Ian Wiid

Boza, a natural source of probiotic lactic acid bacteria

Aims:  To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals.

Total antioxidant levels are low during active TB and rise with anti-tuberculosis therapy.

In tuberculosis, oxidative stress is a result of tissue inflammation, poor dietary intake of micronutrients due to illness, free radical burst from activated macrophages, and anti‐tuberculosis drugs. These free radicals may in turn contribute towards pulmonary inflammation if not neutralized by antioxidants. The total antioxidant status (TAS) of individuals is a function of dietary, enzymatic, and other systemic antioxidants and is therefore an indicator of the free radical load. Our aim was to evaluate the TAS of healthy and M. tuberculosis‐infected persons from a high TB incidence community, as well as tuberculosis patients at various stages of antituberculosis drug treatment and to correlate results with plasma micronutrient levels. Blood plasma samples from TB infected patients and following antituberculosis drug treatment were assayed for TAS, vitamins A, E and Zinc. Statistical analysis of results was by one‐way ANOVA and the Tukey multiple comparison post test. Active TB patients showed a significantly lower TAS (P < 0.001) compared to the community controls. We also show that TAS values increase during therapy. Results correlated with micronutrients vitamin A and zinc but vitamin E remained unaffected. We suggest that total antioxidant status of TB patients should be considered for more effective disease control and that diets low in antioxidants may render individuals susceptible to tuberculosis. IUBMB Life, 56: 101‐106, 2004

Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

ABSTRACT Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH.

Glutamate dehydrogenase and glutamine synthetase are regulated in response to nitrogen availability in Myocbacterium smegmatis.

BackgroundThe assimilation of nitrogen is an essential process in all prokaryotes, yet a relatively limited amount of information is available on nitrogen metabolism in the mycobacteria. The physiological role and pathogenic properties of glutamine synthetase (GS) have been extensively investigated in Mycobacterium tuberculosis. However, little is known about this enzyme in other mycobacterial species, or the role of an additional nitrogen assimilatory pathway via glutamate dehydrogenase (GDH), in the mycobacteria as a whole. We investigated specific enzyme activity and transcription of GS and as well as both possible isoforms of GDH (NAD+- and NADP+-specific GDH) under varying conditions of nitrogen availability in Mycobacterium smegmatis as a model for the mycobacteria.ResultsIt was found that the specific activity of the aminating NADP+-GDH reaction and the deaminating NAD+-GDH reaction did not change appreciably in response to nitrogen availability. However, GS activity as well as the deaminating NADP+-GDH and aminating NAD+-GDH reactions were indeed significantly altered in response to exogenous nitrogen concentrations. Transcription of genes encoding for GS and the GDH isoforms were also found to be regulated under our experimental conditions.ConclusionsThe physiological role and regulation of GS in M. smegmatis was similar to that which has been described for other mycobacteria, however, in our study the regulation of both NADP+- and NAD+-GDH specific activity in M. smegmatis appeared to be different to that of other Actinomycetales. It was found that NAD+-GDH played an important role in nitrogen assimilation rather than glutamate catabolism as was previously thought, and is its activity appeared to be regulated in response to nitrogen availability. Transcription of the genes encoding for NAD+-GDH enzymes seem to be regulated in M. smegmatis under the conditions tested and may contribute to the changes in enzyme activity observed, however, our results indicate that an additional regulatory mechanism may be involved. NADP+-GDH seemed to be involved in nitrogen assimilation due to a constitutive aminating activity. The deaminating reaction, however was observed to change in response to varying ammonium concentrations which suggests that NADP+-GDH is also regulated in response to nitrogen availability. The regulation of NADP+-GDH activity was not reflected at the level of gene transcription thereby implicating post-transcriptional modification as a regulatory mechanism in response to nitrogen availability.

Synthesis and evaluation of SQ109 analogues as potential anti-tuberculosis candidates

As part of an ongoing project to develop highly potent anti-tuberculosis therapeutics, six SQ109 derivatives were synthesized and screened in vitro for their anti-tuberculosis activity against the ATCC strain H37Rv and the extensively drug-resistant clinical strain XDR 173. Compound 16 with an extended alkene chain was the most active against both strains of Mycobacterium tuberculosis within a MIC range of 0.5-0.25 microM. Compound 12 and SQ109 were potent within a MIC range of 1-0.5 microM, whilst compound 18 displayed an activity within the MIC range of 0.5-2 microM against both Mycobacterium tuberculosis strains.

Pentacyclo-undecane derived cyclic tetra-amines: Synthesis and evaluation as potent anti-tuberculosis agents

As part of an ongoing effort to develop highly potent anti-tuberculosis agents, fourteen pentacyclo-undecane (PCU) tetra-amine compounds were synthesized and screened for their in vitro anti-mycobacterial activity against two TB strains, H37Rv and XDR 194 [an extensively drug-resistant strain of tuberculosis]. Using the broth macrodilution method, nitrofuranylamide based compounds (6a and 6b) showed almost similar activities against the H37Rv strain of Mycobacterium tuberculosis when compared with the control drug, ethambutol. N-Geranyl piperazine PCU (8a) and trans-trans farnesyl piperazine PCU (8b) were 3.2 and 3.7 times more potent than commercially available ethambutol. Both isoprenyl PCU tetra-amine derivatives and N-decyl piperazine PCU (9a) were highly active against the XDR 194 strain of tuberculosis with MICs in the range of 0.63-3.02 microM. Cytotoxicities (IC(50)) of isoprenyl based compounds (8a, 8b) and compound 9a were tested on a mammalian cell line [MDBK (Madin Darby bovine kidney epithelium)] with values of 30, 24 and 25 microM respectively.

Regulation of nitrogen metabolism in Mycobacterium tuberculosis: A comparison with mechanisms in Corynebacterium glutamicum and Streptomyces coelicolor

The mechanisms governing the regulation of nitrogen metabolism in Corynebacterium glutamicum and Streptomyces coelicolor have been extensively studied. These Actinomycetales are closely related to the Mycobacterium genus and may therefore serve as a models to elucidate the cascade of nitrogen signalling in other mycobacteria. Some factors involved in nitrogen metabolism in Mycobacterium tuberculosis have been described, including glutamine synthetase and its adenylyltransferase, but not much data concerning the other components involved in the signalling cascade is available. In this review a comparative study of factors involved in nitrogen metabolism in C. glutamicum and S. coelicolor is made to identify similarities with M. tuberculosis on both a genomic and proteomic level. This may provide insight into a potential global mechanism of nitrogen control in Mycobacterium tuberculosis.

Melatonin prevents the free radical and MADD metabolic profiles induced by antituberculosis drugs in an animal model

Abstract:  The objective was to determine the effect of combined antituberculosis (anti‐TB) drug therapy and an antioxidant, melatonin, on the free radical and organic acid profiles in an experimental rat model. A combined anti‐TB drug, Rifater, consisting of 12.0 mg rifampicin, 0.8 mg isoniazid, and 23.0 mg pyrazinamide and 18.56 μg melatonin/kg body weight per day (corresponding to average physiological human intake) were orally administered to Sprague–Dawley rats. Hydroxyl radical production was monitored by quantifying 2,3‐dihydroxybenzoic acid produced after intraperitonial sodium salicylate injections. Organic acid extractions and gas chromatography‐coupled mass spectrometry analyses were performed on collected urine samples. The results show hydroxyl radicals (P = 0.0019) and organic acids (P‐value range: 0.037 to <0.001), characteristic of a multiple acyl‐CoA dehydrogenase defect (MADD), were elevated with Rifater treatment and these elevations were significantly lowered with melatonin pretreatment (P‐value range: 0.031 to <0.001), probably because of its inherent antioxidant activity. We conclude that hydroxyl radical production and an increased organic acid profile induced by anti‐TB medication indicates inhibition of the electron transport chain. We also conclude that free radicals leading to clinical symptoms associated with an MADD metabolic profile induced by anti‐TB treatment could be alleviated by melatonin intervention.

Sulfamethoxazole enhances the antimycobacterial activity of rifampicin

OBJECTIVES To investigate the effect of trimethoprim/sulfamethoxazole on the survival of Mycobacterium tuberculosis and trimethoprim and sulfamethoxazole individually and combined with the first-line tuberculosis drugs (isoniazid, rifampicin and ethambutol). METHODS M. tuberculosis strains were exposed to either trimethoprim/sulfamethoxazole combination or sulfamethoxazole and trimethoprim alone at various concentrations. The strains were also exposed to sulfamethoxazole in combination with existing antibiotics to assess the combined effect on the growth of M. tuberculosis in the BACTEC 460TB system. The effect of the drugs was compared with vehicle-treated controls. Drug interactions were interpreted using quotient values obtained from the growth index of cultures treated with a single drug or the combination. RESULTS Trimethoprim showed a negligible effect on the growth of M. tuberculosis while sulfamethoxazole inhibited 80% of the growth of M. tuberculosis at 4.75 mg/L. There was no synergistic activity between sulfamethoxazole and trimethoprim, although an additive effect was observed. A statistically significant synergistic effect was observed between sulfamethoxazole and rifampicin. Sulfamethoxazole also had an additive effect with ethambutol, but there was no interaction with isoniazid. CONCLUSIONS Sulfamethoxazole is the main active compound against M. tuberculosis in the combination trimethoprim/sulfamethoxazole and has a synergistic effect with rifampicin. These findings suggest that sulfamethoxazole has potential in the multidrug regimen against M. tuberculosis.

Measuring minimum inhibitory concentrations in mycobacteria.

An agar dilution method for measuring minimum inhibitory concentrations (MICs) of Mycobacterium tuberculosis, based on the method of proportion, is described. Mycobacterium strains are grown on Middlebrook 7H10 (or 7H11) agar medium with twofold serially diluted drug concentrations in order to determine specific inhibitory values. The proportion of bacilli resistant to a given drug is determined by comparing the number of colony-forming units (CFU) on a drug-free control with those growing in the presence of drug within a specific concentration range. The MIC is defined as the lowest drug concentration that inhibits growth of more than 99% of a bacterial population of M. tuberculosis on solid Middlebrook medium within 21 days of incubation at 37 degrees C. The proportion method, the absolute concentration method, and the resistant ratio method have traditionally been used as standard procedures for antimycobacterial drug-susceptibility testing (DST), and reference data are mainly based on these methods. DST concepts and alternative procedures that have been adopted for DST are also briefly discussed.