Dynamics of Extensive Drug Resistance Evolution of Mycobacterium tuberculosis in a Single Patient During 9 Years of Disease and Treatment.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Diagnostic Performance of the New Version (v2.0) of GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study.

Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.

Rifampin heteroresistance in Mycobacterium tuberculosis cultures as detected by phenotypic and genotypic drug susceptibility test methods.

Tuberculosis patients may harbor both drug-susceptible and -resistant bacteria, i.e., heteroresistance. We used mixtures of rifampin-resistant and -susceptible Mycobacterium tuberculosis strains to simulate heteroresistance in patient samples. Molecular tests can be used for earlier discovery of multidrug resistance (MDR), but the sensitivity to detect heteroresistance is unknown. Conventional phenotypic drug susceptibility testing was the most sensitive, whereas two line probe assays and sequencing were unable to detect the clinically important 1% resistant bacteria.

Pyrazinamide resistance and pncA gene mutations in Mycobacterium tuberculosis.

Thirty-four pyrazinamide-resistant and 37 pyrazinamide-susceptible Mycobacterium tuberculosis complex strains were analyzed for pncA gene mutations. None of the sensitive strains had any mutations, apart from silent mutations, whereas all but one resistant strain showed pncA mutations. By using sequencing as a means of early resistance detection, the inconsistency of phenotypic pyrazinamide assays can be circumvented.

Enhanced susceptibility of multidrug resistant strains of Mycobacterium tuberculosis to granulysin peptides correlates with a reduced fitness phenotype.

Previously it was shown that the antimicrobial protein granulysin possesses potent membranolytic activity against Mycobacterium tuberculosis. Here we demonstrate that granF2 and G13, which are two short synthetic peptides derived from granulysin, inhibited the in vitro growth of clinical isolates of both multidrug resistant and drug susceptible strains of M. tuberculosis. Importantly, a particularly high activity against multidrug resistant M. tuberculosis correlated with a reduced growth rate compared to drug susceptible strains. A synergistic antibacterial effect of granF2 was further observed in combination with ethambutol, a compound with a documented effect on cell wall permeability. This finding suggests that granF2 and ethambutol exert their functions at different levels of the mycobacterial surface. Upon infection of macrophages in vitro, granF2 but not G13 efficiently reduced the intracellular growth of multidrug resistant M. tuberculosis in the presence of the pore-forming protein streptolysin O. The apoptotic function of granF2 apparently promoted destruction of host cells whereby the peptide gained access to and killed intracellular bacteria. Thus, a cost of resistance and a subsequent reduced fitness, measured as decreased growth among multidrug resistant strains of M. tuberculosis, could be associated with increased susceptibility to natural immune defense mechanisms, such as antimicrobial peptides of granulysin. However, a robust cell wall and the membrane of cells still provide physical shelter for the bacteria that may spare sensitive M. tuberculosis stains from being killed.

Associated risks with the use of surgical face mask in children during the COVID-19 pandemic / Riesgos asociados al uso de la mascarilla quirúrgica en niños durante la pandemia por COVID-19

The SARS-CoV-2 virus affects all age groups. According to the Colombian National Institute of Health, by November 5, 2020, nearly 83,698 children under 18 years of age had been infected by the virus in Colombia. The probability of viral transmission in this age group is similar to that found in adults, even in asymptomatic individuals 1,2. The World Health Organization has advocated social distancing, hand washing and the use of face masks as effective measures to mitigate contagion, and healthcare institutions have implemented measures for the protection of patients and healthcare workers in order to cope with this "new normal" at work 3-5. In accordance with national and international recommendations, our institution has implemented a new preoperative care protocol during the COVID-19 pandemic.

El virus SARS-CoV-2 afecta a todos los grupos de edad. Según el Instituto Nacional de Salud de Colombia, al 5 de noviembre de 2020, cerca de 83.698 niños menores de 18 años habían sido infectados por el virus en Colombia. La probabilidad de transmisión viral en este grupo de edad es similar a la encontrada en adultos, incluso en individuos asintomáticos 1 , 2 . La Organización Mundial de la Salud ha abogado por el distanciamiento social, el lavado de manos y el uso de mascarillas como medidas efectivas para mitigar el contagio, y las instituciones de salud han implementado medidas para la protección de pacientes y trabajadores de la salud para hacer frente a esta "nueva normalidad" en el trabajo. 3 - 5. De acuerdo con las recomendaciones nacionales e internacionales, nuestra institución ha implementado un nuevo protocolo de atención preoperatoria durante la pandemia de COVID-19.

Assessment of the Activity of Selected Indian Medicinal Plants against Mycobacterium tuberculosis: A Preliminary Screening Using the Microplate Alamar Blue Assay.

Aim: Identification of anti-Mycobacterium tuberculosis agents of plant origin, against sensitive and multidrug resistant (MDR) strains. Study Design: Assessing anti-M. tuberculosis activity of five Indian medicinal plants, which have been reported in traditional literature for various uses including respiratory ailments. Place and Duration of Study: Mumbai, India; May 2009 – December 2011. Methodology of Study: The reference strain (H37Rv), three susceptible and three MDR clinical isolates of M. tuberculosis were used. Acetone, ethanol and aqueous extracts (prepared sequentially) of Acorus calamus L. (rhizome), Andrographis paniculata Nees. (leaf), Ocimum sanctum L. (leaf), Piper nigrum L. (seed) and Pueraria tuberosa DC. (tuber) were tested at 1, 10 and 100 μg/ml using the Microplate Alamar Blue Assay. The active extracts were assessed for cytotoxicity on the human lung epithelial cell line (A549) using the neutral red assay and a phytochemical analysis was made using High Performance Thin Layer Chromatography (HPTLC). Results: Among the plants tested, the acetone extract of P. nigrum appears promising. It was effective against H37Rv, all susceptible isolates and one MDR isolate at 100 μg/ml. The ethanol extract caused some inhibition of growth, though less than the cut-off of 99%. A combination of acetone and ethanol extracts at 50 μg/ml each was effective against all isolates tested. The known active phytoconstituent of P. nigrum, piperine (also an efflux pump inhibitor), was effective against H37Rv in the presence of suboptimal concentration of Rifampicin, but not against the clinical isolates tested. Presence of piperine in the acetone and ethanol extracts was confirmed by HPTLC. Extracts of P. nigrum and piperine were not cytotoxic to the A549 cell line. Conclusion: Amongst the five plants tested, P. nigrum was active. The acetone extract may have active components in addition to piperine. It is possible that the class and expression of efflux pumps in H37Rv is different from that in the clinical isolates, and hence piperine did not inhibit these isolates. Thus, it is necessary to screen clinical isolates in addition to reference strains. The observation of the increased efficacy of the combination of acetone and ethanol extracts is interesting.

Glycine and alanine dehydrogenase activities are catalyzed by the same protein in Mycobacterium smegmatis: upregulation of both activities under microaerophilic adaptation.

Microaerophilic adaptation has been described as one of the in vitro dormancy models for tuberculosis. Studies on Mycobacterium tuberculosis adapted to low oxygen levels showed an enhancement of glycine dehydrogenase (deaminating) activity. We studied the physiology of the fast-growing, nonpathogenic strain of Mycobacterium smegmatis ATCC 607 under low oxygen by shifting the actively growing M. smegmatis cells to static microaerophilic growth conditions. This shifting of M. smegmatis culture resulted in a similar phenomenon as seen with M. tuberculosis, i.e., elevated glycine dehydrogenase activity. Further purification of glycine dehydrogenase from M. smegmatis demonstrated glyoxylate amination, but failed to demonstrate glycine deamination, even in the purified fraction. Moreover, the purified protein showed pyruvate amination as well as L-alanine deamination activities. By activity staining, the protein band positive for glyoxylate amination demonstrated only pyruvate amination in the presence of NAD. Absence of glycine deamination activity strongly suggested that alanine dehydrogenase of M. smegmatis was responsible for glyoxylate amination in the cell lysate. This was further confirmed by demonstrating the similar level of upregulation of both glyoxylate and pyruvate amination activities in the cell lysate of the adapted culture.