A genuine mycobacterial thermophile: Mycobacterium hassiacum growth, survival and GpgS stability at near-pasteurization temperatures.

Mycobacterium hassiacum is so far the most thermophilic among mycobacteria as it grows optimally at 50 °C and up to 65 °C in a glycerol-based medium, as verified in this study. Since this and other nontuberculous mycobacteria (NTM) thrive in diverse natural and artificial environments, from where they may access and infect humans, we deemed essential to probe M. hassiacum resistance to heat, a strategy routinely used to control microbial growth in water-supply systems, as well as in the food and drink industries. In addition to possibly being a threat in its own right in rare occasions, M. hassiacum is also a good surrogate for studying other NTM species more often associated with opportunistic infection, namely Mycobacterium avium and Mycobacterium abscessus as well as their strictly pathogenic counterparts Mycobacterium tuberculosis and Mycobacterium leprae. In this regard, this thermophilic species is likely to be useful as a source of stable proteins that may provide more detailed structures of potential drug targets. Here, we investigate M. hassiacum growth at near-pasteurization temperatures and at different pHs and also characterize its thermostable glucosyl-3-phosphoglycerate synthase (GpgS), an enzyme considered essential for M. tuberculosis growth and associated with both nitrogen starvation and thermal stress in different NTM species.

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum.

BACKGROUND: Androstenedione (AD) is an important steroid medicine intermediate that is obtained via the degradation of phytosterols by mycobacteria. The production process of AD is mainly the degradation of the phytosterol aliphatic side chain, which is accompanied by the production of propionyl CoA. Excessive accumulation of intracellular propionyl-CoA produces a toxic effect in mycobacteria, which restricts the improvement of production efficiency. The 2-methylcitrate cycle pathway (MCC) plays a significant role in the detoxification of propionyl-CoA in bacterial. The effect of the MCC on phytosterol biotransformation in mycobacteria has not been elucidated in detail. Meanwhile, reducing fermentation cost has always been an important issue to be solved in the optimizing of the bioprocess. RESULTS: There is a complete MCC in Mycobacterium neoaurum (MNR), prpC, prpD and prpB in the prp operon encode methylcitrate synthase, methylcitrate dehydratase and methylisocitrate lyase involved in MCC, and PrpR is a specific transcriptional activator of prp operon. After the overexpression of prpDCB and prpR in MNR, the significantly improved transcription levels of prpC, prpD and prpB were observed. The highest conversion ratios of AD obtained by MNR-prpDBC and MNR-prpR increased from 72.3 ± 2.5% to 82.2 ± 2.2% and 90.6 ± 2.6%, respectively. Through enhanced the PrpR of MNR, the in intracellular propionyl-CoA levels decreased by 43 ± 3%, and the cell viability improved by 22 ± 1% compared to MNR at 96 h. The nitrogen transcription regulator GlnR repressed prp operon transcription in a nitrogen-limited medium. The glnR deletion enhanced the transcription level of prpDBC and the biotransformation ability of MNR. MNR-prpR/ΔglnR was constructed by the overexpression of prpR in the glnR-deleted strain showed adaptability to low nitrogen. The highest AD conversion ratio by MNR-prpR/ΔglnR was 92.8 ± 2.7% at low nitrogen level, which was 1.4 times higher than that of MNR. CONCLUSION: Improvement in phytosterol biotransformation after the enhancement of propionyl-CoA metabolism through the combined modifications of the prp operon and glnR of mycobacteria was investigated for the first time. The overexpress of prpR in MNR can increase the transcription of essential genes (prpC, prpD and prpB) of MCC, reduce the intracellular propionyl-CoA level and improve bacterial viability. The knockout of glnR can enhance the adaptability of MNR to the nitrogen source. In the MNRΔglnR strain, overexpress of prpR can achieve efficient production of AD at low nitrogen levels, thus reducing the production cost. This strategy provides a reference for the economic and effective production of other valuable steroid metabolites from phytosterol in the pharmaceutical industry.

Can phenotypic data complement our understanding of antimycobacterial effects for drug combinations?

OBJECTIVES: To demonstrate how phenotypic cell viability data can provide insight into antimycobacterial effects for the isoniazid/rifampicin treatment backbone. METHODS: Data from a Mycobacterium komossense hollow-fibre infection model comprising a growth control group, rifampicin at three different exposures (Cmax = 0.14, 0.4 and 1.47 mg/L with t½ = 1.57 h and τ = 8 h) and rifampicin plus isoniazid (Cmax rifampicin = 0.4 mg/L and Cmax isoniazid = 1.2 mg/L with t½ = 1.57 h and τ = 8 h) were used for this investigation. A non-linear mixed-effects modelling approach was used to fit conventional cfu data, quantified using solid-agar plating. Phenotypic proportions of respiring (alive), respiring but with damaged cell membrane (injured) and 'not respiring' (dead) cells data were quantified using flow cytometry and Sytox Green™ (Sigma-Aldrich, UK) and resazurin sodium salt staining and fitted using a multinomial logistic regression model. RESULTS: Isoniazid/rifampicin combination therapy displayed a decreasing overall antimicrobial effect with time (θTime1/2 = 438 h) on cfu data, in contrast to rifampicin monotherapy where this trend was absent. In the presence of isoniazid a phenotype associated with cell injury was displayed, whereas with rifampicin monotherapy a pattern of phenotypic cell death was observed. Bacterial killing onset time on cfu data correlated negatively (θTime50 = 28.9 h, θLAGRIF50 = 0.132 mg/L) with rifampicin concentration up to 0.165 mg/L and this coincided with a positive relationship between rifampicin concentration and the probability of phenotypic cell death. CONCLUSIONS: Cell viability data provide structured information on the pharmacodynamic interaction between isoniazid and rifampicin that complements the understanding of the antibacillary effects of this mycobacterial treatment backbone.

In vitro efficacy of combinations of eight antimicrobial agents against Mycobacteroides abscessus complex.

OBJECTIVES: A standard treatment regimen against Mycobacteroides abscessus complex (MABC) infections has not yet been established, making MABC difficult to treat successfully. In this study, we sought to develop an active ingredient for the clinical treatment of MABC infections. METHODS: We screened 102 MABC strains isolated from clinical specimens using DNA sequence analysis with the housekeeping genes hsp65 and rpoB. Drug susceptibility testing was performed against two subspecies-Mycobacteroides abscessus subsp. abscessus (M. abscessus) and Mycobacteroides abscessus subsp. massiliense (M. massiliense)-using eight antimicrobial agents (clarithromycin, amikacin, doxycycline, imipenem, linezolid, moxifloxacin, faropenem, and rifampicin). The combined efficacy of the antimicrobial agents was investigated using a checkerboard method. RESULTS: We identified 51 isolates as M. abscessus, 46 as M. massiliense, and five as others. Most of the M. abscessus isolates (83.0 %) exhibited inducible resistance to clarithromycin via the expression of the erm(41) gene. Combinations of imipenem with linezolid, moxifloxacin, and rifampicin exhibited additive effects against 81.0 %, 40.7 %, and 26.9 % of M. abscessus, respectively, and against 54.5 %, 69.2 %, and 30.8 % of M. massiliense, respectively. CONCLUSIONS: These results demonstrated the potential efficacy of a regimen containing imipenem against M. abscessus and M. massiliense infections.

Novel MscL agonists that allow multiple antibiotics cytoplasmic access activate the channel through a common binding site.

The antibiotic resistance crisis is becoming dire, yet in the past several years few potential antibiotics or adjuvants with novel modes of action have been identified. The bacterial mechanosensitive channel of large conductance, MscL, found in the majority of bacterial species, including pathogens, normally functions as an emergency release valve, sensing membrane tension upon low-osmotic stress and discharging cytoplasmic solutes before cell lysis. Opening the huge ~30Å diameter pore of MscL inappropriately is detrimental to the cell, allowing solutes from and even passage of drugs into to cytoplasm. Thus, MscL is a potential novel drug target. However, there are no known natural agonists, and small compounds that modulate MscL activity are just now being identified. Here we describe a small compound, K05, that specifically modulates MscL activity and we compare results with those obtained for the recently characterized MscL agonist 011A. While the structure of K05 only vaguely resembles 011A, many of the findings, including the binding pocket, are similar. On the other hand, both in vivo and molecular dynamic simulations indicate that the two compounds modulate MscL activity in significantly different ways.

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory.

BACKGROUND: The development of DNA amplification for the direct detection of M. tuberculosis from clinical samples has been a major goal of clinical microbiology during the last ten years. However, the limited sensitivity of most DNA amplification techniques restricts their use to smear positive samples. On the other hand, the development of automated liquid culture has increased the speed and sensitivity of cultivation of mycobacteria. We have opted to combine automated culture with rapid genotypic identification (ARDRA: amplified rDNA restriction analysis) for the detection resp. identification of all mycobacterial species at once, instead of attempting direct PCR based detection from clinical samples of M. tuberculosis only. RESULTS: During 1998-2000 a total of approx. 3500 clinical samples was screened for the presence of M. tuberculosis. Of the 151 culture positive samples, 61 were M. tuberculosis culture positive. Of the 30 smear positive samples, 26 were M. tuberculosis positive. All but three of these 151 mycobacterial isolates could be identified with ARDRA within on average 36 hours. The three isolates that could not be identified belonged to rare species not yet included in our ARDRA fingerprint library or were isolates with an aberrant pattern. CONCLUSIONS: In our hands, automated culture in combination with ARDRA provides with accurate, practically applicable, wide range identification of mycobacterial species. The existing identification library covers most species, and can be easily updated when new species are studied or described. The drawback is that ARDRA is culture-dependent, since automated culture of M. tuberculosis takes on average 16.7 days (range 6 to 29 days). However, culture is needed after all to assess the antibiotic susceptibility of the strains.

Investigation of opportunistic pathogens in municipal drinking water under different supply and treatment regimes.

Changing regulations to lower disinfectant byproducts in drinking water is forcing utilities to switch disinfection from chlorine to monochloramine. It is generally unknown whether this will impact positively or negatively on the microbiological quality of drinking water. A utility in Florida, using water with relatively high organic carbon levels from deep wells in several wellfields, made the decision to change its disinfection regime from chlorine to chloramine in order to meet the new regulations. To assess the impacts of such a change on the microbiology of its water supplies, it undertook a number of studies before and after the change. In particular, the presence of the opportunistic pathogens Legionella and Mycobacterium, and also the composition of drinking-water biofilms, were examined. A preliminary synthesis and summary of these results are presented here. Legionella species were widely distributed in source waters and in the distribution system when chlorine was the disinfectant. In some samples they seemed to be among the dominant biofilm bacteria. Following the change to monochloramine, legionellae were not detected in the distribution system during several months of survey; however, they remained detectable at point of use, although with less species diversity. A variety of mycobacteria (21 types) were widely distributed in the distribution system when chlorine was the disinfectant, but these seemed to increase in dominance after chloramination was instituted. At point of use, only four species of mycobacteria were detected. Other changes occurring with chloramination included (a) an altered biofilm composition, (b) increased numbers of total coliforms and heterotrophs and (c) nitrification of water storage tanks. The results suggested that consideration should be given to the microbiological effects of changing disinfection regimes in drinking-water and distribution system biofilms.

Enzymatic conversion of the clavan exopolysaccharide by Streptomyces sp. YSDL-20.

A screening programme was set up to isolate microorganisms able to hydrolyse the complex biopolymer clavan produced by Clavibacter michiganensis subsp. michiganensis LMG 5604. This valuable exopolysaccharide is very rich in L-fucose (37.5% w/w), a rare sugar, used in the medical field (Vanhooren, 1999). A microorganism capable of depolymerizing the polymer may decrease the high viscosity during clavan batch fermentations and remove the limitations of the oxygen transfer and consequently increase the clavan yield. It could also release free L-fucose or L-fucose rich oligosaccharides. An actinomycete, designated YSDL-20, isolated from a soil sample, was able to depolymerize this biopolymer. Based on its morphology and molecular characteristics, this strain could only be identified as Streptomyces sp.. On clavan, this strain displays good growth (17.5 g DCW/l after 96 h of cultivation) characterized by filamentous growth during the earlier days of cultivation followed by sporulation after 4 days. The flow behaviour of the Clavibacter broth was characterized, the fermentation culture broth behaves as a pseudoplastic fluid. The viscosity of the culture broth as well as of the purified clavan EPS, decreases when lyophilised supernatant of Streptomyces sp. YSDL-20 was added, indicating clavanase action. The viscosity decreases by 26% when the Clavibacter culture broth was incubated during 18 h with the crude Streptomyces enzyme source, whereas a 82% viscosity drop was observed, when the purified clavan EPS (10 g/l) was incubated with the lyophilised Streptomyces supernatant for 5 h.

Alpha-1-antitrypsin (AAT) anomalies are associated with lung disease due to rapidly growing mycobacteria and AAT inhibits Mycobacterium abscessus infection of macrophages.

Rapidly growing mycobacteria (RGM) are ubiquitous in the environment but cause lung disease in only a fraction of exposed individuals. This variable susceptibility to disease implies vulnerability to RGM infection due to weakness in host defense. Since most persons who contract RGM lung disease have no known host defense defect, it is likely that uncharacterized host deficiencies exist that predispose to RGM infection. Alpha-1-antitrypsin (AAT) is a host factor that may protect individuals from respiratory infections. Therefore, we assessed AAT protein anomalies as a risk factor for RGM lung disease. In a cohort of 100 patients with RGM lung disease, Mycobacterium (M.) abscessus was the most prevalent organism, isolated in 64 (64%) subjects. Anomalous AAT proteins were present in 27% of the cohort, which is 1.6 times the estimated prevalence of anomalous AAT proteins in the United States population (p=0.008). In in vitro studies, both AAT and a synthetic inhibitor of serine proteases suppressed M. abscessus infection of monocyte-derived macrophages by up to 65% (p<0.01). AAT may be an anti-RGM host-defense factor, and anomalous AAT phenotypes or AAT deficiency may constitute risk factors for pulmonary disease due to RGM.

Developmental potential of Frankia vesicles.

The ability of nitrogenase-containing Frankia sp. strain CpI1 vesicles to regrow vegetative hyphae is demonstrated. Vesicles attached to hyphae in N2-fixing CpI1 cultures and sucrose gradient-isolated vesicles exhibited hyphal outgrowths when incubated in certain defined liquid media. Single or multiple hyphal extensions grew out from the vesicles.

[Species composition of mycococci]. / O vidovom sostave mikokokkov.

The phenotypical similarity between 34 microbial strains was determined, and they were classified on the principle of maximal general similarity in 70 traits. All the strains were subdivided into three groups within which the similarity of the strains was not less than 85%. The groups correspond to the species Mycococcus capsulatus, M. ruber, and Mycobacterium brevicale. The results of the investigation suggest that the group of mycococci possesses a considerable number of characteristics which make it possible to consider it as a separate microbial genus. However, the principle of equal weight can be hardly applied to the taxonomy of mycococci since sharp variations in many traits do not permit to differentiate between specific and strain differences. Morphological and cultural properties still remain the most reliable criteria of the genus Mycococcus.

Potentially pathogenic, slow-growing mycobacteria released into workplace air during the remediation of buildings.

Construction workers' exposure to airborne viable mycobacteria was studied during the remediation of three moldy and two nonmoldy buildings. Furthermore, the concentrations of airborne fungal and actinobacterial spores were determined. The samples for the microbial analyses were collected using a six-stage impactor and an all-glass impinger sampler, and by filter sampling. Specific mycobacteria media and nonselective media were used for the cultures. The samples were cultured for the total numbers of rapidly growing and slow-growing mycobacteria, and the isolates obtained were identified to the genus or species level. Mycobacteria were recovered from the air during the remediation of two of the moldy buildings and one nondamaged building. Concentrations of mycobacteria up to 160 cfu/m3 were detected. A total of 43 mycobacterial isolates was recovered. Most of the isolates were slow-growers, only two rapid-growing strains being detected. The 38 identified isolates belonged to potentially pathogenic species, including Mycobacterium avium complex, M. scrofulaceum, and M. fortuitum, and to saprophytic species, including M. nonchromogenicum and M. terrae. Mycobacteria were the most often detected in samples taken with a six-stage impactor. They were found in buildings with both high and low concentrations of fungi. In conclusion, mycobacteria, both potentially pathogenic and saprophytic species, may be released into the indoor air during the remediation of buildings.