sodium-dodecyl-sulfate and Tuberculosis

Serine/threonine protein kinases (STPK) play a major role in the physiology and pathogenesis of Mycobacterium tuberculosis. Here, we have examined the role of pknE, a STPK in the adaptive responses of M. tuberculosis using a deletion mutant ΔpknE. The survival of ΔpknE was assessed in the presence of stress (pH, surfactant and cell wall-damaging agents) and anti-tuberculosis drugs. ΔpknE had a defective growth in pH 7.0 and lysozyme (a cell wall-damaging agent) with better survival in pH 5.5, SDS and kanamycin (a second-line anti-tuberculosis drug). Furthermore, ΔpknE was reduced in cell size during growth in liquid media and exhibited hypervirulence in a guinea pig model of infection. In conclusion, our data suggest that pknE plays a role in adaptive response of M. tuberculosis regulating cellular integrity and survival.

Topics: Animals; Antitubercular Agents; Drug Resistance, Bacterial; Guinea Pigs; Hydrogen-Ion Concentration; Muramidase; Mycobacterium tuberculosis; Polymorphism, Restriction Fragment Length; Protein Serine-Threonine Kinases; Sequence Deletion; Sodium Dodecyl Sulfate; Stress, Physiological; Tuberculosis

Pathogenic mycobacteria possess two homologous chaperones encoded by cpn60.1 and cpn60.2. Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study, we show that inactivation of the Mycobacterium bovis BCG cpn60.1 (Mb3451c) gene does not significantly affect bacterial growth in 7H9 broth, but that this knockout mutant (Δcpn60.1) forms smaller colonies on solid 7H11 medium than the parental and complemented strains. When growing on Sauton medium, the Δcpn60.1 mutant exhibits a thinner surface pellicle and is associated with higher culture filtrate protein content and, coincidentally, with less protein in its outermost cell envelope in comparison with the parental and complemented strains. Interestingly, in this culture condition, the Δcpn60.1 mutant is devoid of phthiocerol dimycocerosates, and its mycolates are two carbon atoms longer than those of the wild-type, a phenotype that is fully reversed by complementation. In addition, Δcpn60.1 bacteria are more sensitive to stress induced by H(2)O(2) but not by SDS, high temperature or acidic pH. Taken together, these data indicate that the cell wall of the Δcpn60.1 mutant is impaired. Analysis by 2D gel electrophoresis and MS reveals the upregulation of a few proteins such as FadA2 and isocitrate lyase in the cell extract of the mutant, whereas more profound differences are found in the composition of the mycobacterial culture filtrate, e.g. the well-known Hsp65 chaperonin Cpn60.2 is particularly abundant and increases about 200-fold in the filtrate of the Δcpn60.1 mutant. In mice, the Δcpn60.1 mutant is less persistent in lungs and, to a lesser extent, in spleen, but it induces a comparable mycobacteria-specific gamma interferon production and protection against Mycobacterium tuberculosis H37Rv challenge as do the parental and complemented BCG strains. Thus, by inactivating the cpn60.1 gene in M. bovis BCG we show that Cpn60.1 is necessary for the integrity of the bacterial cell wall, is involved in resistance to H(2)O(2)-induced stress but is not essential for its vaccine potential.

Topics: Animals; Anti-Bacterial Agents; Bacterial Load; Bacterial Proteins; Cell Wall; Culture Media; Disease Models, Animal; Electrophoresis, Gel, Two-Dimensional; Gene Knockout Techniques; Genetic Complementation Test; Hydrogen Peroxide; Lipids; Lung; Mass Spectrometry; Mice; Mice, Inbred BALB C; Molecular Chaperones; Mycobacterium bovis; Mycolic Acids; Oxidative Stress; Proteome; Rodent Diseases; Sodium Dodecyl Sulfate; Spleen; Tuberculosis

The ability of physicians to diagnose tuberculosis is impacted by the use of smear and culture techniques combined with specimen processing methods. The objective of this study was to evaluate the effects of specimen processing on smear and culture sensitivity by comparing the specimen processing method that uses C(18)-carboxypropylbetaine with the method that combines sodium dodecyl sulfate and sodium hydroxide. A total of 1,201 specimens were entered into this study. Specimens were split approximately equally such that one-half of each specimen was processed with sodium dodecyl sulfate-sodium hydroxide, while the other half was processed with C(18)-carboxypropylbetaine. All sediments were subjected to acid-fast staining and then analyzed using the MB/BacT liquid culture system (bioMérieux, France) and solid media. The sensitivity of smear following processing with sodium dodecyl sulfate-sodium hydroxide and C(18)-carboxypropylbetaine was 61.2% and 58.6% (P>0.05), respectively, while the specificities were identical (99.7%). The sensitivity of culture was 84.2% and 96.1% (P<0.05), respectively. The time to detection in the MB/BacT liquid culture system was 13.2+/-5.6 and 15.0+/-8.8 days (P>0.05), respectively, and 20.0+/-7.6 and 15.7+/-8.9 days (P<0.05), respectively, on solid media. The contamination rates in the MB/BacT system were 0.8% and 8.7%, respectively, whereas the contamination rates on solid media were 2.6% and 4.3%, respectively. C(18)-carboxypropylbetaine specimen processing was less labor-intensive than sodium dodecyl sulfate-sodium hydroxide processing and improved the ability of laboratory staff to detect the presence of mycobacteria by culture.

Topics: Bacteriological Techniques; Betaine; Chi-Square Distribution; Culture Media, Conditioned; Female; Humans; Male; Mycobacterium tuberculosis; Probability; Reagent Kits, Diagnostic; Sensitivity and Specificity; Sodium Dodecyl Sulfate; Sodium Hydroxide; Specimen Handling; Tuberculosis

A total of 52 mycobacterial isolates were recovered from 1,197 clinical specimens decontaminated by a sodium dodecyl (lauryl) sulfate (SDS)-NaOH protocol. Of these, 94% were recovered with the BacT/Alert 3D system (Organon Teknika, Durham, N.C.) and 79% were recovered on Löwenstein-Jensen (LJ) medium. Mean times to detection of organisms of the Mycobacterium tuberculosis complex (n = 47) were 22.8 days with LJ medium and 16.2 days with the system. The BacT/Alert 3D system is a rapid and efficient detection system which can be used with an SDS-NaOH decontamination procedure.

Topics: Bacteriological Techniques; Culture Media; Disinfection; Humans; Mycobacterium; Mycobacterium Infections; Mycobacterium tuberculosis; Sodium Dodecyl Sulfate; Sodium Hydroxide; Tuberculosis

To evaluate specimen processing with sodium dodecyl (lauryl) sulfate (SDS) on the detection of Mycobacterium tuberculosis by PCR-reversed dot hybridization.. Totally 266 specimens were collected from sputum, pleural fluid, ascites, urine or blood of patients with active tuberculosis and were treated with SDS or routine method. All specimens handled were tested for detecting M. tuberculosis by PCR-reversed dot hybridization.. The detection rate of M. tuberculosis from specimens prepared by SDS method was 65.0% (173/266) as compared with 51.5% (136/168) by routine method. Results from two methods were considered statistically significant (chi(2) = 35.03, P < 0.001).. The SDS processing method may be used to detect M. tuberculosis from clinical specimens by PCR and enhances the detection rate of M. tuberculosis.

Topics: Detergents; Humans; Mycobacterium tuberculosis; Peritonitis, Tuberculous; Polymerase Chain Reaction; Sodium Dodecyl Sulfate; Tuberculosis; Tuberculosis, Meningeal; Tuberculosis, Pleural; Tuberculosis, Pulmonary; Tuberculosis, Renal

Mycobacterium bovis BCG vaccine strains were compared with Mycobacterium tuberculosis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 25-kDa protein observed in the BCG strains was absent in M. tuberculosis. Rabbit antibodies specific to the 25-kDa protein uniquely identified this protein in BCG strains but not in M. tuberculosis. It is suggested that the 25-kDa protein and polyclonal antibodies directed against this antigen can be exploited to distinguish BCG strains from M. tuberculosis.

Topics: Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; BCG Vaccine; Electrophoresis, Polyacrylamide Gel; Humans; Immunocompromised Host; Molecular Weight; Mycobacterium bovis; Mycobacterium tuberculosis; Sodium Dodecyl Sulfate; Species Specificity; Tuberculosis

Between 1974 and 1978 there were carried out comparative studies concerning the value of several detergents used to homogenization of diagnostic specimens in six countries (Bulgaria, Czechoslovakia, German Democratic Republic, Poland, The Soviet Union and Yugoslavia). In the first study it was confirmed that the homogenization of diagnostic materials by detergents gives good results and is more economical than conventional Petroff's method. In the majority of centers the highest detection of tubercle bacilli was found after homogenization by Sodium-Laurylsulphated-technique. Remarkable differences in the time of growth of tubercle bacilli after homogenization of the same samples by Laurylsulphate, Laurosept and Petroff's lye method were not observed. While, after homogenization by Nekal BX the time of growth was a little bit retarded. In the lowest percentage of contamination was observed after homogenization by Nekal BX. In the second study the value of Polish detergents (Chlorhexidinum gluconicum and Laurosept) was compared with Ditalan OW hc made in GDR and with other routinely in the participating laboratories used techniques. The best results were obtained after homogenization of specimens by Chlorhexidinum gluconicum. But the comparison to Laurosept and Ditalan WO hc showed no significant differences. Homogenization of sputa was better and number of contaminations lower after using laurosept or Ditalan WO hc than by means of Chlorhexidinum gluconicum.

Topics: Chlorhexidine; Detergents; Microbiological Techniques; Naphthalenesulfonates; Pyridinium Compounds; Sodium Dodecyl Sulfate; Sputum; Surface-Active Agents; Tuberculosis