Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.     

Characterization of binding sites for a bacteriocin produced by Mycobacterium smegmatis.

A bacteriocin from Mycobacterium smegmatis ATCC 14468 which adsorbed to the cell wall-enriched fraction and the crude lipopolysaccharide preparation from Mycobacterium diernhoferi ATCC 19340 was inhibited by D-glucose and alpha-linked glucosylated derivatives.     

分枝杆菌特征性蛋白的筛选

目的研究分枝杆菌蛋白表达,筛选分枝杆菌特征性蛋白,为分枝杆菌的快速鉴定奠定基础。方法选取分枝杆菌标准菌株,提取细菌蛋白,干化学法测蛋白浓度,应用表面增强激光解吸电离飞行时间质谱技术（SELDI-TOF-Ms）检测分枝杆菌蛋白表达,和非分枝杆菌蛋白指纹图谱比较,筛选分枝杆菌特征性蛋白。重复测定20次分枝杆菌蛋白标本,评价SELDI检测分枝杆菌蛋白分子量的重复性。结果耻垢分枝杆菌ATCC 14468有约20个蛋白峰,结核分枝杆菌ATCC 25177、土地分枝杆菌ATCC 15755、胞内分枝杆菌ATCC 13950、耻垢分枝杆菌ATCC 607有近40个蛋白峰。与非分枝杆菌蛋白指纹图谱相比,4个蛋白峰为分枝杆菌所特有。SELDI重复检测20次分枝杆菌蛋白标本显示同一蛋白峰的分子量变异系数≤0．05％。结论分枝杆菌有其特征性蛋白峰,可能有助于分枝杆菌的快速鉴定。     

Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to species status: Mycobacterium abscessus comb. nov.

We studied the taxonomic positions of the rapidly growing organism Mycobacterium fortuitum and phenotypically related organisms. We confirmed that "Mycobacterium peregrinum" ATCC 14467T (T = type strain) is genetically independent of M. fortuitum ATCC 6841T by using various DNA hybridization conditions. Strains that were genetically identified as "M. peregrinum" were phenotypically differentiated from M. fortuitum ATCC 6841T. Thus, we propose that "M. peregrinum" should be revived as an independent species, Mycobacterium peregrinum sp. nov., nom. rev. The type strain is strain ATCC 14467. M. fortuitum subsp. acetamidolyticum ATCC 35931T exhibited a high level of DNA relatedness to M. fortuitum ATCC 6841T. The hybridized DNAs maintained stable heteroduplexity at high stringency; thus, we confirmed that M. fortuitum subsp. acetamidolyticum is identical to M. fortuitum ATCC 6841T. We found that M. chelonae subsp. abscessus ATCC 19977T is genetically different from M. chelonae subsp. chelonae NCTC 946T on the basis of the results of quantitative hybridization even under optimal conditions. There was no reason to maintain this organism as a subspecies of M. chelonae. Thus, we propose that M. chelonae subsp. abscessus should be elevated to species status as Mycobacterium abscessus (Kubica et al.) comb. nov. The type strain is strain ATCC 19977.     

Distribution of a novel mycolic acid in species of the genus Mycobacterium

We found that Mycobacterium porcinum ATCC 33776T (T = type strain) contains a new kind of mycolic acid with a methoxy group at the omega-1 position. This mycolic acid was identified by comparing it with the previously described methoxymycolic acids. The patterns of mycolic acid methyl esters from 418 strains belonging to 44 species of mycobacteria were studied by using thin-layer chromatography. In addition to M. procinum ATCC 33776T, representative strains of M. porcinum, Mycobacterium fortuitum, "Mycobacterium peregrinum," Mycobacterium senegalense, and a recently isolated Mycobacterium sp. contained appreciable amounts of the newly described mycolic acid.     

Synthesis and antimycobacterial activity of new S-alkylisothiosemicarbazone derivatives

A new series of S-alkylisothiosemicarbazones of 3- and 4-pyridincarboxaldehyde and 4-fluoro- and 4-trifluoromethylbenzaldehyde was synthesized and evaluated for biological activity against various Mycobacterium strains. Inhibitory activity against Mycobacterium tuberculosis H37Rv ATCC 27294 and INH-R ATCC 35822 was compared with activity against clinical isolated Mycobacteria as well as against MOTT. Some of newly prepared compounds showed best inhibitory values against clinical isolated Mycobacteria, besides to low citotoxicity values.     

Synthesis and biological evaluation of novel 1,2,3-triazole derivatives as anti-tubercular agents

A library of seventeen novel 1,2,3-triazole derivatives were efficiently synthesized in excellent yields by the popular ‘click chemistry’ approach and evaluated in vitro for their anti-tubercular activity against Mycobacterium tuberculosis H37Ra (ATCC 25177 strain). Among the series, six compounds exhibited significant activity with minimum inhibitory concentration (MIC) values ranging from 3.12 to 0.78 μg/mL and along with no significant cytotoxicity against MBMDMQs (mouse bone marrow derived macrophages). Molecular docking of the target compounds into the active site of DprE1 (Decaprenylphosphoryl-β-d-ribose-2′-epimerase) enzyme revealed noteworthy information on the plausible binding interactions.     

Antituberculous drug susceptibility testing of Mycobacterium bovis BCG strain Montréal

Testing of Mycobacterium bovis BCG strain Montréal for susceptibility to four primary antituberculous drugs (isoniazid, ethambutol, streptomycin, and rifampin) and to one secondary drug (p-aminosalicylic acid) showed the strain to be susceptible to all five substances. Mycobacterium bovis strains ATCC 35735, which is isoniazid sensitive, and ATCC 35747, which is isoniazid resistant, were included in the test; with the exception of their respective susceptibility to isoniazid, both were inhibited by the other four drugs.     

Complete genome sequence of Mycobacterium intracellulare strain ATCC 13950(T)

Here we report the first complete genome sequence of Mycobacterium intracellulare ATCC 13950(T), a Mycobacterium avium complex (MAC) strain. This genome sequence will serve as a valuable reference for understanding the epidemiologic, biological, and pathogenic aspects of the disparity between MAC members.     

Alterations in lipid constituents during growth of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354.

Phospholipids of Mycobacterium phlei ATCC 354 and Mycobacterium smegmatis CDC 46 consist of cardiolipin, phosphatidyl ethanolamine, tri-acylated dimannophosphoinositide, tetra-acylated dimannophosphoinositide and tetra-acylated pentamannosphosphoinositide. A comparative study of lipid patterns of M. phlei ATCC 354 and of M. smegmatis CDC 46 in relation to age of culture revealed higher total lipid level and increased activity of malate-vitamin K reductase, a phospholipid requiring enzyme, during the early logarithmic growth phase of the former. No appreciable change occurred in the latter. The high total lipid content coincides with an increase in phospholipid, brought about apparently by the increase in malate-vitamin K reductase. Changes in cardiolipin and phosphatidyl ethanolamine appeared to be unique to M. phlei ATCC 354. However, in both bacterial species, a decrease in glyceride and a progressive increase in tuberculostearic acid with a concomitant decrease in oleic acid, occurred with ageing.     

Synthesis and antimycobacterial activity of isoniazid derivatives from renewable fatty acids

This work describes the synthesis of a series of fatty acid hydrazide derivatives of isoniazid (INH). The compounds were tested against Mycobacterium tuberculosis H37Rv (ATCC 27294) as well as INH-resistant (ATCC 35822 and 1896 HF) and rifampicin-resistant (ATCC 35338) M. tuberculosis strains. The fatty acid derivatives of INH showed high antimycobacterial potency against the studied strains, which is desirable for a pharmaceutical compound, suggesting that the increased lipophilicity of isoniazid plays an important role in its antimycobacterial activity.     

Complete annotated genome sequence of Mycobacterium tuberculosis Erdman

We report the completely annotated genome sequence of Mycobacterium tuberculosis Erdman (TMC 107; ATCC 35801), which is a well-known laboratory strain of M. tuberculosis.     

Benzothiazole analogs as potential anti-TB agents: computational input and molecular dynamics

Biotin is very important for the survival of Mycobacterium tuberculosis. 7,8-Diamino pelargonic acid aminotransaminase (DAPA) is a transaminase enzyme involved in the biosynthesis of biotin. The benzothiazole title compounds were investigated for their in vitro anti-tubercular activity against two tubercular strains: H37Rv (ATCC 25,177) and MDR-MTB (multidrug-resistant M. tuberculosis, resistant to isoniazid, rifampicin, and ethambutol) by an agar incorporation method. The possible binding mode and predicted affinity were computed using a molecular docking study. Among the synthesized compounds in the series, the title compound {2-(benzo[d]thiazol-2-yl-methoxy)-5-fluorophenyl}-(4-chlorophenyl)-methanone was found to exhibit significant activity with minimum inhibitory concentrations of 1 μg/mL and 2 μg/mL against H37Rv and MDR-MTB, respectively; this compound showed the highest binding affinity (–24.75 kcal/mol) as well.     

Characterization of the susceptibility of mycobacteria in BACTEC 12B media containing PANTA that had been supplemented with ceftazidime, and characterization of the individual components of PANTA in the presence of C18-carboxypropylbetaine

C(18)-carboxypropylbetaine (CB-18) specimen processing has enhanced the diagnosis of mycobacterioses by smear, culture, and nucleic acid amplification. However, toxic side effects of CB-18 in liquid culture, especially in the presence of antibiotics, have been reported. The interaction of CB-18 at 20-25 microg/ml with the individual components of the antibiotic supplement PANTA that had been fortified with ceftazidime (PANTA-caz) was characterized in BACTEC 12B cultures using four mycobacterial isolates. When the Mycobacterium tuberculosis isolate ATCC 27294 was examined CB-18 plus PANTA-caz did not significantly alter the time-to-positive (i.e., time to a growth index (GI) of 15 (GI(15))), but did significantly increase the time to a GI of 500 (GI(500)) by approximately 8.5 days. This result could be attributed primarily to nalidixic acid, but also to ceftazidime to a lesser degree. Statistically significant increases in GI(15) of 12.5 days and GI(500) of 16.5 days were observed in the presence of CB-18 plus PANTA-caz with the Mycobacterium avium isolate ATCC 25291. These increases were due exclusively to trimethoprim. Statistically significant increases of approximately 2.5 and 9 days in GI(15) and GI(500), respectively, were observed with Mycobacterium kansasii ATCC 12478 in CB-18 plus PANTA-caz. The presence of nalidixic acid and ceftazidime were responsible for these alterations. When the behavior of the Mycobacterium fortuitum isolate ATCC 6841 was investigated in CB-18 plus PANTA-caz, significant increases in GI(15) of 8.5 days and GI(500) of 13 days were observed. The additive effects of nalidixic acid and azlocillin were responsible for these results. No single component of the PANTA-caz formulation was responsible for the interaction between CB-18 and PANTA-caz, although nalidixic acid contributed to these effects most often. These findings are consistent with the previous recommendation that CB-18 specimen processing follow a dilution-based format to ensure that the concentration of CB-18 carried-over into liquid media falls below 5-10 microg/ml.     

Synthesis of triazole-oxazolidinones via a one-pot reaction and evaluation of their antimicrobial activity

C-5-substituted triazole-oxazolidinones were synthesized using a bromide catalyzed cycloaddition between aryl isocyanates and epibromohydrin followed by a three-component Huisgen cycloaddition. The library of compounds was screened for antibacterial activity against Mycobacterium smegmatis ATCC 14468, Bacillus subtilis ATCC 6633, and Enterococcus faecalis ATCC 29212. Notably, the 3-(4-acetyl-phenyl)-5-(1H-1,2,3-triazol-1-yl)methyl)-oxazolidin-2-one (18) showed an MIC of 1 μg/mL against M. smegmatis ATCC 14468, fourfold lower than the MIC measured for isoniazid.     

Identification of antimicrobial activity among new sulfonamide metal complexes for combating rapidly growing mycobacteria

Mycobacteriosis is a type of infection caused by rapidly growing mycobacteria (RGM), which can vary from localized illness, such as skin disease, to disseminated disease. Amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem and sulfamethoxazole are antimicrobial drugs chosen to treat such illnesses; however, not all patients obtain the cure. The reason why the treatment does not work for those patients is related to the fact that some clinical strains present resistance to the existing antimicrobial drugs; thereby, the research of new therapeutic approaches is extremely relevant. The coordination of antimicrobial drugs to metals is a promising alternative in the development of effective compounds against resistant microorganisms. Sulfonamides complexed with Au, Cd, Ag, Cu, and Hg have shown excellent activity against a variety of microorganisms. Considering the importance of fighting against infections associated with RGM, the objective of this study is to evaluate the antimycobacterial activity of metal complexes of sulfonamides against RGM. Complexed sulfonamides activity were individually tested and in association with trimethoprim. The minimum inhibitory concentration (MIC) and time-kill curve of compounds against the standard strains of RGM [Mycobacterium abscessus (ATCC 19977), Mycobacterium fortuitum (ATCC 6841) and Mycobacterium massiliense (ATCC 48898)] was determined. The interaction of sulfonamides with trimethoprim was defined by inhibitory concentration index fractional for each association. The results showed that sulfonamides complexed whit metals have outstanding antimicrobial activity when compared to free sulfamethoxazole, bactericidal activity and synergistic effect when combined with trimethoprim.     

Acetylation and nitrosation of ciprofloxacin by environmental strains of mycobacteria

To determine the ability of environmental bacteria to metabolize the frequently prescribed fluoroquinolone drug ciprofloxacin, eight Mycobacterium spp. cultures were grown for 4 days in a medium containing sorbitol and yeast extract with 100 mg x L(-1) ciprofloxacin. After the cultures had been centrifuged and the supernatants extracted with ethyl acetate, two metabolites were purified by using high-performance liquid chromatography. They were identified with liquid chromatography/electrospray ionization mass spectrometry and proton nuclear magnetic resonance spectroscopy. Ciprofloxacin was transformed to both N-acetylciprofloxacin (2.5%-5.5% of the total peak area at 280 nm) and N-nitrosociprofloxacin (6.0%-8.0% of the peak area) by Mycobacterium gilvum PYR-GCK and Mycobacterium sp. PYR100 but it was transformed only to N-acetylciprofloxacin by Mycobacterium frederiksbergense FAn9, M. gilvum ATCC 43909, M. gilvum BB1, Mycobacterium smegmatis mc2155, Mycobacterium sp. 7E1B1W, and Mycobacterium sp. RJGII-135. The results suggest that biotransformation may serve as a ciprofloxacin resistance mechanism for these bacteria.     

Some 3-thioxo/alkylthio-1,2,4-triazoles with a substituted thiourea moiety as possible antimycobacterials.

A series of novel N-alkyl/aryl-N'-[4-(4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thione-5-yl)phenyl]thioureas 1-19 and three S-alkylated representatives of the former, N-alkyl/aryl-N'-[4-(3-aralkylthio-4-alkyl/aryl-4H-1,2,4-triazole-5-yl)phenyl]thioureas 20-22, were synthesized and tested for antimycobacterial activity against Mycobacterium tuberculosis H37Rv as well as Mycobacterium fortuitum ATCC 6841 which is a rapid growing opportunistic pathogen. Compounds 4 and 9-11 were found to possess the same MIC value with that of Tobramycin against M. fortuitum ATCC 6841 whereas 1-3 and 21 had positive response against M. tuberculosis H37Rv at varying degrees. Compound 21 was identified as the most potent derivative of the 1-22 series by an MIC value of 6.25 microg/mL and selectivity index of 1.6.     

Comparative study with two different enrichments in the culture media used in the disinfectant efficacy assay

Recent changes in Brazilian legislation for commercial disinfectants have been published due to the recent epidemic of nosocomial infections caused by rapidly growing mycobacteria (RGM) in many states of Brazil over the last 8 years. One of these documents requires that all the manufacturers provide evidence of efficacy of sterilizing and disinfectant products, used for semi critical medical devices, against the Mycobacterium bovis BCG Moreau and Mycobacterium abscessus subsp. bolletii INCQS 00594 strains by using the Confirmative in vitro Test for Determining Tuberculocidal Activity of Disinfectants recommended by the Association of Official Analytical Chemists. These changes have caused additional costs and increased problems for importation of enrichment products at national laboratories where disinfectant efficacy assay service is performed. Middlebrook ADC Enrichment (ADC) is provided by a unique manufacturer and used in the official protocol. The aim of the present study was to evaluate an alternative in house low-cost enrichment composed of fetal bovine serum and glucose (FBSG) with ADC for performance of disinfectant efficacy assay against mycobacteria. After obtaining the growth curves for M. abscessus ATCC 19977, M. abscessus subsp. bolletii INCQS 00594, Mycobacterium chelonae ATCC 35752, and Mycobacterium fortuitum ATCC 6841 by using ADC enrichment and FBSG in Kirchners and 7H9 culture media. Through statistical analysis via the Kruskal-Wallis test on the evaluation of microorganism growth rate, it was observed that there was no inhibition of RGM growth by any of the enrichments used. These results suggest that low-cost enrichment FBSG may be used as a potential substitute of ADC for composition of media for mycobacterial growth, including in disinfectant tests.     

Purification of bovine milk lactoperoxidase and investigation of antibacterial properties at different thiocyanate mediated

Bovine lactoperoxidase (LPO) was purified with amberlite CG 50 H+ resin, CM sephadex C-50 ion-exchange chromatography, and sephadex G-100 gel filtration chromatography from skim milk. The activity of lactoperoxidase was measured by using 2.2-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH 6.0. Purification degree for the purified enzyme was controlled with SDS-PAGE and Rz value (A412/A280). Rz value for the purified LPO was 0.8. Km value at pH 6.0 at 20 degrees C for the LPO was 0.20 mM. Vmax value was 7.87 micromol/ml min at pH 6.0 at 20 degrees C. Bovine LPO showed high antibacterial activity in 100 mM thiocyanate--100 mM H2O2 medium for some pathogenic bacteria, such as Aeromonas hydrophila ATCC 7966, Micrococcus luteus LA 2971, Mycobacterium smegmatis RUT, Bacillus subtilis IMG 22, Pseudomonas pyocyanea, Bacillus subtilis var. niger ATCC 10, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 15753, Bacillus brevis FMC3, Klebsiella pneumoniae FMC 5, Corynebacterium xerosis UC 9165, Bacillus cereus EU, Bacillus megaterium NRS, Yersinia enterocolytica, Listeria monocytogenes scoot A, Bacillus megaterium EU, Bacillus megaterium DSM32, Klebsiella oxytocica, Staphylococcus aerogenes, Streptococcus faecalis, Mycobacterium smegmatis CCM 2067 and compared with well known antibacterial substances such as penicilline, ampicilline, amoxicillin-clavulanate and ceftriaxon. The LPO--100 mM thiocyanate--100 mM H2O2 system was purposed as an effective agent against many of the diseases causing organisms in human and animals.