Screening of antimicrobial activities in red, green and brown macroalgae from Gran Canaria (Canary Islands, Spain)

Extracts from 44 species of seaweed from Gran Canaria (Canary Islands, Spain) were screened for the production of antibacterial and antifungal compounds against a panel of gram-negative and gram-positive bacteria, mycobacteria, yeasts and fungi. A total of 28 species displayed antibacterial activity, of which six also showed antifungal activity. Asparagopsis taxiformis and Cymopolia barbata were the species with the strongest activities against the broadest spectrum of target microorganisms. All the species with antibacterial activity were active against gram-positive bacteria, whereas only two species, A. taxiformis and Osmundea hybrida, were active against mycobacteria. The production of secondary metabolites with antimicrobial activities by the macroalgae was also studied under different conditions, although no common trend for bioactivity was observed.     

Quinones as antimycobacterial agents

Mycobacterium tuberculosis is a serious worldwide health threat, killing almost 3 million people per year. Other mycobacterial species, especially Mycobacterium avium, are emerging pathogens in the immunocompromised population, most notably AIDS patients. These nontuberculous mycobacteria (NTM) are ubiquitous in the environment, and naturally resistant to many disinfection procedures. Treatment options are limited, and no new antibiotics have been developed against mycobacteria since the 1970s. There is a desperate need for new biocides and antibiotics to prevent and treat mycobacterial infections. A small aromatic compound library has been screened for effectiveness in growth inhibition or killing of mycobacteria. Four species, representing the M. tuberculosis complex, the slow-growing NTM, and the rapid-growing NTM were used. Active compounds had minimal inhibitory concentrations as low as 12.5 microg/mL, with the active component being a quinone. The primarily bactericidal activity observed represents a unique mechanism of action. A fluorescent assay involving M. smegmatis expressing gfp was analyzed as a rapid assay for predicting inhibitory activity, but failed to predict activity well. Our compounds may have significant utility as soluble biocides against mycobacteria and other hardy nosocomial pathogens.     

PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp

A method based on PCR-restriction fragment length polymorphism analysis (PRA) using a novel region of the hsp65 gene was developed for the rapid and exact identification of mycobacteria to the species level. A 644 bp region of hsp65 in 62 mycobacteria reference strains, and 4 related bacterial strains were amplified, and the amplified DNAs were subsequently digested with restriction enzymes, namely, AvaII, HphI, and HpaII. Most of the mycobacteria species were easily differentiated at the species level by the developed method. In particular, the method enabled the separation of M. avium, M. intracellulare and M. tuberculosis to the species level by AvaII digestion alone. An algorithm was constructed based on the results and a blind test was successfully performed on 251 clinical isolates, which had been characterized by conventional biochemical testing. Our results suggest that this novel PRA offers a simple, rapid, and accurate method for the identification of mycobacteria culture isolates at the species level.     

Mycobacterial ecology of the Rio Grande

This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.     

Liposomes-mycobacteria incubation systems as a partial model of host-parasite interaction at cell membrane level

The multilamellar lecithin-cholesterol liposomes entrapping carboxyfluorescence as a specific marker were prepared to be incubated with mycobacteria. The extent of resulting liposomal lysis was measured by marker release. Mycobacteria were highly active in this respect regardless of the species. Staphylococci were much less active and E. coli was completely inactive. Molecular species of lecithin and their ratio to cholesterol were related with the liposome sensitivity to mycobacteria. Ultrastructural and biochemical study showed that liposomes can be in close contact with mycobacteria and lecithins are degraded so that released fatty acids are incorporated into bacterial lipids, especially in virulent species. Liposomes-mycobacteria interaction was discussed as a partial model of the phagocyte-parasite interaction at the membrane level.     

Environmental mycobacteria: a threat to human health?

In many cases, bacterial pathogens are close relatives to nonpathogens. Pathogens seem to be limited lineages within nonpathogenic bacteria. Nonpathogenic isolates are generally more diverse and widespread in the environment and it is generally considered that environmental bacteria do not pose a risk to human health as clinical isolates do; this may not be the case with mycobacteria, but environmental mycobacteria have not been well studied. It is documented that several environmental mycobacteria constitute a source for human infections. Diverse mycobacterial environmental isolates are rarely involved in human disease. Environmental mycobacteria may have a role in degradation of different compounds. Environmental mycobacteria have had a long interaction with humans, maybe as long as the human species, and may have contributed to human evolution.     

A Comparative Study of Mycobacteria from Patients' Room Dusts and from Sputa of Tuberculous Patients

The source of mycobacteria other than Mycobacterium tuberculosis occurring in sputa of tuberculous patients as casual isolates was investigated by comparing the occurrence rate of mycobacterial species between patient's room dust and patient's sputum. Almost all species of mycobacteria recovered from sputa could be found in dusts obtained from the rooms. However, the percentage of occurrence of the mycobacterial species in dusts differed from that in sputa. In dusts, Mycobacterium fortuitum (39.6%), Mycobacterium nonchromogenicum (23.5%) and Mycobacterium gordonae (16.7%) occurred in high frequencies, whereas Mycobacterium intracellulare (69.6%), M. gordonae (5.9%), Mycobacterium scrofulaceum (5.2%), and Gordona bronchialis (10.4%) were the main species found in sputa. The patterns of occurrence of mycobacterial species as illustrated above may suggest that pathogenic mycobacteria survive in the respiratory tract while nonpathogenic ones are destroyed there, thus the human body acts as a selective medium for the pathogenic mycobacterial species. Serotype studies on strains of M. intracellular as casual isolates indicated that those from sputa of tuberculous patients were different from those derived from patients with lung diseases due to this species of mycobacteria. These results led us to the conclusion that mycobacteria in patients' room dusts were the source of mycobacteria occurring in sputa as casual isolates, particularly the pathogenic mycobacteria in dusts are more likely to survive in the human respiratory tract, occasionally multiplying and causing disease under favorable circumstances.     

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.     

Isolation and automated ribotyping of Mycobacterium lentiflavum from drinking water distribution system and clinical specimens

Automated ribotyping as a tool for identifying of nontuberculous mycobacteria was evaluated. We created a database comprising of riboprints of 60 strains, representing 32 species of nontuberculous mycobacteria. It was shown that combined ribopatterns generated after digestion with EcoRI and PvuII were distinguishable between species of both slow-growing and rapid-growing mycobacteria. The findings were in good agreement with the 16S rRNA gene sequencing results, allowing correct identification of Mycobacterium lentiflavum isolated from clinical specimens and from biofilms growing in public water distribution system. The automated ribotyping was powerful in discriminating between M. lentiflavum and closely related species M. simiae and M. palustre. Mycobacterium lentiflavum strains from drinking water biofilms were resistant to two to four antimycobacterial drugs. The drinking water distribution system may, thus, be a source of nontuberculous mycobacteria resistant to multiple drugs.     

Acetophenones with selective antimycobacterial activity

AIMS: Mycobacteria are a serious cause of infections in humans, with limited treatment options, as no new antibiotics have been developed against mycobacteria since the 1960s. In this study, the antimycobacterial activity of a small library of acetophenone (AP) compounds was analysed. METHODS AND RESULTS: Twenty-three AP derivatives were examined for activity against mycobacteria using a microbroth assay. The compounds were bacteriostatic, with the most effective (cyclohexylacetophenone and piperidinoacetophenone) having minimal inhibitory concentrations of 246 microM. Active compounds tended to be more hydrophobic, and may work by alkylation of as yet undetermined intracellular target protein(s). Cytotoxicity against eukaryotic cells was also determined and appears to be unrelated to the bacteriostatic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: AP may serve as a novel group of useful therapeutics against the mycobacteria.     

Trafficking pathways of mycolic acids: structures, origin, mechanism of formation, and storage form of mycobacteric acids

Mycolic acids, the hallmark of mycobacteria and related bacteria, are major and specific components of their cell envelope and essential for the mycobacterial survival. Mycobacteria contain structurally related long-chain lipids, but the metabolic relationships between these various classes of compounds remain obscure. To address this question a series of C(35) to C(54) nonhydroxylated fatty acids (mycobacteric acids), ketones, and alcohols structurally related to the C(70-80) dicyclopropanated or diethylenic mycolic acids were characterized in three mycobacterial strains and their structures compared. The relationships between these long-chain acids and mycolic acids were established by following the in vivo traffic of (14)C labeled alpha-mycolic acids purified from the same mycobacterial species. The labeling was exclusively found in mycobacteric acids. The mechanism of this degradation was established by incorporation of (18)O(2) into long-chain lipids and shown to consist in the rupture of mycolic acids between carbon 3 and 4 by a Baeyer-Villiger-like reaction. We also demonstrated that mycobacteric acids occur exclusively in the triacylglycerol (TAG) fraction where one molecule of these acids esterifies one of the three hydroxyl groups of glycerol. Altogether, these data suggest that these compounds represent a pathway of metabolic energy that would be used by mycobacteria in particular circumstances.     

Sensitivity to levofloxacin of various types of non-tuberculosis Mycobacterium]

Activity of levofloxacin (Tavanic) against 10 species of nontuberculosis of mycobacteria was investigated by indirect method of absolute concentrations on Levenstain-Jensen media (levofloxacin concentration 5 and mcg/mL). The investigation was performed on 71 strains of nontubercolosis mycobacteria: Mycobacterium avium-intracellulare--24 strains, M. fortuitum--17 strains, M. chelonae--10 strains, M. malmoense--13 strains and 6 other species of mycobacteria. Susceptible to critical levofloxacin concentration were 8 species of 10. Resistance to levofloxacin (10 mcg/mL) was estimated for 16.7 per cent of M. avium-intracellulare and 30 per cent of M. chelonei strains. It is concluded that levofloxacin may be a drug of choice for management of mycobacteriosis caused by M. fortuitum, M. kansasii, M. xenopi, M. malmoense, and in the most of cases due to M. avium-intracellulare and M. chelonae.     

Mycobacterial Ecology of the Rio Grande

This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.     

Differentiation of mycobacterial species by investigation of petroleum ether-soluble sulfolipids using thin-layer chromatography after incubation with [35S]sulfate

The method of detecting petroleum ether-soluble sulfolipids by thin-layer chromatography after incubation with [35S]sulfate is useful for differentiation between mycobacterial species. Rapidly growing mycobacteria, including two subspecies of Mycobacterium chelonei, were differentiated by this method. Most species of slowly growing mycobacteria were characterized by the pattern of distribution of radioactive sulfolipids in the thin-layer chromatograms. Mycobacterium nonchromogenicum was clearly differentiated from Mycobacterium terrae and Mycobacterium triviale by the presence of a sulfolipid. The latter two did not contain any significant amount of sulfolipids. Mycobacterium avium contained a large amount of sulfolipids, whereas Mycobacterium scrofulaceum did not contain any detectable sulfolipid. Rapidly growing, scotochromogenic mycobacteria, except for Mycobacterium flavescens, did not contain any remarkable amounts of sulfolipids.     

Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.     

Environmental mycobacteria in Korea. I. Distribution of the organisms

Environmental mycobacteria in Korea have been investigated by examining 54 soil, 111 house dust, 63 well water, and 98 sewage samples collected from 123 randomly selected areas in Korea during the fourth nationwide tuberculosis prevalence survey in 1980. A variety of mycobacteria were isolated from 76% of soil, 67% of sewage, 43% of well water, and 7% of house dust samples. Some samples yielded more than one species; thus 56 strains were obtained from soil, 107 strains from sewage, 48 strains from well water and 8 strains from house dust. Mycobacterium fortuitum was the most common species of environmental mycobacteria in Korea and the species was distributed equally in all types of samples tested. The M. terrae complex was also one of the common species of environmental mycobacteria and it seemed to be more abundant in water samples than in soil. Scotochromogenic slow growers M. scrofulaceum and M. gordonae were common microbes in soil and water samples, although the latter was more frequently detected in water samples. Scotochromogenic rapid growers M. flavescens and M.phlei, and photochromogenic rapid grower M. vaccae were isolated more frequently from sewage or water samples than from soil. Nonphotochromogenic rapid growers M. chelonei (chelonae) and M. smegmatis were isolated mostly from sewage and the former was rarely found in soil and well water samples. The clinically important species M. avium-intracellulare complex was found less frequently in all types of test samples.     

Identification of mycobacteria in peat moss processing plants: application of molecular biology approaches

Peat moss processing plant workers are exposed to high concentrations of bioaerosols. Although mycobacteria have been cultured from peat moss, no study has examined the workers' exposure to mycobacterial bioaerosols. We evaluated the presence of mycobacteria in air samples from peat moss processing plants using molecular biology approaches (cloning-sequencing and polymerase chain reaction (PCR)) and the workers exposure using immunoglobulin G (IgG) complexes to mycobacteria. In addition, species detected in air samples and in peat moss were compared. Two peat moss processing plants were chosen among 14 previously studied. A total of 49 clones were sequenced. Real-time PCR was also performed on the same air samples to evaluate the airborne concentration of mycobacteria and estimate exposure levels. Several Mycobacterium species were present in the air samples (M. malmoense, M. smegmatis, M. graceum, M. bohemicum, and M. interjectum). Mycobacterium avium was recovered by culture in peat moss but not in the air using the molecular approach. Total airborne Mycobacterium concentration was estimated at 8.2 x 10(8)/m3. Workers had IgG against the mycobacterial mix and M. avium, suggesting significant exposure. The findings from air samples, supported by IgG measurements, demonstrate that peat moss processing plant workers are exposed to mycobacteria in addition to other biological agents.     

Catecholates and mixed catecholate hydroxamates as artificial siderophores for mycobacteria

Different mono-, bis- or triscatecholates and mixed mono- or biscatecholate hydroxamates were synthesized as potential siderophores for mycobacteria. Siderophore activity was tested by growth promotion assays using wild type strains and iron transport mutants of mycobacteria as well as Gram-negative bacteria. Some triscatecholates and biscatecholate hydroxamates were active in mutants of Mycobacterium smegmatis deficient in mycobactin and exochelin biosynthesis or exochelin permease, respectively, indicating an uptake route independent of the exochelin/mycobactin pathway. Structure activity relationships were studied. Ampicillin conjugates of some of these compounds were inactive against mycobacteria but active against Gram-negative bacteria.     

Role of mycobacterial efflux transporters in drug resistance: an unresolved question

Two mechanisms are thought to be involved in the natural drug resistance of mycobacteria: the mycobacterial cell wall permeability barrier and active multidrug efflux pumps. Genes encoding drug efflux transporters have been isolated from several mycobacterial species. These proteins transport tetracycline, fluoroquinolones, aminoglycosides and other compounds. Recent reports have suggested that efflux pumps may also be involved in transporting isoniazid, one of the main drugs used to treat tuberculosis. This review highlights recent advances in our understanding of efflux-mediated drug resistance in mycobacteria, including the distribution of efflux systems in these organisms, their substrate profiles and their contribution to drug resistance. The balance between the drug transport into the cell and drug efflux is not yet clearly understood, and further studies are required in mycobacteria.     

Real-time PCR detection of environmental mycobacteria in house dust

Analysing the number and species of microbes in indoor dust is needed for assessment of human exposure to microbes in dwellings. Environmental mycobacteria are common heterotrophic bacteria in both natural and man-made environments and potential inducers of human immune system. Culture of mycobacteria from samples rich with other microbes is difficult due to the slow growth rate of mycobacteria and this has hampered the studies on their role in indoor human exposure. A quantitative, real-time 5'-nuclease (TaqMan) PCR assay was developed to detect environmental mycobacteria in indoor dust samples. The specificity of the primers and the probe targeting the 16S rDNA of mycobacteria, tested with 26 mycobacterial and 10 non-mycobacterial but related species, proved to be high. When tested on 20 indoor dust samples collected from five homes, the assay gave counts varying between 4.8 × 10⁴ and 7.2 × 10⁶ cell/g, being on average 1.1 × 10³ times higher than culture. Seasonal variation in the dust counts of mycobacteria was observed by both culture and qPCR. Total of 140 isolates considered as mycobacteria by Ziehl-Neelsen staining and GLC-analyses were subjected to PCR analysis with the mycobacterial primers, and 39 isolates to partial 16S rDNA sequencing. All proved to be mycobacteria and revealed high diversity of mycobacterial species in the dust samples. Majority of the sequences were related to M. terrae and M. avium complexes.