Genetic Diversity of Mycobacterium tuberculosis Isolates From an Amerindian Population in Chiapas, México.

This is the first report of the genetic diversity of the Mycobacterium tuberculosis complex isolates found in a Mexican-Amerindian setting. In this study, we analyzed isolates collected from the Highlands region of Chiapas, Mexico, by using spoligotyping and whole-genome sequencing analyses. Seventy-three M. tuberculosis isolates were analyzed initially by spoligotyping; no new spoligotypes were identified. Nineteen percent of the isolates were identified as SIT53 (T1) (n = 14), followed by SIT42 (14%, n = 10, LAM9) and SIT119 (11%; n = 8, X1). SIT53, SIT42, and orphan isolates (16.4%, n = 12) constituted about 50% of the isolates studied and were subjected to whole-genome sequencing (WGS) analysis. Most SIT53 (10/12) isolates belonged to the Euro-American sub-lineage 4.8. Most SIT42 isolates (4/7) as .well as most orphan isolates (5/8) belonged to the lineage 4.3.3 LAM group. By comparing the single-nucleotide polymorphism (SNP) patterns of the SIT53 isolates, we found one clone (<7 SNPs) and four clustered isolates (<15 SNPs). In isolates from the SIT42 and orphan groups, we did not find any clones or clusters. This work demonstrates the success of sub-lineage 4.8 to predominate in Mexico and confirms the dominion of sub-lineage 4.3.3 in Central and South America.

Mycobacterium leprae Infection in a Wild Nine-Banded Armadillo, Nuevo León, Mexico.

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and are implicated in the zoonotic transmission of leprosy in the United States. In Mexico, the existence of such a reservoir remains to be characterized. We describe a wild armadillo infected by M. leprae in the state of Nuevo León, Mexico.

Complete Genome Sequences of Mycobacterium tuberculosis Isolates Subjected to 200 Continuous Passages.

We report 6 draft genome sequences corresponding to Mycobacterium tuberculosis H37Rv and M. tuberculosis DR689, a Beijing isolate, plus their counterparts subjected to 200 continuous passages in Middlebrook 7H9 broth, either alone or with ox bile.

Antimycobacterial Activity of Laurinterol and Aplysin from Laurencia johnstonii.

Marine environments represent a great opportunity for the discovery of compounds with a wide spectrum of bioactive properties. Due to their large variety and functions derived from natural selection, marine natural products may allow the identification of novel drugs based not only on newly discovered bioactive metabolites but also on already known compounds not yet thoroughly investigated. Since drug resistance has caused an increase in infections by Mycobacterium tuberculosis and nontuberculous mycobacteria, the re-evaluation of known bioactive metabolites has been suggested as a good approach to addressing this problem. In this sense, this study presents an evaluation of the in vitro effect of laurinterol and aplysin, two brominated sesquiterpenes isolated from Laurencia johnstonii, against nine M. tuberculosis strains and six nontuberculous mycobacteria (NTM). Laurinterol exhibited good antimycobacterial activity, especially against nontuberculous mycobacteria, being remarkable its effect against Mycobacterium abscessus, with minimum inhibitory concentration (MIC) values lower than those of the reference drug imipenem. This study provides further evidence for the antimycobacterial activity of some sesquiterpenes from L. johnstonii, which can be considered interesting lead compounds for the discovery of novel molecules to treat NTM infections.

Mycobacterium lepromatosis Infections in Nuevo León, Mexico.

The frequency of infection caused by the recently described pathogen Mycobacterium lepromatosis is unknown. Here, we describe the demographics, clinical characteristics, and therapeutic outcomes of five lepromatous leprosy patients suffering from M. lepromatosis infection in Nuevo Léon, Mexico. Diagnosis was facilitated by a new highly specific PCR procedure.

Comparative Mycobacterium tuberculosis spoligotype distribution in Mexico.

In the present work, we studied the genetic diversity of Mycobacterium tuberculosis clinical isolates from patients according to their gender, age, and geographic location in Mexico. We did not observe any statistically significant differences in regard to age or gender. We found that spoligo international type 53 (SIT53) is more frequent in the northern states and that SIT119 predominates in central Mexico.

Intracellular activity of tedizolid phosphate and ACH-702 versus Mycobacterium tuberculosis infected macrophages.

BACKGROUND: Due to the emergency of multidrug-resistant strains of Mycobacterium tuberculosis, is necessary the evaluation of new compounds. FINDINGS: Tedizolid, a novel oxazolidinone, and ACH-702, a new isothiazoloquinolone, were tested against M. tuberculosis infected THP-1 macrophages. These two compounds significantly decreased the number of intracellular mycobacteria at 0.25X, 1X, 4X and 16X the MIC value. The drugs were tested either in nanoparticules or in free solution. CONCLUSION: Tedizolid and ACH-702 have a good intracellular killing activity comparable to that of rifampin or moxifloxacin.

In vitro activity of a new isothiazoloquinolone, ACH-702, against Mycobacterium tuberculosis and other mycobacteria.

In this work, we describe the activity of ACH-702 against clinical isolates of Mycobacterium tuberculosis and six different nontuberculous mycobacteria. The MIC(50) and MIC(90) of both susceptible and drug-resistant M. tuberculosis strains tested were 0.0625 and 0.125 microg/ml, respectively. The MIC(50) and MIC(90) values for Mycobacterium fortuitum isolates were 0.0625 microg/ml in both cases; Mycobacterium avium complex isolates showed MIC(50) and MIC(90) values of 0.25 and 4 microg/ml, respectively.

Mycobacterium tuberculosis spoligotypes in Monterrey, Mexico.

Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n = 8), SIT92 (n = 3), SIT70 (n = 2), and SIT3038 (n = 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico.

Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates.

A simplified amplified-fragment length polymorphism (AFLP) method was developed and applied to genotype 52 Mycobacterium tuberculosis isolates. This method can be carried out using only one restriction enzyme (XhoI), one double strand adapter, and one PCR primer. The amounts of DNA and DNA polymerase, and the concentrations of primer and Mg(2+) in the PCR step were optimized using the Basic Sequential Simplex method. AFLP analysis of the isolates generated a total of 24 differently sized bands ranging from 1537 to 121 bp, and 52 different band patterns, with a minimum of 2 and a maximum of 13 bands. The results were compared with the well-established IS6110 restriction fragment length polymorphism (IS6110-RFLP) typing method, which rendered a total of 32 differently sized bands from 1 to 12 kbp, and 52 different band patterns, with a minimum of 3 and a maximum of 15 bands. Therefore, both genotyping methods showed a discriminatory power of samples of 100%. Nevertheless, pairwise comparisons of the 1326 similarity indexes calculated for both typing methods showed a total absence of correlation between the similarity indexes of the two methods. The simplified AFLP method is expected to be more useful for genotyping M. tuberculosis isolates compared to the IS6110-RFLP method, since the former evaluates genetic variations throughout the M. tuberculosis genome. Furthermore, the relatively rapid and low-cost simplified AFLP method compares favorably to the IS6110-RFLP or conventional AFLP methods, and shows great promise for genotyping M. tuberculosis isolates, especially in developing countries or for preliminary screening.

Detection and identification of mycobacteria by mycolic acid analysis of sputum specimens and young cultures.

Ziehl-Neelsen acid-fast staining and mycolic acid analysis of concentrated samples and Middlebrook 7H9 cultures were carried out on 127 sputum specimens to evaluate a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures and compare with mycobacterial detection using Lowenstein-Jensen (LJ) cultures. All samples were classified into five groups according to the number of acid-fast bacilli observed in the smear. The group of samples with 3+ acid-fast bacilli in the smear had the highest number of positive detections of mycolic acids in the concentrated samples and the Middlebrook 7H9 cultures (81.8 and 100%, respectively). The overall percentages of mycolic acid detection for both sample types were 43.2 and 91.3%, respectively. The mycolic acid analysis of the Middlebrook 7H9 cultures had the fewest false negative detections with respect to the LJ cultures. The analysis of fluorescent derivatives of mycolic acids, using HPLC, is useful for concentrated sputum samples with large number of bacilli (3+) and is preferred for Middlebrook 7H9 cultures, even for clinical specimens with a low number of bacilli. Furthermore, with this analytical method, the simultaneous detection and identification of mycobacteria is usually possible.

Use of a colorimetric assay to measure differences in cytotoxicity of Mycobacterium tuberculosis strains.

Several techniques have been used to quantify the cytotoxicity produced by Mycobacterium tuberculosis bacilli on cell monolayers; however, they are semi-quantitative or time consuming. Herein, a method based on crystal violet (CV) uptake by THP-1 cell monolayers is described. This colorimetric method quantifies the cytotoxic effect as a function of the number of remaining cells after the infection with M. tuberculosis. Since this micro-organism is not stained by the dye, it does not produce a background that affects absorbance readings. As determined by CV assay (CVA), M. tuberculosis strain H37Rv destroyed 10.5 % of THP-1 cell monolayers at 24 h and 50.52 % at 72 h, while M. tuberculosis strains lacking the complete phospholipase C locus produced a reduced cytotoxic effect. The damage estimated by microscopy corresponded to the effect quantified by CVA. The results show that the use of CVA is a rapid, sensitive and reliable quantitative assay to measure the cytotoxicity of different M. tuberculosis strains.

Genetic characterization of Mycobacterium tuberculosis clinical isolates with deletions in the plcA-plcB-plcC locus.

SETTING: The basis for Mycobacterium tuberculosis virulence is not completely understood. Analysis of the genomic structure of clinical isolates will give information that can be related to biological activities involved in virulence. OBJECTIVE: To determine the extension of the deletion in the plcA-plcB-plcC locus of selected M. tuberculosis isolates, as well as other changes in the chromosome. DESIGN: In the present work we characterized a group of M. tuberculosis isolates devoid of the plcA-plcB-plcC locus by PCR, sequencing and microarrays. RESULTS: PCR amplification of this region demonstrated a complete lack of plcA and plcB ORF's in all of the isolates. The plcC gene was completely deleted in one of the strains (DR-689) and the other three isolates still conserved part of this ORF. The loss of lateral DNA sequences ranged from 3723 to 7646bp. An IS6110 element was present in all tested strains cases, and some isolates presented the insertion of ORF's coding for proteins homologous to the ESAT-6 and QILSS families. Genomic DNA of all the strains was extracted and analyzed with an in-house microarray system to observe loss of other genes possibly implicated in attenuated virulence. Two of the strains presented novel deletions; the rest of the isolates showed deletions already reported for other M. tuberculosis strains. DR-689, a Beijing type M. tuberculosis strain isolated in Canada, showed an IS6110 RFLP and a genomic deletion pattern similar to a San Francisco family of strains, although completely unrelated epidemiologically. CONCLUSION: Genomic changes in M. tuberculosis seem to occur in a controlled manner and they are possibly related to changes in its pathogenic properties.

In vitro activities of the novel oxazolidinones DA-7867 and DA-7157 against rapidly and slowly growing mycobacteria.

DA-7867 and DA-7157 are oxazolidinones active against pathogenic aerobic actinomycetes including Nocardia spp. and Mycobacterium tuberculosis. However, the activity of these drugs against nontuberculous mycobacterium (NTM) species is not known. In this work, we compared the susceptibilities of 122 clinical isolates and 29 reference species of both rapidly growing and slowly growing mycobacteria to linezolid, DA-7867, and DA-7157 by the broth microdilution method. The MICs for 50 and 90% of the strains tested (MIC50s and MIC90s, respectively) of DA-7867 and DA-7157 were lower than those of linezolid. In all of the cases, a MIC90 of <8 microg/ml was observed for all of the species tested in both groups of NTM. For M. kansasii and M. marinum isolates, the MIC90s of both DA-7867 and DA-7157 were less than 0.5 microg/ml. These results demonstrate the potential of these compounds to treat NTM infections.