Middlebrook 7H9 Research Articles

Cryopreservation of Mycobacterium tuberculosis Complex Cells

Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.

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.

Filterable forms and L-forms of Mycobacterium bovis BCG

Bacterial L-form conversion, or existence without cell walls, is assumed a universal phenomenon in nature. An interesting aspect of this phenomenon is occurrence of L-forms in vaccine strains. Since BCG is currently a widely used and extensively studied live vaccine for tuberculosis, understanding L-form conversion of M. bovis BCG bacilli can provide new insight into behavior of BCG vaccine. In this respect, specific features, concerning the ability of BCG vaccine to produce viable filterable forms and L-forms, were studied by filtration and starvation stress experiments in vitro. The filterable forms obtained after filtration of BCG suspension, grew on Middlebrook 7H9 semisolid agar and formed typical “fried eggs” L-form colonies. Electron microscopy clearly demonstrated presence of L-form elements with size smaller than the size of bacterial filter pores of 0.2 µm in M. bovis BCG strains. Development of L-form subpopulation with typical morphological appearance of self-replicating cell wall-defective forms was observed after filtration, as well as after starvation stress. Specific DNA detection of pncA gene in derived L-form cultures from filterable and stressed BCG strains verified their identity as M. bovis BCG. In conclusion, the results confirm existence of filterable forms in commercial BCG vaccine, which are able to develop L-form population under appropriate conditions. L-form transformation of BCG bacilli displays a new intriguing aspect concerning exhibition of unusual features and atypical behavior of live BCG vaccine. Further research is requested to explore the influence of L-form phenomenon on BCG vaccine effects in vivo

Primary anti-tuberculous drugs resistance of pulmonary tuberculosis in Southwestern Saudi Arabia

The emergence of drug-resistant tuberculosis (TB) is an increasing problem which adversely affects patient care and public health. This study aimed to determine the rates and patterns of primary anti-TB drugs resistance in Najran, Southwestern Saudi Arabia. The study included 80 smear-positive new pulmonary TB patients. Sputum samples were cultured on Lowenstein-Jensen and Middle-Brook 7H10 media. Mycobacterium tuberculosis susceptibility testing was done by the conventional agar proportion method for isoniazide (INH), rifampicin (RIF), streptomycin (SPM) and ethambutol (EMB). Out of the 68 M. tuberculosis isolates, 42 (61.8%) were sensitive to all 4 drugs and 26 (38.2%) were resistant to one or more drugs. The most common resistance was found to INH (33.8%), followed by RIF (23.5%), SPM (13.2%) and EMB (2.9%). Eight (11.8%) isolates were resistant to one drug, 14 (20.6%) were resistant to 2 drugs, 3 (4.4%) were resistant to 3 drugs and one (1.5%) was resistant to 4 drugs. Multi-drug resistant (MDR) isolates were found in 14 (20.6%) cases. In conclusion, the primary resistance rate to four first-line anti-tuberculous drugs and MDR-TB rate are worryingly high, representing an alarming situation in Najran. Further studies are necessary for continuous surveillance of M. tuberculosis resistance patterns.

Improved productivity of recombinant human-like collagen ii by supplying amino acids encoded by rare codons

The spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis has become a challenge to the global tuberculosis control and prevention program.Nevertheless, no adequate information has been made available on the drug susceptibility status of M. tuberculosis in Ethiopia. Hence, this study aimed atevaluating the drug resistance patterns of M. tuberculosis in the three sites of Ethiopia and its association with socio-demographic factors. A total of 102 M. tuberculosis isolates obtained from tuberculosis (TB) patients from three different sites were tested for drug susceptibility against the first and second-line anti-TB drugs using the proportion method on enriched Middle Brook 7H10 agar. The result showed that 36.3% of the isolates were resistant to at least one of the first line anti-TB drugs used in this study. The proportions of mono-resistance were 29.4, 13.7, 13.7 and 23.5% to isoniazid, rifampicin, streptomycin and ethambutol, respectively. The proportion of MDR M. tuberculosis was 11.8%. Only 55% of the isolates were susceptible to all the second line anti-TB drugs used in this study. One isolate (1%) was extensively drug-resistant (XDR). The occurrence of MDR M. tuberculosisisolates was not associated with sex, age, anti-TB treatment history, HIV status andlocation. The findings of this study showed moderate number of MDR M. tuberculosis isolates in the study area, warranting the necessity of the surveillance and monitoring. Key words: Mycobacterium tuberculosis, drug resistance, Ethiopia

Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods

Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 106MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

Efficacy of Cryptolepis sanguinolenta root extract on slow-growing rifampicin resistant Mycobacterium tuberculosis

We report here, the results from total crude methanol extract as well as serial ether, chloroform, and methanol extracts of the root of Cryptolepis sanguinolenta (Lindl) Schltr that were screened against three strains of Mycobacterium tuberculosis. The strains used included the pan sensitive H37Rv, the rifampicin-resistant TMC-331 and a wild strain of Mycobacetrium avium (MA) isolated from a Ugandan patient. The disc diffusion method was used for susceptibility tests on solid Middle brook 7H10 while the minimum inhibitory concentration(MIC) and minimum bactericidal concentration (MBC) were determined by the microtitre plate method using Middle brook 7H9 broth. We report that the total crude methanol extract showed the highest activity against H37Rv and TM-331 with complete clearance of quadrants at 50 mg/ml and zones of inhibition of 10.0 to 11 mm at 25 mg/ml concentration although it was not effective against M. avium. The corresponding MIC values were 1.17 mg/ml for H37Rv and 1.56 mg/ml for TMC-331. The values for isoniazid were 0.25 and 9.38 µg/ml for H37Rv and TMC-331, respectively, while for rifampicin the MIC value was 0.25 µg/ml for H37Rv but it was not active on TMC-331. Acute toxicity test gave an LD50 of 758.5 mg/kg body weight while the phytochemical analysis showed the presence of alkaloids, tannins and flavonoids. Key words: Anti-mycobacterial, in vitro activity, medicinal plant, Cryptolepis sanguinolenta, Mycobacterium tuberculosis, Mycobacterium avium, rifampicin, isoniazid.

Synthesis, characterization and in-vitro antitubercular activity of isoniazid-gelatin conjugate

A novel and simple method to synthesize antitubercular-protein conjugate by solid phase synthesis was developed employing a carboxypolystyrene resin. The aim was to covalently bind a drug with antitubercular activity, isoniazid, to a biomacromolecule, gelatin, widely used in the pharmaceutical, cosmetic and food industry. Calorimetric and (1) H NMR analyses were performed to verify the bond formation between the antitubercular drug and gelatin. After absorption isoniazid delivers toxic metabolites and so an oxidation test with tert-butyl hydroperoxide was performed to assess the amount of toxic metabolites released from the prodrug (gelatin linked to isoniazid), compared with isoniazid itself. Spectrophotometric analysis revealed that the protein derivative was an excellent isoniazid prodrug since there was a 40% reduction in release of toxic metabolites (isonicotinic acid) by the prodrug. The results clearly showed that antitubercular moieties, covalently linked to a natural polymer, allowed the introduction of peculiar features for specific pharmaceutical applications into the macromolecule. In addition, antitubercular activity of the new polymer was determined by Middlebrook 7H11 medium against Mycobacterium tuberculosis complex. The new isoniazid-gelatin conjugate showed significant antitubercular activity and for this reason should be useful as an efficacious tool in the treatment of tuberculosis.

The performance of tofu-whey as a liquid medium in the propagation of mycobacterium tuberculosis strain H37Rv

ObjectiveTo investigate the performance of “tofu-whey liquid medium” for the propagation of Mycobacterium tuberculosis (MTB) strain H37Rv. MethodTwo hundred micro liters (200μl) of 1 McFarland standard (1mg/ml-bacillary suspension) were inoculated into different batches of tofu-whey liquid medium. Each series contained three trials of test (tofu-whey liquid medium) and control media (Middlebrook 7H9 medium). Turbidity was measured within three weeks of inoculation using a nephelometer. The combinations of various tofu-whey liquid culture media were as follows; T1 (tofu-whey+ADC+glycerol+Potassium sulfate+Magnesium citrate+Sodium glutamate); T2 (tofu-whey+ADC+glycerol+Potassium sulfate+Magnesium citrate); T3 (tofu-whey+ADC+glycerol+Potassium sulfate); T4 (tofu-whey+ADC+glycerol); T5 (tofu-whey+ADC); and T6 (tofu-whey only). ResultsIn all test and control liquid culture media, the multiplication of M. tuberculosis was documented under light and fluorescence microscopy. Of various tofu-whey medium used, T1 demonstrated the most potential for MTB propagation. The increased turbidity reading represented by the value in “unit drop of % transmittance” was higher (25 scores) in the T1 tofu-whey medium, compared with the T6 tofu-whey medium (8 scores). The overall growth was significantly better in Middlebrook 7H9 culture media, although by the third day of incubation, the bacillary growth was superior in the T1 tofu-whey culture media. Sub-cultures in Lowenstein–Jensen (L–J) medium yielded between 87% (47 of 54) and 89% (48 of 54) recovery rate with between 7% and 13% contamination rate with coagulase-negative staphylococci. ConclusionTofu-whey media can be used as an economical alternative to Middlebrook 7H9 in resource-limited settings.

Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium bovis

This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10(5) CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.

Comparison of the Lowenstein-Jensen Medium, the Middlebrook 7H10 Medium and MB/BacT for the Isolation of Mycobacterium Tuberculosis (MTB) from Clinical Specimens

Tuberculosis (TB) is the most common cause of death due to a single infectious agent worldwide in adults. India alone accounts for 30% of the global tuberculosis burden. There is a need for a method of cultivation of mycobacteria that is reliable and economical and has a short turnaround time. The present study was attempted to assess the feasibility of using MB BACT and Middlebrook 7h10(MB7H10) as primary isolation media for mycobacteria. They were compared with the LJ medium, which was the gold standard. Various clinical specimens from a total of 236 clinically suspected cases of TB were studied. All the samples were decontaminated by using the modified Petroff's method. Each sample was subjected to ZN staining and it was simultaneously inoculated onto the LJ medium, the MB7H10 medium and MB BACT. The growth from the cultures were confirmed by ZN staining and they were speciated by using biochemical reactions. Out of the 236 samples which were screened, 116 isolates were obtained. All the 116 were isolated from MB BACT, 82 were isolated from the LJ medium and 62 were isolated from MB7H10. 82 isolates were obtained from MB BACT and the LJ medium, 62 were obtained from MB7H10 and MB BACT, 58 were isolated from LJ and MB7H10 and 58 were isolated from LJ medium, MB7H10 and MB bact. Neither the L J medium nor the Middlebrook 7h 10 medium could isolate mycobacteria exclusively. It showed that the combination of media did not prove to be superior over the use of MB BACT alone. The average isolation time of L J, the MB7H10 medium and MB BACT was 30.81 days, 31.06 days and 18.70 days. MB BACT is a better medium as compared to the L J medium and the MB7H10 medium, both in terms of the number of isolates and the isolation rate. The MB BACT method proved to be a very speedy method and it could isolate mycobacteria 7-10 days earlier as compared to the L J medium and the Middlebrook 7 H10 medium.

Preliminary Evaluation of Mycobacterium tuberculosis Detection in Culture and Artificial Sputum Using a BioNanoPore Membrane and Realtime PCR

The rapid detection and diagnosis of Mycobacterium tuberculosis is critical to evaluate disease severity, efficacy of treatments and therapeutics, and public health monitoring. This study evaluated a BioNanoPore technology (BNP™ Middlebrook agar) to detect and quantitate M. tuberculosis in less time than traditional plate counting methods. BNP™ Middlebrook enabled visual detection of M. tuberculosis from actively-growing cultures and inoculated artificial sputum within 5 days; however, colonies were not visible on Middlebrook 7H10 agar. For cultures incubated in the presence or absence of artificial sputum for 19 days on BNP™ Middlebrook, M. tuberculosis ranged from 5.81-5.86 log10CFU/mL from liquid culture and 6.39-6.50 log10CFU/mL in artificial sputum; counts for M. tuberculosis in liquid culture ranged from 5.70-5.85 log10CFU/mL on Middlebrook 7H10. All colonies from 19 day-old cultures evaluated from the BNP™ Middlebrook and Middlebrook 7H10 media were positive for the Mycobacterium insertion sequence (IS) 6110 by real-time PCR. This study demonstrates that BNP™ Middlebrook can detect M. tuberculosis faster than standard plating techniques in the presence or absence of a simulated biological matrix (artificial sputum). Moreover, the BNP™ Middlebrook color development step does not interfere with real-time PCR detection of IS 6110. This study provides a preliminary assessment of the potential use of BNP™ Middlebrook for a more rapid screening and detection of viable M. tuberculosis with respect to clinical specimen evaluation, therapeutic treatment/vaccine efficacy, or epidemiological surveillance.

Rapid Diagnosis of Genital Tuberculosis by Real-time Polymerase Chain Reaction

ABSTRACT Objective Rapid diagnosis of genital tuberculosis (GTB) is essential as it is an important cause of infertility among women. Diagnosis can be done by imaging techniques, direct visualization by endoscopy, serology, histopathology, culture and polymerase chain reaction (PCR) tests. Laparoscopy detects macroscopic changes only; histology is only suggestive and not confirmatory unless acid fast bacilli (AFB) are demonstrated in the lesion but sensitivity is poor in paucibacillary disease. Culture methods are the gold standard but slow growth of most pathogenic mycobacteria delays the diagnosis. PCR can rapidly detect even few copies of DNA with high sensitivity and specificity. Aim Comparative evaluation of AFB smear examination, culture in Middlebrook 7H9 media and IS6110 based real-time PCR assay for the detection of mycobacteria in various female genital samples. Materials and methods A total of 555 female genital samples like endometrial and fallopian tube biopsies, menstrual blood and vaginal discharge were processed by modified Petroff's method. The deposit was used for detection of mycobacteria by AFB smear, culture on Middlebrook 7H9 media and IS6110 based real-time PCR. Results Out of 555 samples, 25.22 % (140/555) were positive by the combination of all the methods used. Overall positivity by real-time PCR alone was 23.78% (132/555), by culture 8.28% (46/555) and 2.70% (15/555) by AFB smear examination. Out of total positives, 94.28% (132/140) were positive by PCR alone, 32.85% (46/140) by culture and 10.71% (15/140) by AFB smear. Eight (5.71%) culture positive samples were negative by smear and PCR, six of these were nontubercular mycobacteria (NTM) and two samples had PCR inhibitors as confirmed by spiking with positive DNA. Contamination was observed in 25/555 (4.5%) which were reported negative by culture but three of these were PCR positive. AFB smear results were available in 1 hour, PCR in 1 day and culture in 4 to 6 weeks. Conclusion PCR was found to be the most rapid and sensitive (94.28%) method, 9-fold more sensitive than smear examination and 3-fold than the culture for detection of mycobacteria. Results were available in 4 to 6 weeks time for culture but in only 1 day by PCR. IS6110 PCR can detect only MTB and not the NTM. Use of multiplex PCR with genus and MTB specific primers will increase the sensitivity of test but care needs to be taken to prevent false positivity due to cross contamination and false negative due to PCR inhibitors. How to cite this article Malhotra B, Sinha P, Hooja S, Vyas L. Rapid Diagnosis of Genital Tuberculosis by Real-time Polymerase Chain Reaction. J South Asian Feder Obst Gynae 2012;4(1):39-42.

In vitro culture medium influences the vaccine efficacy of Mycobacterium bovis BCG

The varied rates of protection induced by Mycobacterium bovis BCG vaccine against tuberculosis has been attributed to many factors such as genetic variability among BCG strains, rapid clearance of BCG in some populations, and different levels of previous exposure of vaccinated populations to environmental mycobacteria. However, the methods and conditions employed to prepare this vaccine for human usage by various manufacturers have not been investigated as potential factors contributing to the variation in vaccine efficacy. A review of the literature indicates discrepancies between the approach for growing BCG vaccine in the laboratory to assess immune responses and protective ability in animal models, and that employed for production of the vaccine for administration to humans. One of the major differences is in the growth medium used for routine propagation in the laboratory and the one used for bulk vaccine production by manufacturers. Here we compared the immunogenicity of the BCG vaccine grown in Middlebrook 7H9 medium, the most commonly used medium in laboratory studies, against that grown in Sauton medium, which is used for growing BCG by most manufacturers. Our results showed clear differences in the behavior of BCG grown in these different culture media. Compared to BCG grown in Middlebrook 7H9 medium, BCG grown in Sauton media was more persistent inside macrophages, more effective at inhibiting apoptosis of infected cells, induced stronger inflammatory responses and stimulated less effective immunity against aerosol challenge with a virulent Mtb strain. These findings suggested that the growth medium used for producing BCG vaccine is an important factor that deserves increased scrutiny in ongoing efforts to produce more consistently effective vaccines against Mtb.

Application of the Alamar blue assay to determine the susceptibility to anti-tuberculosis pharmaceuticals

The aim of this study was to apply the Alamar Blue assay to determine the susceptibility to anti-tuberculosis pharmaceuticals, in Mycobacterium tuberculosis isolated strains from patients with tuberculosis (TB). In this study, a TB diagnostic was done by bacilluscope as an essential diagnostic tool for pulmonary TB using the Ziehl-Neelsen (ZN) stain. Furthermore, a culture was prepared in Lowenstein-Jensen (L-J) solid media, Middlebrook 7H9, and Middlebrook 7H10. For all the clinical samples investigated, five reference strains of M. tuberculosis were used to standardize the sensibility to streptomycin (SM), isoniazid (INH), rifampicin (RIF), and ethambutol (EMB) of the ATCC. Additionally, 97Mycobacterium strains were analyzed from clinical isolations. The microplate Alamar Blue assay (MABA) was standardized and applied. The resistance levels of this disease were obtained in La Comarca Region (Lagunera). The results indicated that the MABA test is fast and easy to apply, however the most important aspect is the reliability of the method to determine the drug sensibility to pharmaceuticals. The MABA colorimetric test can be used in different regional as well as state TB diagnostic laboratories. Key words: Tuberculosis, Alamar blue, drug resistance.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

Primary isolation of Mycobacterium bovis from bovine tissues: Conditions for maximising the number of positive cultures

In studies of Mycobacterium bovis infection in animals a definitive diagnosis requires the isolation of the organism. However, the optimum conditions for the primary isolation of M. bovis have not been determined. The aim of this study was to determine for primary isolation of M. bovis, (a) the incubation time required to achieve maximum sensitivity (i.e., the number of positive samples identified), (b) the effect of decontaminants on bacterial growth rates, and (c) the influence of media and the number of slopes of media on the number of positive samples detected. Two agar-based media, modified Middlebrook 7H11 (7H11) and tuberculosis blood agar (B83), and an egg-based medium, Stonebrink's (SB) were compared. Three decontaminants, 2% (w/v) sodium hydroxide (NaOH), 0.75% (w/v) and 0.075% (w/v) cetylpyridinium chloride (CPC, also called hexadecylpyridinium chloride, HPC) and 0.5% (w/v) benzalkonium chloride (BC) were evaluated against treatment with sterile distilled water. The inoculated media slopes were incubated for up to 15weeks. Colonies first appeared after 2weeks on all media types and 75% of positive slopes were identified by 8weeks. An incubation time of 15weeks was required to identify all positive samples. The slowest growth was associated with inocula that contained the fewest viable bacilli. The time to the appearance of colonies was influenced by medium type: the median time to detection of colonies was 28 days on 7H11 and B83, and 36 days on SB. However, SB returned the greatest number of positive samples. Decontamination procedures increased the minimum incubation time required to detect positive cultures, probably due to the toxic effect of the decontaminants. Increasing the number of inoculated slopes resulted in an increased number of positive samples and a decreased time to the detection of colonies. Overall, the detection of M. bovis was significantly influenced by the choice of media, the decontaminant and the duration of incubation of cultures.

Evaluation of INNO-LiPA mycobacteria v2 assay for identification of rapidly growing mycobacteria

A total of 54 rapidly growing mycobacteria (RGM) isolated from patients attended in the two hospitals of Cádiz Bay (Spain) were selected during a seven-year-period (2000-2006) in order to evaluate the INNO-LiPA Mycobacteria v2 assay for mycobacterial identification, based on the reverse hybridization principle. The strains were cultured in Löwenstein-Jensen and Middlebrook 7H9 media and identified to the species level by sequencing of the 16S rRNA, PCR-restriction enzyme analysis of the hsp65 gene, conventional tests and INNO-LiPA Mycobacteria v2 assay. By the molecular methods we identified a total of 12 different species: 23 Mycobacterium fortuitum, 11 M. chelonae, 10 M. abscessus, 2 M. senegalense, 1 M. alvei, 1 M. brumae, 1 M. mageritense, 1 M. mucogenicum, 1 M. neoaurum, 1 M. peregrinum, 1 M. septicum and 1 M. smegmatis. Fifty two strains (96.3%) were correctly identified by conventional techniques and 47 strains (87.0%) by INNO-LiPA Mycobacteria v2 assay. We find INNO-LiPA Mycobacteria v2 assay simple to perform but it provides few advantages in comparison with conventional methods and sometimes needs complementary tests to identify Mycobacterium fortuitum complex, M. chelonae complex and specific species due to the great heterogeneity in the RGM group.

Nasal Fish Tank Granuloma: An Uncommon Cause for Epistaxis

An immunocompetent 28-year-old male aquarium store employee presented with 1 month of intermittent epistaxis. A bleeding granulomatous lesion was identified over the right nasal septum (Figure 1). Histopathology from the incisional biopsy revealed granulomatous inflammation with the presence of acid-fast bacilli (Figure 2). Culture of the homogenized specimen on Middlebrook 7H11 agar after 14 days displayed white clones of non-tuberculous Mycobacterium (NTM) that turned brilliant yellow upon light exposure (Figure 3). Polymerase chain reaction (PCR) amplification of the 65-kDa heat shock protein gene with restriction enzyme analysis using BstE II and Hae III confirmed the diagnosis of Mycobacterium marinum infection. The lesion regressed completely with 3 months of doxycycline treatment. Figure 1. Anterior rhinoscopy revealed a granulomatous lesion with active oozing (arrow) on the right nasal septum. Inferior turbinate was identified as an asterisk. Figure 2. Histopathology of the specimen disclosed acid-fast bacilli (arrow) with the granulation inflammation. Figure 3. Mycobacterium marinum clones on Middlebrook 7H11 agar plates showed the photochromogenic characteristic, which are white when grown in the dark (A) and turn a brilliant yellow soon after exposure to light (B). Fish tank granuloma is a rare granulomatous skin infection caused by Mycobacterium marinum, an opportunistic NTM, commonly found in aquatic environments.1 Extremities are most frequently affected; nasal cavity infection is seldom reported. The diagnosis is generally based on a history of aquatic exposure (occupational or recreational), histopathology of granulomatous inflammation with or without the presence of acid-fast bacilli, and a slow-growing photochromogenic NTM incubated at low temperature (28–30°C). Furthermore, PCR facilitates an early identification of Mycobacterium marinum infection. Superficial lesion sometimes resolves spontaneously, but several months of antimicrobial drug are usually necessary. Various monotherapies, including doxycycline, are effective.2

Impaired fitness of Mycobacterium tuberculosis resistant isolates in a cell culture model of murine macrophages

We analysed the ability of Mycobacterium tuberculosis clinical isolates to penetrate and grow inside murine macrophages as a surrogate of fitness. Thirty-five drug-resistant and 10 drug-susceptible M. tuberculosis isolates were studied in a murine macrophage model from the J774.2 cell line in a 6 day protocol, performing semi-quantitative counts in Middlebrook 7H11 medium. The mycobacterial penetration index (MPI) after infection and the mycobacterial growth ratio (MGR) inside the macrophages were determined to evaluate the fitness of isolates. Isolates with the katG S315T mutation and multidrug-resistant (MDR) isolates had a significantly lower MGR compared with drug-susceptible isolates. The MPI of the isolates with the katG S315T mutation showed a significant decrease compared with the MPI of those without this mutation. A trend to significantly lower values was also observed on comparing the MPI of the MDR isolates with that of the drug-susceptible isolates and the isolates resistant to isoniazid. The isoniazid-resistant and MDR isolates with mutations in the katG gene showed decreased multiplication inside murine macrophages, suggesting a lower fitness of M. tuberculosis with these resistance patterns.