 83bp片段 (猿猴分枝杆菌与结核分枝杆菌复合体相同 )。与上述相比 ,分枝杆菌感染的临床标本分别增加了一条 2 6 8bp片段。对 2 0 9例临床初步诊断为淋巴结结核病人的石蜡包埋组织标本进行了多重PCR检测 ,1 93例病理诊断为淋巴结结核、结核性肉芽组织、结核性肉芽肿性炎症病人的标本 ,检测结果符合结核分枝杆菌复合体感…     

Possibility of existence and multiplication of Mycobacteria in the soil

The time of the survival of Mycobacterium bovis in the soil of pastures depended on the physicochemical composition and biological activity of the soil. In clay soils M. bovis retained their virulence for as long as 2 months (at the depth of 2 cm in high- and medium-posted solonetz) to 12 months (at the depth of 10 cm in crust solonetz). The M. bovis strains under study multiplied in soil extracts (the term of observation was 8 months). The cultures grown at 20-24 degrees C were found to have the prolonged exponential phase in comparison with that of the cultures grown at 37.5-38.5 degrees C. In the deep litter and the soil under it M. avium remained viable for 10 years.     

Antibody bound to the surface antigen MPB83 of Mycobacterium bovis enhances survival against high dose and low dose challenge

Tuberculosis caused by infection with Mycobacterium tuberculosis or Mycobacterium bovis is a significant disease of man and animals. Whilst cellular immunity is the major immunological component required for protection against these organisms, recent reports have suggested that monoclonal antibodies can modify infection with M. tuberculosis. To test whether the same was true for M. bovis infection, we determined the effect of preincubation of M. bovis with a monoclonal antibody on subsequent intravenous infection of mice. Antibodies bound to the surface of M. bovis increased the survival time of mice infected with M. bovis and changed the morphology of granulomas and the distribution of acid-fast bacilli in the lung. These studies suggest that antibodies directed to the surface of virulent mycobacteria can modulate their virulence in vivo.     

Mannosylated lipoarabinomannans inhibit IL-12 production by human dendritic cells: evidence for a negative signal delivered through the mannose receptor

IL-12 is a key cytokine in directing the development of type 1 Th cells, which are critical to eradicate intracellular pathogens such as Mycobacterium tuberculosis. Here, we report that mannose-capped lipoarabinomannans (ManLAMs) from Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis inhibited, in a dose-dependent manner, the LPS-induced IL-12 production by human dendritic cells. The inhibitory activity was abolished by the loss of the mannose caps or the GPI acyl residues. Mannan, which is a ligand for the mannose receptor (MR) as well as an mAb specific for the MR, also inhibited the LPS-induced IL-12 production by dendritic cells. Our results indicate that ManLAMs may act as virulence factors that contribute to the persistence of M. bovis bacillus Calmette-Guérin and M. tuberculosis within phagocytic cells by suppressing IL-12 responses. Our data also suggest that engagement of the MR by ManLAMs delivers a negative signal that interferes with the LPS-induced positive signals delivered by the Toll-like receptors.     

Rapid and simple approach for identification of Mycobacterium tuberculosis and M. bovis by detection of regulatory gene whiB7

Identification of Mycobacterium tuberculosis and M. bovis is necessary for the application of adequate drug therapy. PCR amplification is a good tool for this purpose, but choosing proper target is of a great concern. We describe a PCR assay for fast detection of M. tuberculosis and M. bovis.As a BLAST and BLASTP search we selected regulatory gene whiB7 that encodes multi-drug resistance in this bacterium. Thirty clinical isolates of M. tuberculosis were sequenced and all the mutations in gene whiB7 were detected. The best set of several pairs of primers was selected and used in comparison by rpoB gene for differentiation of M. bovis, M. avium, M. kansasii, M. phlei, M. fortuitum, M. terrae, seven non-pathogenic Mycobacterium isolates and 30 clinical isolates of M. tuberculosis.It was proved that only clinical isolates of M. tuberculosis and M. bovis have positive bands of 667 bp whiB7. Other non-tuberculous and non-pathogenic isolates did not show any positive sign. Furthermore, 667-bp PCR products of whiB7 gene were observed for ten positive sputum samples (preliminarily approved to be positive for M. tuberculosis by commercially real-time based method), but no bands were detected in 5 negative sputum samples. RpoB gene could not differentiate non-tuberculous strains and non-pathogenic isolates from pathogenic clinical isolates. We concluded that PCR amplification of the gene coding for the WhiB7 protein could be successfully used as a good tool for rapid identification of M. tuberculosis and M. bovis. We propose application of this method as a rapid and simple approach in mycobacteriological laboratories.