Infection of mice with aerosolized Mycobacterium tuberculosis: use of a nose-only apparatus for delivery of low doses of inocula and design of an ultrasafe facility

Aerosolized delivery of virulent or hypervirulent Mycobacterium tuberculosis requires careful consideration of methodology and safety. To maximize safety, we installed a nose-only aerosol apparatus that can reproducibly deliver a low dose (<100 CFU per mouse) of M. tuberculosis in a carefully designed biohazard facility.     

A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis

With the advent of HIV and the widespread emergence of drug-resistant strains of Mycobacterium tuberculosis, newer control strategies in the form of a better vaccine could decrease the global incidence of tuberculosis. A desirable trait in an effective live attenuated vaccine strain is an ability to persist within the host in a limited fashion in order to produce important protective antigens in vivo. Attenuated M. tuberculosis vaccine candidates have been constructed by deleting genes required for growth in mice. These candidate vaccines did not elicit adequate protective immunity in animal models, due to their inability to persist sufficiently long within the host tissues. Here we report that an auxotrophic mutant of M. tuberculosis defective in the de novo biosynthesis of pantothenic acid (vitamin B5) is highly attenuated in immunocompromised SCID mice and in immunocompetent BALB/c mice. SCID mice infected with the pantothenate auxotroph survived significantly longer (250 days) than mice infected with either bacille Calmette-Guerin (BCG) vaccine or virulent M. tuberculosis (77 and 35 days, respectively). Subcutaneous immunization with this auxotroph conferred protection in C57BL/6J mice against an aerosol challenge with virulent M. tuberculosis, which was comparable with that afforded by BCG vaccination. Our findings highlight the importance of de novo pantothenate biosynthesis in limiting the intracellular survival and pathogenesis of M. tuberculosis without reducing its immunogenicity in vaccinated mice.     

Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress

Bacteria can, under certain conditions, enter into a cell-less state known as L-form conversion. This phenomenon is universal, but also recognized with difficultly by microbiologists. The current study addresses several aspects concerning the ability of tubercle bacilli to use L-form conversion as a unique adaptive strategy to survive and reproduce under unfavorable conditions. Nutrient starvation of M. tuberculosis in vitro followed by passages in Middlebrook 7H9 semisolid medium was used for stress induction and the selective isolation of mycobacterial L-form variants. Light and electron microscopy images evidence the peculiar characteristics of mycobacterial L-forms. For example, mycobacterial L-forms were observed to lose their acid-fastness and change their morphology. In addition, wide morphological variability, the presence of large and elementary bodies, coccoids and small granular forms, as well as the appearance of unusual modes of irregular cell division were observed. Unlike classical tubercle bacilli, L-form variants grew and developed typical "fried-egg" colonies faster. L-forms were verified as M. tuberculosis by spoligotyping. The results provide insights into the nature of L-form phenomena in M. tuberculosis and link them to the mechanisms allowing mycobacterial survival under stress.     

Bioaerosol mass spectrometry for rapid detection of individual airborne Mycobacterium tuberculosis H37Ra particles

Single-particle laser desorption/ionization time-of-flight mass spectrometry, in the form of bioaerosol mass spectrometry (BAMS), was evaluated as a rapid detector for individual airborne, micron-sized, Mycobacterium tuberculosis H37Ra particles, comprised of a single cell or a small number of clumped cells. The BAMS mass spectral signatures for aerosolized M. tuberculosis H37Ra particles were found to be distinct from M. smegmatis, Bacillus atrophaeus, and B. cereus particles, using a distinct biomarker. This is the first time a potentially unique biomarker was measured in M. tuberculosis H37Ra on a single-cell level. In addition, M. tuberculosis H37Ra and M. smegmatis were aerosolized into a bioaerosol chamber and were sampled and analyzed using BAMS, an aerodynamic particle sizer, a viable Anderson six-stage sampler, and filter cassette samplers that permitted direct counts of cells. In a background-free environment, BAMS was able to sample and detect M. tuberculosis H37Ra at airborne concentrations of >1 M. tuberculosis H37Ra-containing particles/liter of air in 20 min as determined by direct counts of filter cassette-sampled particles, and concentrations of >40 M. tuberculosis H37Ra CFU/liter of air in 1 min as determined by using viable Andersen six-stage samplers. This is a first step toward the development of a rapid, stand-alone airborne M. tuberculosis particle detector for the direct detection of M. tuberculosis bioaerosols generated by an infectious patient. Additional instrumental development is currently under way to make BAMS useful in realistic environmental and respiratory particle backgrounds expected in tuberculosis diagnostic scenarios.     

A personal sampler to monitor airborne particles of biological origin

Aerobiological studies are important in many fields, specifically in medicine as they enable the identification of those airborne particles of biological origin coming into contact with man. These data are useful to evaluate the cause-effect ratio, to prevent allergy, to adjust the dose of drugs. A personal sampler for aerobiological particles (PARTRAP FA 52) was handed over to eleven atopic patients for a monitoring day. The sampling room was placed at about 15 cm from nose and mouth. The patients also underwent the Prick test and RAST test. The personal sampler for aerobiological particles enables a qualitative and quantitative sampling of the air close to the patient. More allergenic particles were found with the aerobiological personal sampling as compared to the sensitisation shown by Prick test and RAST in the same patient. This sampling method can be useful to demonstrate the cause-effect ratio directly near the patient and, moreover and can be indicative for further clinical and immunological investigations.     

Synthesis of gatifloxacin derivatives and their biological activities against Mycobacterium leprae and Mycobacterium tuberculosis

Novel 3′-piperazinyl derivatives of the 8-hydrogeno and 8-methoxy-6-fluoro-1-cyclopropyl-4-quinolone-3-carboxylic acid scaffolds were designed, synthesized and characterized by ¹H, ¹³C and ¹⁹F NMR, and HRMS. The activity of these derivatives against pathogenic mycobacteria (M. leprae and M. tuberculosis), wild-type (WT) strains or strains harboring mutations implicated in quinolone resistance, were determined by measuring drug concentrations inhibiting cell growth (MIC) and/or DNA supercoiling by DNA gyrase (IC₅₀), or inducing 25% DNA cleavage by DNA gyrase (CC₂₅). Compound 4 (with a methoxy in R₈ and a secondary carbamate in R₃′) and compound 5 (with a hydrogen in R₈ and an ethyl ester in R₃′) displayed biological activities close to those of ofloxacin but inferior to those of gatifloxacin and moxifloxacin against M. tuberculosis and M. leprae WT DNA gyrases, whereas all of the compounds were less active in inhibiting M. tuberculosis growth and M. leprae mutant DNA gyrases. Since R₃′ substitutions have been poorly investigated previously, our results may help to design new quinolone derivatives in the future.     

Characteristics of clinical isolates of Mycobacterium tuberculosis using molecular biological methods

A total of 230 clinically drug-resistant and 3 drug-sensitive isolates of M. tuberculosis obtained from patients in Tula and Tula region in 1998-2001 were studied. The RFLP-IS6110 genotyping showed that 52 (30.2%) of isolates had unique patterns, and 120 (69.8%) of them were grouped to form 16 clusters. 95 (55.2%) and 53 (30.8%) of isolates were attributed to groups A1 and W (Beijing), respectively. Double mycobacterium cultures were detected in 4.1% of isolates. 2 to 4 clinical isolates were obtained from each of 55 patients during 1.5 years. A replacement of mycobacterium isolates was registered in the course of treatment in 12 (21.8%) patients. No replacement of clinical isolates occurred in 43 (78.2%) patients during the whole follow-up period. Repeatedly obtained isolates acquired the determinants of drug-resistance in 5 (11.6%) patients. Changes in the quantity of IS6110 elements were registered only in 1.05% of isolates during a 3-year follow-up.     

The use of molecular biological methods for the individual characterization of Mycobacterium tuberculosis strains

The expediency of using molecular biological methods for the evaluation of M. tuberculosis clinical strains by individual genetic certification of circulating M. tuberculosis strains has been substantiated. Considerable genetic heterogeneity of M. tuberculosis strains isolated from patients in different regions of the Russian Federation has been established; this heterogeneity is due to the presence of differences in the number of copies (5-26) of element IS6110 in M. tuberculosis cells.     

Preparation and biological evaluation of ethionamide-mesoporous silicon nanoparticles against Mycobacterium tuberculosis

Ethionamide (ETH) is an important second-line antituberculosis drug used for the treatment of patients infected with multidrug-resistant Mycobacterium tuberculosis. Recently, we reported that the loading of ETH into thermally carbonized-porous silicon (TCPSi) nanoparticles enhanced the solubility and permeability of ETH at different pH-values and also increased its metabolization process. Based on these results, we synthesized carboxylic acid functionalized thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi NPs) conjugated with ETH and its antimicrobial effect was evaluated against Mycobacterium tuberculosis strain H37Rv. The activity of the conjugate was increased when compared to free-ETH, which suggests that the nature of the synergy between the NPs and ETH is likely due to the weakening of the bacterial cell wall that improves conjugate-penetration. These ETH-conjugated NPs have great potential in reducing dosing frequency of ETH in the treatment of multidrug-resistant tuberculosis (MDR-TB).     

A data-mining approach to spacer oligonucleotide typing of Mycobacterium tuberculosis

MOTIVATION: The Direct Repeat (DR) locus of Mycobacterium tuberculosis is a suitable model to study (i) molecular epidemiology and (ii) the evolutionary genetics of tuberculosis. This is achieved by a DNA analysis technique (genotyping), called sp acer oligo nucleotide typing (spoligotyping ). In this paper, we investigated data analysis methods to discover intelligible knowledge rules from spoligotyping, that has not yet been applied on such representation. This processing was achieved by applying the C4.5 induction algorithm and knowledge rules were produced. Finally, a Prototype Selection (PS) procedure was applied to eliminate noisy data. This both simplified decision rules, as well as the number of spacers to be tested to solve classification tasks. In the second part of this paper, the contribution of 25 new additional spacers and the knowledge rules inferred were studied from a machine learning point of view. From a statistical point of view, the correlations between spacers were analyzed and suggested that both negative and positive ones may be related to potential structural constraints within the DR locus that may shape its evolution directly or indirectly. RESULTS: By generating knowledge rules induced from decision trees, it was shown that not only the expert knowledge may be modeled but also improved and simplified to solve automatic classification tasks on unknown patterns. A practical consequence of this study may be a simplification of the spoligotyping technique, resulting in a reduction of the experimental constraints and an increase in the number of samples processed.     

Protein tyrosine phosphatase PtpA is not required for Mycobacterium tuberculosis growth in mice

Mycobacterium tuberculosis (Mtb) alters the host response to infection by secreting protein factors. Mtb produces two secreted protein tyrosine phosphatases, PtpA and PtpB, which are thought to interfere with host signaling. Deletion of ptpA or ptpB attenuates bacterial growth in activated macrophages. To address the in vivo function of PtpA, we generated a genetic deletion mutant, DeltaptpA. The mutant was not defective when grown in vitro, consistent with the presumed role of PtpA in the host. The ptpA mutant, however, also showed no growth defect in a mouse infection model. The absence of a growth defect in mice suggests that the requirement for PtpA differs in mouse and human infections, and that mice are not a suitable infection model for the study of PtpA.     

Aging and immunity to tuberculosis: prolonged survival of old mice infected with Mycobacterium tuberculosis by adoptive immunization with memory-immune T lymphocytes

This study shows that memory immune T lymphocytes, harvested from young (3-month-old) mice infected intravenously with Mycobacterium tuberculosis and then exposed to protracted chemotherapy with isoniazid, are capable of adoptively protecting old (24 month) mice from a subsequent fatal challenge infection. Survival of such adoptively protected animals was prolonged, but did not extend beyond the mean survival time of uninfected old control mice. During this time the passively transferred memory T cell population retained their functional capacity to protect against subsequent tuberculosis infection. These data indicate, therefore, that reconstitution of decayed cell-mediated antimicrobial immunity in old mice in vivo with memory T cells is technically feasible, although the life span of the animal is not extended over that of control animals. They indicate, moreover, that the memory T cells remain functional in what some reports have considered a suppressive environment and show further that the macrophages with which the infused T cells interact in the aged host remain functionally able to express acquired resistance.     

Peptide deformylase inhibitors of Mycobacterium tuberculosis: Synthesis,structural investigations,and biological results

Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalyzing the removal of the N-terminal formyl group from newly synthesized proteins. We report the synthesis and biological activity of highly potent inhibitors of Mycobacterium tuberculosis (Mtb) PDF enzyme as well as the first X-ray crystal structure of Mtb PDF. Structure–activity relationship and crystallographic data clarified the structural requirements for high enzyme potency and cell based potency. Activities against single and multi-drug-resistant Mtb strains are also reported.     

A continuous method to compute model parameters for soft biological materials

Developing appropriate mathematical models for biological soft tissues such as ligaments, tendons, and menisci is challenging. Stress-strain behavior of these tissues is known to be continuous and characterized by an exponential toe region followed by a linear elastic region. The conventional curve-fitting technique applies a linear curve to the elastic region followed by a separate exponential curve to the toe region. However, this technique does not enforce continuity at the transition between the two regions leading to inaccuracies in the material model. In this work, a Continuous Method is developed to fit both the exponential and linear regions simultaneously, which ensures continuity between regions. Using both methods, three cases were evaluated: idealized data generated mathematically, noisy idealized data produced by adding random noise to the idealized data, and measured data obtained experimentally. In all three cases, the Continuous Method performed superiorly to the conventional technique, producing smaller errors between the model and data and also eliminating discontinuities at the transition between regions. Improved material models may lead to better predictions of nonlinear biological tissues' behavior resulting in improved the accuracy for a large array of models and computational analyses used to predict clinical outcomes.