Mycobacterium Tuberculosis In Guinea Pigs Research Articles

Disruption of MenT2 toxin impairs the growth of Mycobacterium tuberculosis in guinea pigs.

Toxin-antitoxin (TA) systems are abundantly present in the genomes of various bacterial pathogens. TA systems have been implicated in either plasmid maintenance or protection against phage infection, stress adaptation or disease pathogenesis. The genome of Mycobacterium tuberculosis encodes for more than 90 TA systems and 4 of these belong to the type IV subfamily (MenAT family). The toxins and antitoxins belonging to type IV TA systems share sequence homology with the AbiEii family of nucleotidyl transferases and the AbiEi family of putative transcriptional regulators, respectively. Here, we have performed experiments to understand the role of MenT2, a toxin from the type IV TA system, in mycobacterial physiology and disease pathogenesis. The ectopic expression of MenT2 using inducible vectors does not inhibit bacterial growth in liquid cultures. Bioinformatic and molecular modelling analysis suggested that the M. tuberculosis genome has an alternative start site upstream of the annotated menT2 gene. The overexpression of the reannotated MenT2 resulted in moderate growth inhibition of Mycobacterium smegmatis. We show that both menT2 and menA2 transcript levels are increased when M. tuberculosis is exposed to nitrosative stress, in vitro. When compared to the survival of the wild-type and the complemented strain, the ΔmenT2 mutant strain of M. tuberculosis was more resistant to being killed by nitrosative stress. However, the survival of both the ΔmenT2 mutant and the wild-type strain was similar in macrophages and when exposed to other stress conditions. Here, we show that MenT2 is required for the establishment of disease in guinea pigs. Gross pathology and histopathology analysis of lung tissues from guinea pigs infected with the ∆menT2 strain revealed significantly reduced tissue damage and inflammation. In summary, these results provide new insights into the role of MenT2 in mycobacterial pathogenesis.

Evaluation of Mycobacterium tuberculosis Early Secreted Antigenic Target 6 Recombinant Protein as a Diagnostic Marker in Skin Test

ObjectivesTuberculosis (TB) is the leading infectious disease in the developing world. Delayed-type hypersensitivity skin test diagnoses TB using tuberculin purified protein derivative (PPD), but this test is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacillus Calmette–Guérin (BCG) vaccination or an infection caused by nontuberculous mycobacteria (NTM). This study was performed to evaluate the use of recombinant early secretory antigenic target 6 (rESAT-6), a secretory protein found only in MTB, Mycobacterium bovis, and few other mycobacterial species, as a skin marker for MTB in guinea pigs. MethodsWe prepared recombinant MTB ESAT-6 and evaluated its use as a specific antigen for MTB in guinea pigs. ResultsOur results show that the purified MTB rESAT-6 antigen is capable of inducing a positive reaction only in guinea pigs sensitized to MTB. No such reaction was observed in the animals sensitized to M. bovis, BCG vaccination, or NTM (Mycobacterium avium). ConclusionOur study results confirm that the ESAT-6 antigen is more specific to MTB infection than PPD and could be used in more specific skin tests for detection of MTB in large animals and in humans.

Cortisone-Forced Reactivation of Weakly Acid Fast Positive Mycobacterium Tuberculosis in Guinea Pigs Previously Treated With Chemotherapy

Cortisone-Forced Reactivation of Weakly Acid Fast Positive Mycobacterium Tuberculosis in Guinea Pigs Previously Treated With Chemotherapy

Guinea Pig Skin, a Model for Epidermal Cellular and Molecular Changes Induced by UVR in vivo and in vitro: Effects on Mycobacterium bovis Bacillus Calmette–Guérin Vaccination

Previously, we reported that ultraviolet B-radiation (UVR) suppressed Bacillus Calmette-Guérin (BCG) vaccine-induced resistance to Mycobacterium tuberculosis in guinea pigs (GP). Herein, we investigated the cellular and molecular changes within the irradiated GP epidermis and the in vivo effect of supernatants from UV-irradiated (200 J m(-2)) epidermal cells (UV-sup) on M. bovis BCG vaccination. UVR increased the number of nucleated keratinocytes in the skin, but caused a decrease in the proportions of CD25(+)T cells. In the spleen, UVR resulted in a decrease in the proportions of T-cell subsets including CD25(+)T cells, and major histocompatibility complex (MHC) class II(+) and CD14(+) cells. Similarly, significant up-regulation of several cytokine mRNAs including IL-10 was also observed. Furthermore, UV-sup significantly reduced the MHC class II expression in peritoneal cells and reduced T-cell proliferation to ConA. The proliferation to purified protein derivative (PPD) was restored to normal levels by anti-IL-10 antibody. The UV-sup when injected into BCG-vaccinated GP significantly diminished the skin test response and T-cell proliferation to PPD and up-regulated the expression of IL-10, IL-4, IL-1β and Foxp3 mRNAs in the lymph node or spleen. Thus, whole body UVR induces profound cellular and molecular changes and injection of UV-sup from epidermal cells mimics the effect of whole body UVR in BCG-vaccinated GP.

UVB-induced suppression of resistance to Mycobacterium tuberculosisin guinea pigs after BCG vaccination: cellular and molecular changes in the skin (99.19)

Abstract Previously, we reported that ultraviolet-B radiation (UVR) suppressed BCG vaccine-induced resistance to Mycobacterium tuberculosis in guinea pigs as it induced immuno-suppression and increased pathogenesis. In this study, we investigated the cellular and molecular changes within the guinea pig epidermis.The dorsal skin of guinea pigs was exposed to 5.6 kJ/m2 UVR and the epidermal cells were harvested 24 h later. Although UVR did not induce any gross morphological changes in the epidermis, a significant increase in the angular nucleated keratinocytes and a reduction in the number of cells with high nuclear to cytoplasmic ratio and binucleated cells were observed. Flow cytometry studies indicated that the proportions of CD3+ and CD4+ T cells increased in the epidermis while spleen showed a decrease in the CD3+, CD4+, CD8+, CD25+, MHC class II+ and CD14+ cells. Upregulation of IL-10, IL-4, IL-12p40, TGF-β, IL-1β and INOS mRNA expression was observed in the UV-irradiated epidermal cells. In addition, IL-4, IL-12p40, IFN-γ, and IL-1β mRNAs were increased in the spleen cells after UVR. Furthermore, culture supernatants from in vitro UV-irradiated keratinocytes reduced the MHC class II expression in the peritoneal cells and reduced the spleen cell proliferation to ConA. Future studies will address the identity of the suppressive factors induced by UVR and the systemic effect of culture supernatants from UV-irradiated epidermal cells on M. bovis BCG or M. tuberculosis infection.

The potent bactericidal activity of streptomycin in the guinea pig model of tuberculosis ceases due to the presence of persisters

The biphasic kill curve of isoniazid against Mycobacterium tuberculosis in guinea pigs is due to the presence of persisters rather than selection of isoniazid-resistant mutants. To determine whether this phenomenon is common to other bactericidal drugs, we studied the activity of streptomycin and its ability to select for streptomycin-resistant mutants in the guinea pig model of tuberculosis. Pharmacokinetic studies were performed to establish the human-equivalent dose of streptomycin. Guinea pigs were aerosol-infected with M. tuberculosis and 2 weeks later streptomycin was given for 5 days/week via intramuscular injection. Bactericidal activity was assessed by homogenizing and plating lungs for cfu until 10 weeks of treatment. At each timepoint, cfu were isolated, suspended in normal saline and re-plated on plates containing 0.5, 1.0, 2.0 or 10.0 mg/L streptomycin. The human-equivalent dose of streptomycin was determined to be 70 mg/kg. Streptomycin showed potent activity during the first 14 days of treatment, rescuing all animals from acute tuberculosis-related death and reducing lung cfu by ∼4 log(10). However, streptomycin activity was dramatically reduced thereafter, as lung cfu declined by only ∼1 log(10) over the next 56 days of treatment. Although streptomycin-resistant mutants were detectable, their frequency of isolation was identical at treatment initiation and after 70 days of treatment. The reduced activity of streptomycin during the second phase of monotherapy is not associated with the selection of streptomycin-resistant mutants but, rather, with the presence of phenotypically tolerant 'persisters'.

Biphasic Kill Curve of Isoniazid Reveals the Presence of Drug‐Tolerant, Not Drug‐Resistant,Mycobacterium tuberculosisin the Guinea Pig

The marked reduction in the potent early bactericidal activity of isoniazid during the initial phase of antituberculosis (anti-TB) therapy has been attributed not only to the depletion of logarithmically growing bacilli but also to the emergence of isoniazid resistance. We studied the anti-TB activity of isoniazid and its ability to select for drug-resistant mutant strains in guinea pigs, in which the histopathology of TB closely resembles that of human TB. Prior mouse passage did not appear to enhance the virulence of Mycobacterium tuberculosis in guinea pigs. The human-equivalent dose of isoniazid was determined to be 60 mg/kg. Although isoniazid therapy caused rapid killing of bacilli in guinea pig lungs during the first 14 days of administration and rescued guinea pigs from acute death, its activity was dramatically reduced thereafter. This reduction in activity was not associated with the emergence of isoniazid-resistant mutant strains but, rather, with the selection of phenotypically tolerant "persisters."

Dietary Polyunsaturated Fatty Acids Modulate Resistance to Mycobacterium tuberculosis in Guinea Pigs

It is well established that the nutritional status of the host affects resistance to disease. The impact of dietary lipids on experimental pulmonary infection with mycobacteria has not been investigated. Therefore, the purpose of this study was to determine the role of dietary (n-3) and (n-6) fatty acids on immunity and resistance to aerosol infection with virulent Mycobacterium tuberculosis in guinea pigs. Weanling guinea pigs were fed purified, isocaloric diets differing only in lipid source, and the effects of diet on specific immune cell functions were evaluated after 3 or 6 wk. Dietary (n-3) fatty acid consumption reduced in vivo skin test and in vitro lympho-proliferative responses (P < 0.05) relative to (n-6) fatty acid consumption. The effect of diet on resistance to mycobacterial infection was assessed by enumerating viable mycobacteria in the lungs and spleens of guinea pigs infected with virulent M. tuberculosis by the aerosol route. (n-3) Fatty acid-fed guinea pigs had more bacteria in the lungs compared with (n-6) fatty acid-fed guinea pigs at 3 (P < 0.05) and 6 wk postinfection (P < 0.01). These data document the immunomodulatory effects of (n-3) fatty acid consumption in the context of tuberculosis resistance. The loss of antigen-specific T-cell functions in addition to impaired resistance to mycobacterial disease suggests a susceptible phenotype in (n-3) fatty acid-fed guinea pigs.

Mycobacterium bovis BCG vaccination modulates TNF- α production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs

Tumor necrosis factor-alpha (TNF-alpha) plays critical and opposing roles in the pathogenesis of tuberculosis (TB). We examined the effects of Mycobacterium bovis BCG vaccination on TNF-alpha production in three distinct guinea pig leukocyte populations before and after pulmonary infection with M. tuberculosis H37Rv. Following BCG vaccination alone, and following challenge, bronchoalveolar lavage cells (BALC), resident peritoneal cells (PC), and splenocytes (SPC) were stimulated with purified protein derivative (PPD). Before virulent challenge, BCG vaccination clearly enhanced the ability of BALC, PC and SPC to produce TNF-alpha in response to PPD stimulation ex vivo. Following challenge, the TNF-alpha production of all three leukocyte populations from BCG-vaccinated animals remained relatively constant at pre-challenged levels. In sharp contrast, 5 weeks post-challenge, all three leukocyte populations from unvaccinated animals produced very high amounts of TNF-alpha in response to PPD. Three weeks post-challenge, SPC from one of the unvaccinated animals produced higher levels of TNF-alpha but the others produced lower levels of TNF-alpha than BCG-vaccinated animals. As expected, BCG vaccination reduced the levels of virulent mycobacteria in both the lungs and spleens. Thus, BCG vaccination allows guinea pigs to modulate TNF-alpha levels in conjunction with a reduction in bacillary loads in their tissues.

Importance of Catalse Enzyme in Virulence of Isoniazid Resistant Strains of Mycobacterium Tuberculosis in Guinea-Pigs

Importance of Catalse Enzyme in Virulence of Isoniazid Resistant Strains of Mycobacterium Tuberculosis in Guinea-Pigs

Growth characteristics of recent sputum isolates of Mycobacterium tuberculosis in guinea pigs infected by the respiratory route.

The consideration of virulence must distinguish between infectivity and the ability to cause progressive disease once the infection is established. Several investigators have reported the presence of naturally occurring isolates which differ in virulence for guinea pigs. Isolates from south India which differed with respect to gross disease and number of bacilli recovered from spleen after an intramuscular infection also differed in their efficiencies to initiate an infection, once inhaled and retained. Also, this difference was correlated with differences in the rate of multiplication at the site of implantation and rate of multiplication at sites of hematogenous seeding, as well as the extent of hematogenous seeding. The number of metastatic foci was identified as a quantitative measure of hematogenous seeding, which was not confounded by the rate of multiplication of bacilli. Even allowing for the fourfold-reduced efficiency of low-virulence tubercle bacilli to produce a lesion, this measure clearly revealed a significantly reduced ability of the low-virulence tubercle bacilli to disseminate via the bloodstream.

Structural requirements of muramylpeptides for induction of necrosis at sites primed with Mycobacterium tuberculosis in guinea pigs.

Intracutaneous injection of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) in guinea pigs caused an extensive necrotic reaction in footpads prepared by injection of heat-killed Mycobacterium tuberculosis in water-in-mineral-oil emulsion. We examined a variety of analogs and derivatives of muramylpeptides for their ability to provoke this reaction. A maximum and a minimum structure responsible for the necrotic reaction were found to be N-acetylglycosaminyl-beta(1-4)-N-acetylmuramyl-tripeptide (GlcNAc-MurNAc-L-Ala-D-isoGln-meso-A2pm) and MDP, respectively. An unexpected finding was that GlcNAc-MurNAc-tetrapeptides having L-amino acids at their C termini, unlike comparable compounds having C-terminal D-amino acids, exhibited definite necrosis-inducing activity, probably due to their tendency to undergo in vivo degradation to GlcNAc-MurNAc-tripeptide. Introduction of some acyl groups, especially the stearoyl group, to the 6-O position of the muramic acid or the peptide moiety of muramylpeptides increased the necrosis-inducing activity of the parent molecules. However, this was not observed with 1-thio-muramic acid analogs of MDP. Modification of the alpha- or gamma-carboxyl groups of the glutamic acid residues of muramylpeptides tended to decrease their necrosis-inducing ability. Analogs and derivatives of muramylpeptides which are capable of inducing necrosis at a primed site, with few exceptions, exhibited powerful adjuvanticity against ovalbumin in guinea pigs. However, the reverse was not necessarily true.

Observations on the measurement of skin hypersensitivity to tuberculin

Summary In tuberculin positive subjects there is a linear relationship between the area of skin induration and the log-dose of the intradermal tuberculin injected in a constant volume. The area of skin induration is influenced by the volume of injected intradermal fluid as well as the tuberculin dose. A mathematical form is deduced to enable these variations to be predicted. A linear relationship is found to exist between the area of skin induration and the dose of infecting organisms. A study of the results occuring with BCG organisms in human subjects and Mycobacterium tuberculosis in guinea pigs is used to compare the sensitising ability of these organisms and calibrate the skin reaction in terms of the antigenic stimulus.

STREPTOMYCIN IN TUBERCULOUS MENINGITIS

Tuberculous meningitis has always carried a hopeless prognosis. The use of streptomycin in the treatment of this disease has already altered the outlook. Waksman and his associates 1 reported the isolation of a new antibiotic agent in 1944. Feldman and Hinshaw reported in 1944 2 its apparent ability to suppress the growth of Mycobacterium tuberculosis in guinea pigs. Since that time several reports in the literature have confirmed the bacteriostatic action of streptomycin on Myco. tuberculosis. Its first successful use in tuberculous meningitis was reported by Cooke, Dumphy and Blake, 3 who treated an infant 1 year of age. Krafchik 4 reported another apparent arrest in a child 5 months of age, significant in that no residual neural damage was noted. Recently Hinshaw, Feldman and Pfeutze 5 reported a series of 9 cases of combined miliary and meningeal tuberculosis in 4 of which the patients survived. In all their successful