Aims:  To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals. Methods and Results:  The strains survived low pH conditions (pH 3·0), grew well at pH 9·0 and were not inhibited by the presence of 0·3% (w/v) oxbile. Cytotoxicity levels of the bacteriocins, expressed as CC50, ranged from 38 to 3776 μg ml−1. Bacteriocin bacST284BZ revealed high activity (EC50 = 735 μg ml−1) against herpes simplex virus type 1. Growth of Mycobacterium tuberculosis was 69% repressed after 5 days in the presence of bacST194BZ. Various levels of auto‐cell aggregation and co‐aggregation with Listeria innocua LMG 13568 were observed. Adhesion of the probiotic strains to HT‐29 cells ranged from 18 to 22%. Conclusions:  Boza is a rich source of probiotic lactic acid bacteria. All strains survived conditions simulating the gastrointestinal tract and produced bacteriocins active against a number of pathogens. Adherence to HT‐29 and Caco‐2 cells was within the range reported for Lactobacillus rhamnosus GG, a well‐known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study:  Boza contains a number of different probiotic lactic acid bacteria and could be marketed as a functional food product.

SUMMARY Restriction endonuclease analysis of specific gene sequences is proving to be a valuable technique for characterisation and diagnosis of inherited disorders. This paper describes detailed protocols for isolation, restriction, and blot hybridisation of genomic DNA. Problems and alternatives in the procedure are discussed and a troubleshooting guide has been provided to help rectify faults.The development of techniques for the cloning1 and analysis2 of genes from complex organisms laid the foundation for the study of mutant genes associated with human inherited disorders. DNA from a person can now be cleaved into fragments of defined length by restriction endonucleases. The fragments are then separated by gel electrophoresis, blotted onto filters,2 and incubated with radioactively labelled gene specific probes. These probes, obtained by molecular cloning techniques, are isolated and characterised DNA sequences which will associate specifically with homologous genomic DNA sequences on the filter. Thus, only fragments containing part or all of the gene of interest will be detected. This new recombinant DNA technology was rapidly applied to the molecular characterisation and antenatal diagnosis of the haemoglobinopathies and thalassaemia.3-7 As cloning techniques have become more sophisticated, the number of purified, cloned human genes has proliferated to the extent that a recently published list8 is already out of date. A considerable number of genetic diseases are therefore amenable to DNA analysis, and the use of linked restriction fragment length polymorphisms9 10 has further extended the applicability of the technique.These advances have brought DNA analysis within the scope of the clinical geneticist, and the techniques will ultimately become part of the routine service provided by human genetics departments. DNA blotting and molecular hybridisation

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dNew drugs to treat drug-resistant tuberculosis are urgently needed. Extensively drug-resistant and probably the totally drugresistant tuberculosis strains are resistant to fluoroquinolones like moxifloxacin, which target gyrase A, and most people infected with these strains die within a year. In this study, we found that a novel aminobenzimidazole, VXc-486, which targets gyrase B, potently inhibits multiple drug-sensitive isolates and drug-resistant isolates of Mycobacterium tuberculosis in vitro (MICs of 0.03 to 0.30 g/ml and 0.08 to 5.48 g/ml, respectively) and reduces mycobacterial burdens in lungs of infected mice in vivo. VXc-486 is active against drug-resistant isolates, has bactericidal activity, and kills intracellular and dormant M. tuberculosis bacteria in a low-oxygen environment. Furthermore, we found that VXc-486 inhibits the growth of multiple strains of Mycobacterium abscessus, Mycobacterium avium complex, and Mycobacterium kansasii (MICs of 0.1 to 2.0 g/ml), as well as that of several strains of Nocardia spp. (MICs of 0.1 to 1.0 g/ml). We made a direct comparison of the parent compound VXc-486 and a phosphate prodrug of VXc-486 and showed that the prodrug of VXc-486 had more potent killing of M. tuberculosis than did VXc-486 in vivo. In combination with other antimycobacterial drugs, the prodrug of VXc-486 sterilized M. tuberculosis infection when combined with rifapentine-pyrazinamide and bedaquiline-pyrazinamide in a relapse infection study in mice. Furthermore, the prodrug of VXc-486 appeared to perform at least as well as the gyrase A inhibitor moxifloxacin. These findings warrant further development of the prodrug of VXc-486 for the treatment of tuberculosis and nontuberculosis mycobacterial infections.

c 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. G lutathione (GSH) is a thiol known for its efficient detoxification of reactive oxygen species, reactive nitrogen species, and free radicals in eukaryotes. Mycobacteria do not synthesize GSH but produce two low-molecular-weight thiols,and ergothioneine (2-mercaptohistidine trimethylbetaine) (ERG) (4, 5). Four genes are involved in MSH biosynthesis in mycobacteria, namely, mshA, mshB, mshC, and mshD, and mutants harboring deletions in mshB, mshC, and mshD produce different levels of MSH due to the ability of other enzymes to partially compensate for their loss (6, 7). MSH-deficient mutants of Mycobacterium smegmatis show increased sensitivity to oxidative stress, alkylating agents, and a range of antibiotics, including erythromycin, azithromycin, vancomycin, penicillin G, streptomycin, and rifampin, but exhibit increased resistance to isoniazid (INH) and ethionamide (ETH) (8, 9). The MSH-deficient ⌬mshA mutant of Mycobacterium tuberculosis requires catalase during in vitro growth, implicating MSH in detoxifying reactive oxygen species (10).ERG biosynthetic genes (egtA, egtB, egtC, egtD, and egtE) were recently identified in M. smegmatis (11). Although several lines of evidence support the cytoprotective and antioxidative role of ERG in eukaryotes (12), bacteria (13), and, recently, fungi (14), nothing is known of its role in mycobacteria. ERG has also been implicated in modulating the immune response (15) and in the inhibition of metalloenzymes, preventing the copper-induced oxidation of DNA and protein due to its metal-chelating properties (16,17). Eukaryotes obtain ERG from their diet, and its accumulation in cells is dependent on the activity of a highly specific transporter, OCTN1, since the zwitterionic nature of ERG prevents it from crossing the plasma membrane (18,19). In an M. smegmatis ⌬mshA mutant, which is MSH deficient, the levels of ERG and the organic hydroperoxide resistance (Ohr) protein are elevated, suggesting that ERG may partly compensate for the loss of MSH (20). This may exp...

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The high acquisition rate of drug resistance by necessitates the ongoing search for new drugs to be incorporated in the tuberculosis (TB) regimen. Compounds used for the treatment of other diseases have the potential to be repurposed for the treatment of TB. In this study, a high-throughput screening of compounds against thiol-deficient strains and subsequent validation with thiol-deficient strains revealed that and mutants had increased susceptibility to azaguanine (Aza) and sulfaguanidine (Su); and mutants had increased susceptibility to bacitracin (Ba); and, , and mutants had increased susceptibility to fusaric acid (Fu). Further analyses revealed that some of these compounds were able to modulate the levels of thiols and oxidative stress in This study reports the activities of Aza, Su, Fu, and Ba against and provides a rationale for further investigations.

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 it's 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.