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We compared the prevalence of levofloxacin (LVX) resistance with that of ofloxacin (OFX) and moxifloxacin (MFX) among multidrug resistant (MDR) MTB clinical isolates collected in Medellin, Colombia, between 2004 and 2009 and aimed at unraveling the underlying molecular mechanisms that explain the correlation between QRDR-A mutations and LVX resistance phenotype. We tested 104 MDR isolates for their susceptibility to OFX, MFX, and LVX. Resistance to OFX was encountered in 10 (9.6%) of the isolates among which 8 (7.7%) were also resistant to LVX and 6 (5.7%) to MFX. Four isolates resistant to the 3 FQ were harboring the Asp94Gly substitution, whilst 2 other isolates resistant to OFX and LVX presented the Ala90Val mutation. No mutations were found in the QRDR-B region. The molecular modeling of the interaction between LVXand the DNA-DNAgyrase complex indicates that the loss of an acetyl group in the Asp94Gly mutation removes the acid base interaction with LVX necessary for the quinolone activity. The Ala90Val mutation that substitutes a methyl for an isopropyl group induces a steric modification that blocks the LVX access to the gyrase catalytic site. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (MTB) is known for its adaptive capability in developing resistance to antibiotics, through the selection of spontaneous mutations that arise during treatment. Generating spontaneous antibiotic-resistant mutants in vitro is challenging but necessary for studying this phenomenon. A protocol was designed and tested to select stable, MTB spontaneous, d-cycloserine (DCS) resistant mutants. Twenty-four colonies resistant to DCS were selected, demonstrating an increase between 1 and 4 times the Minimum Inhibitory Concentration (MIC) set for Mycobacterium tuberculosis H37Rv ATCC 27294 reference strain., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

Background: Childhood tuberculosis continues to be a major public health problem. Although the visibility of the epidemic in this population group has increased, further research is needed., Objective: To design, implement and evaluate an integrated care strategy for children under five years old who are household contacts of bacteriologically confirmed pulmonary tuberculosis patients in Medellín and the Metropolitan Area., Methods: A quasi-experimental study in which approximately 300 children who are household contacts of bacteriologically confirmed pulmonary tuberculosis patients from Medellín and the Metropolitan Area will be evaluated and recruited over one year. A subgroup of these children, estimated at 85, who require treatment for latent tuberculosis, will receive an integrated care strategy that includes: some modifications of the current standardized scheme in Colombia, with rifampicin treatment daily for four months, follow-up under the project scheme with nursing personnel, general practitioners, specialists, professionals from other disciplines such as social work, psychology, and nutritionist. Additionally, transportation and food assistance will be provided to encourage treatment compliance. This strategy will be compared with isoniazid treatment received by a cohort of children between 2015 and 2018 following the standardized scheme in the country. The study was approved by the CIB Research Ethics Committee and UPB., Clinicaltrials: gov identifier NCT04331262., Discussion: This study is expected to contribute to the development of integrated care strategies for the treatment of latent tuberculosis in children. The results will have a direct impact on the management of childhood tuberculosis contributing to achieving the goals proposed by the World Health Organization's End TB Strategy., Trial Registration: ClinicalTrials.gov identifier NCT04331262 . Implementation of an Integrated Care Strategy for Children Contacts of Patients with Tuberculosis. Registered 2 April 2020., (© 2023. The Author(s).)

Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n = 57 ETH-resistant MDR-TB isolates; n = 3 ETH-susceptible MDR-TB isolates; and n = 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Introduction: A variable proportion of isolates of multidrug-resistant Mycobacterium tuberculosis also presents resistance to ethionamide. It is important to determine whether resistance to isoniazid is independent or crossed with resistance to ethionamide, given that this could lead to the re-evaluation of second-line anti-tuberculosis treatment. The GenoType MTBDR plus ® molecular test is used for the detection of MDR-MTB, as it identifies mutations associated with resistance to isoniazide and could detect cross-resistance with ethionamide., Objective: To evaluate the performance of GenoType MTBDR plus ® in comparison with sequencing in the detection of mutations in gene katG and promotor inhA in clinical isolates of multidrug-resistant M. tuberculosis ., Materials and Methods: The GenoType MTBDR plus 1.0® commercial kit and sequencing were used to evaluate mutations in gene katG and promotor inhA in 30 multidrug-resistant M. tuberculosis isolates that were resistant to ethionamide. The laboratory strain H37Rv and three pan-sensitive isolates acted as controls., Results: The kappa index for the comparison between the results of sequencing and GenoType MTBDR plus® was 1. All the isolates resistant to isoniazid and ethionamide had the mutations detected by GenoTypeMTBDR plus® in the katG gene and 40% of them in promotor inhA. Sequencing also revealed katG mutations in positions different to those detected by GenoType MTBDR plus®., Conclusion: GenoType MTBDR plus ® is able to detect resistance to isoniazid rapidly. Our results suggest that it could also be used to screen for cross-resistance with ethionamide.

Background: Phylogeographic composition of M. tuberculosis populations reveals associations between lineages and human populations that might have implications for the development of strategies to control the disease. In Latin America, lineage 4 or the Euro-American, is predominant with considerable variations among and within countries. In Colombia, although few studies from specific localities have revealed differences in M. tuberculosis populations, there are still areas of the country where this information is lacking, as is a comparison of Colombian isolates with those from the rest of the world., Principal Findings: A total of 414 M. tuberculosis isolates from adult pulmonary tuberculosis cases from three Colombian states were studied. Isolates were genotyped using IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and 24-locus Mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTRs). SIT42 (LAM9) and SIT62 (H1) represented 53.3% of isolates, followed by 8.21% SIT50 (H3), 5.07% SIT53 (T1), and 3.14% SIT727 (H1). Composite spoligotyping and 24-locus MIRU- VNTR minimum spanning tree analysis suggest a recent expansion of SIT42 and SIT62 evolved originally from SIT53 (T1). The proportion of Haarlem sublineage (44.3%) was significantly higher than that in neighboring countries. Associations were found between M. tuberculosis MDR and SIT45 (H1), as well as HIV-positive serology with SIT727 (H1) and SIT53 (T1)., Conclusions: This study showed the population structure of M. tuberculosis in several regions from Colombia with a dominance of the LAM and Haarlem sublineages, particularly in two major urban settings (Medellín and Cali). Dominant spoligotypes were LAM9 (SIT 42) and Haarlem (SIT62). The proportion of the Haarlem sublineage was higher in Colombia compared to that in neighboring countries, suggesting particular conditions of co-evolution with the corresponding human population that favor the success of this sublineage.

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6%) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%), five of the 512 patients from Argentina (1.0%), two of the 252 Brazilian cases (0.8%), one of the 166 patients from Paraguay (0.6%) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

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.

Availability of the M. tuberculosis genome sequence and the development of sophisticated systems for genetic manipulation of bacilli offer the potential for new and effective tools to prevent and control tuberculosis. Efficient methods to inactivate mycobacterial genes have been developed. These methods have become the cornerstone for the application and development of mycobacterial functional genomics. Specific mutants are generated to establish the role of targetted genes associated with mycobacterial physiology and pathogenesis. Gene inactivation, supported directly or indirectly by the deciphering of the mycobacterial genome, has permitted the generation of large numbers of M. tuberculosis mutants. Analysis of these mutants has (in some cases) established relationships between gene products and their role in mycobacterial physiology and pathogenesis.