Response of Hypersensitive Mice to the Footpad Injection of Living Homologous or Heterologous Mycobacteria: Preliminary Report

Mice sensitized by the injection of viable mycobacteria into one of the hind footpads responded to a second injection of mycobacteria (3 to 4 weeks later), introduced into the contralateral foot, with a degree of footpad swelling that was both accelerated and exaggerated beyond that observed after the first inoculation. The degree of specificity of this reaction (i.e., response to homologous versus heterologous mycobacteria) was comparable to that previously reported for dermal reactions of hypersensitive guinea pigs to tuberculin or tuberculin-like antigens from mycobacteria. In preliminary studies it was impossible to achieve this state of specific sensitization by vaccinating mice subcutaneously with water-in-oil emulsions of heat-killed mycobacteria; reasons for the failure are discussed. It is suggested that this tool could prove useful in both taxonomic and immunological investigations. Advantages and disadvantages of the mouse footpad test in relation to the dermal skin test in guinea pigs are discussed.     

Purified Protoplasmic Peptides of Mycobacteria: In Vivo and In Vitro Comparison of the Species Specificity of Purified Protoplasmic Peptides and Purified Protein Derivatives of Mycobacterial Culture Filtrates

The specificity of purified protein derivatives (PPD) prepared from the culture filtrates of Mycobacterium tuberculosis (PPD), M. kansasii (PPD-Y), M. intracellulare (PPD-B), and M. scrofulaceum (PPD-G) were compared to comparable protoplasmic extracts (PPP) of the same organisms by gel diffusion and delayed hypersensitivity reactions in sensitized guinea pigs. PPD and, to a lesser degree, PPD-Y demonstrated specificities sufficient to enable identification of homologously sensitized guinea pigs in the above group of four mycobacteria. PPD-B and PPD-G did not always elicit the largest reaction in homologously sensitized animals. The PPP sensitins from M. tuberculosis and M. kansasii produced as good skin reactions at 24 and at 48 hr as did their PPD counterparts. The PPP from M. scrofulaceum and M. intracellulare were more specific and more reactive than corresponding PPD, regardless of the time of comparison. Although based on different immunological mechanisms, the specificity of these two groups of sensitins, as demonstrated by delayed hypersensitivity, correlated well with serological comparisons in the gel diffusion test. The low degree of specificity of PPD-B and PPD-G in contrast to that of corresponding PPP was reflected in the precipitin bands in agar gel.     

High-Throughput Sequencing Enhanced Phage Display Identifies Peptides That Bind Mycobacteria

Bacterial cell wall components have been previously used as infection biomarkers detectable by antibodies. However, it is possible that the surface of the Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), also possesses molecules which might be non-antigenic. This makes the probing of biomarkers on the surface of M. tb cell wall difficult using antibodies. Here we demonstrate the use of phage display technology to identify peptides that bind to mycobacteria. We identified these clones using both random clone picking and high throughput sequencing. We demonstrate that random clone picking does not necessarily identify highly enriched clones. We further showed that the clone displaying the CPLHARLPC peptide which was identified by Illumina sequencing as the most enriched, binds better to mycobacteria than three clones selected by random picking. Using surface plasmon resonance, we showed that chemically synthesised CPLHARLPC peptide binds to a 15 KDa peptide from M.tb H37Rv whole cell lysates. These observations demonstrate that phage display technology combined with high-throughput sequencing is a powerful tool to identify peptides that can be used for investigating potential non-antigenic biomarkers for TB and other bacterial infections.     

Tuberculin Reactions in Bulls And Boars Sensitized with Atypical Mycobacteria from Sawdust

Due to a planned export from a combined bull and boar station, more than 70 boars and 100 bulls were examined by tuberculin tests. Distinct reactions to avian tuberculin appeared in about half of the animals. Many of them also reacted to bovine tuberculin. For diagnostic purposes, many of the reactors were slaughtered, and samples from these and from the environment were examined bacteriologically. Strains of Mycobacterium avium were isolated from only 2 out of 14 reacting boars and from none of the 23 reacting bulls. No isolation of Mycobacterium bovis was made. However, atypical mycobacterial strains, classified as Runyon Group III and IV, were isolated from 3 boars, 2 bulls, 1 pigeon and from many samples of sawdust. The isolation of identical fast-growing mycobacterial strains from the sawdust used in the pens for the reacting boars and bulls, was especially remarkable. The strains differed enzymatically and biochemically from those isolated from other sources. This indicated a possible sensitization of the animals with similar mycobacterial strains. Possible cross-reactions to avian and bovine tuberculin were investigated in tuberculin assays with guniea pigs and pigs sensitized to one of the mycobacterial strains isolated. Distinct reactions to both avian and bovine tuberculin appeared in all the animals. From these results it was concluded that the tuberculin reactions in the boars and the bulls were not due to any tuberculous infection in the herd, but to a sensitization of the animals with atypical mycobacteria.     

New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli

The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.     

Chromatographic analysis and immunobiologic properties of tuberculins]

Immunobiologic activities of tuberculin preparations and their components have been comparatively studied using gel filtration and high-performance liquid chromatography (HPLC) techniques. It is shown that high-molecular weight fraction of protein-purified derivate tuberculin (PPD) had higher activity as compared to nonfractionated preparation in skin tests on guinea pigs sensitized with Mycobacterium bovis BCG as well as in enzyme-linked immunosorbent assay with affinity purified rabbit antibodies against PPD. Using the preparative HPLC-technique we failed to isolate a component of PPD having greater tuberculin test potency than nonfractionated preparation.     

Diagnostic Key to Mycobacteria Encountered in Clinical Laboratories

A diagnostic key has been developed which will permit identification of most mycobacteria encountered in clinical laboratories. The key is based on performance of a few simple tests. The efficiency and accuracy of the key was evaluated in terms of correlation between identifications based on the few tests and those arrived at through application of the techniques of numerical taxonomy, which involves a large battery of tests. Of 679 cultures of mycobacteria other than Mycobacterium tuberculosis, 86.5% were correctly identified by use of the key, and only 1.8% of the cultures were erroneously identified. The remaining cultures required further examination.     

Development of a Paper Strip Test for Detection of Niacin Produced by Mycobacteria

The development of a reagent-impregnated paper strip test for niacin is described. The test system is based on the formation of cyanogen chloride by the reaction of chloramine-T and potassium thiocyanate in the presence of citric acid. Rupture of the pyridine ring of niacin by cyanogen chloride yields gamma-carboxy glutaconic aldehyde and coupling with a primary aromatic amine produces a yellow color. Sensitivity to niacin, both in known solutions and from extracts of 378 clinical mycobacteria isolates, equalled or exceeded that of other methods for detection of niacin. Correlation with other tests for mycobacterial niacin was excellent.     

Evaluation of a tetrazolium salt test to determine absence of live mycobacteria in tuberculin purified protein derivative.

Current methodology to determine absence of live mycobacteria in tuberculin purified protein derivative (PPD) takes up to 8 weeks to perform and may also involve testing on animals. In this paper we describe an in vitro test utilising the tetrazolium salt, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to monitor the absence of live Mycobacterium tuberculosis (Mtb) in PPD. In the presence of live cells XTT is converted to a coloured formazan product that can be measured spectrophotometrically. Live mycobacteria present in spiked PPD were detected by a marked change in optical density above background levels. This test is easy to perform and is complete in just 48 hr.     

Long-Term Preservation and Storage of Mycobacteria

Under contract with the National Institute of Allergy and Infectious Diseases, the Trudeau Mycobacterial Culture Collection has been greatly expanded to provide for the scientific community a collection of representative strains of mycobacteria of biomedical importance. Problems concerned with the preparation, bottling, and distribution of such organisms have been dealt with and are detailed in this paper. Examination of collected data revealed that the temperature of storage and not the suspending menstruum was more important for prolonged survival of mycobacteria stored at subzero temperatures. For optimum results, mycobacteria may be suspended either in Dubos Tween-albumin broth or in Middlebrook 7H-9 liquid medium supplemented with ADC enrichment (commercial sources used) and stored at -70 C. Either of these suspending fluids supplied a growth-supporting medium for cultures which must be shipped long distances, often without refrigeration. To avoid sublimation of suspending medium during prolonged storage at -70 C (a problem inherent in many screw-capped containers), we have chosen to use vaccine-stoppered serum bottles sealed with aluminum crimp caps. The methods described have provided suspensions with (i) excellent viability over prolonged periods of storage, (ii) stable metabolic activities, and (iii) highly reproducible inocula for animal experiments.     

Mycobacteria in the light of modern genetics development

It is generally assumed that genetic research of mycobacteria is delayed as compared with other, more commonly used, bacterial models, particularly in the field of genetic transfers. In the field of mutagenesis the problems have been studied to such an extent that replication maps of the chromosome of M. phlei and M. tuberculosis H37 Rv have already been constructed and a new model of the cell cycle of bacteria exhibiting a slow growth rate has been worked out. When the problems of mycobacterial genetics are looked upon in the light of gene manipulations it may be concluded that mycobacteria belong to a few models whose genes are used for cloning and that problems of practical significance will be studied by means of the most modern approaches.     

Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria: II. Skin Cross-Reactivity in Sensitized Guinea Pigs

A study on skin cross-reactivity between stabilized ¹⁴C-labeled mycobacterial antigens, namely tuberculin purified protein derivative (PPD; from Mycobacterium tuberculosis), PPD-A (M. avium), PPD-Y (M. kansasii), PPD-G (M. scrofulaceum), PPD-B (M. intracellulare), and PPD-F (M. fortuitum), has been carried out in groups of guinea pigs sensitized with one of the following heat-killed mycobacteria: M. tuberculosis, M. avium, M. kansasii, M. scrofulaceum, M. intracellulare, or M. fortuitum. For each type of sensitization, the average response for the corresponding PPD antigen was higher than the average response for any of the other antigens. However, the responses to the heterologous PPD antigens were not necessarily significantly different among themselves, and the significant differences of the heterologous PPD antigens were distributed differently according to the type of sensitization. Therefore, ¹⁴C-PPD antigens skin cross-reacted in guinea pigs essentially in the same manner as reported by others for nonradioactive PPD antigens.     

Interpretation of the PPD skin test in BCG-vaccinated children.

Skin testing with 5 tuberculin units (TU) of purified protein derivative (PPD) of tuberculin stabilized with polysorbate (Tween) 80 was done 3 months and 1 year after immunization with bacille Calmette-Guérin (BCG) vaccine in two groups of children: one group vaccinated at birth and another group at age 6 years. Interpretation of the PPD skin test with 5 TU is possible in children 1 year and older vaccinated with BCG at birth: if the diameter of induration is more than 10 to 12 mm the reaction cannot be ascribed to BCG vaccination and is highly suggestive of supervening infection with Mycobacterium tuberculosis or occasionally atypical mycobacteria. In contrast, the interpretation of a PPD test in children vaccinated at age 6 years is extremely difficult.     

Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.     

Optimized Method for Preparation of DNA from Pathogenic and Environmental Mycobacteria

Genomic studies on pathogenic and environmental mycobacteria are of growing interest for understanding of their evolution, distribution, adaptation, and host-pathogen interaction. Since most mycobacteria are slow growers, material from in vitro cultures is usually scarce. The robust mycobacterial cell wall hinders both experimental cell lysis and efficient DNA extraction. Here, we compare elements of several DNA preparation protocols and describe a method that is economical and practical and reliably yields large amounts—usually 10-fold increased compared to earlier protocols—of highly pure genomic DNA for sophisticated downstream applications. This method was optimized for cultures of a variety of pathogenic and environmental mycobacterial species and proven to be suitable for direct mycobacterial DNA extraction from infected insect specimens.     

Confinement-Induced Drug-Tolerance in Mycobacteria Mediated by an Efflux Mechanism

Tuberculosis (TB) is the world’s deadliest curable disease, responsible for an estimated 1.5 million deaths annually. A considerable challenge in controlling this disease is the prolonged multidrug chemotherapy (6 to 9 months) required to overcome drug-tolerant mycobacteria that persist in human tissues, although the same drugs can sterilize genetically identical mycobacteria growing in axenic culture within days. An essential component of TB infection involves intracellular Mycobacterium tuberculosis bacteria that multiply within macrophages and are significantly more tolerant to antibiotics compared to extracellular mycobacteria. To investigate this aspect of human TB, we created a physical cell culture system that mimics confinement of replicating mycobacteria, such as in a macrophage during infection. Using this system, we uncovered an epigenetic drug-tolerance phenotype that appears when mycobacteria are cultured in space-confined bioreactors and disappears in larger volume growth contexts. Efflux mechanisms that are induced in space-confined growth environments contribute to this drug-tolerance phenotype. Therefore, macrophage-induced drug tolerance by mycobacteria may be an effect of confined growth among other macrophage-specific mechanisms.     

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.     

Staphylococcal Enterotoxin B: Solid-Phase Radioimmunoassay

An immunoassay employing (125)I-labeled enterotoxin B and polystyrene tubes coated with specific antibody was used for assaying purified and crude enterotoxin. Antibody was adsorbed to untreated polystyrene tubes. Unlabeled enterotoxin competed with (125)I-labeled enterotoxin for antibody-combining sites. The uptake of (125)I-labeled toxin reflected the concentration of unlabeled toxin present. The test is sensitive to 1 to 5 ng of purified and crude enterotoxin B per ml, and cross-reactions with heterologous enterotoxins did not interfere with the specificity. This test possesses the combination of sensitivity and objectivity absent in current methods for assaying enterotoxin and provides a model for investigating other enterotoxin serotypes.     

Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.     

First Detection of Mycobacteria in African Rodents and Insectivores, Using Stratified Pool Screening

With the rising number of patients with human immunodeficiency virus (HIV)/AIDS in developing countries, the control of mycobacteria is of growing importance. Previous studies have shown that rodents and insectivores are carriers of mycobacteria. However, it is not clear how widespread mycobacteria are in these animals and what their role is in spreading them. Therefore, the prevalence of mycobacteria in rodents and insectivores was studied in and around Morogoro, Tanzania. Live rodents were trapped, with three types of live traps, in three habitats. Pieces of organs were pooled per habitat, species, and organ type (stratified pooling); these sample pools were examined for the presence of mycobacteria by PCR, microscopy, and culture methods. The mycobacterial isolates were identified using phenotypic techniques and sequencing. In total, 708 small mammals were collected, 31 of which were shrews. By pool prevalence estimation, 2.65% of the animals were carriers of mycobacteria, with a higher prevalence in the urban areas and in Cricetomys gambianus and the insectivore Crocidura hirta. Nontuberculous mycobacteria (Mycobacterium chimaera, M. intracellulare, M. arupense, M. parascrofulaceum, and Mycobacterium spp.) were isolated from C. gambianus, Mastomys natalensis, and C. hirta. This study is the first to report findings of mycobacteria in African rodents and insectivores and the first in mycobacterial ecology to estimate the prevalence of mycobacteria after stratified pool screening. The fact that small mammals in urban areas carry more mycobacteria than those in the fields and that potentially pathogenic mycobacteria were isolated identifies a risk for other animals and humans, especially HIV/AIDS patients, that have a weakened immune system.