Fernanda Sá Spies

Identification of Mutations Related to Streptomycin Resistance in Clinical Isolates of Mycobacterium tuberculosis and Possible Involvement of Efflux Mechanism

ABSTRACT The MIC for streptomycin in the presence of efflux pump (EP) inhibitors and the sequencing of rpsL, rrs, and gidB genes provided evidence for the possible participation of EP in low-level streptomycin (STR) resistance of some isolates without mutations. Mutation in the gidB gene and an EP could act synergistically to confer low STR resistance.

Streptomycin resistance and lineage specific polymorphisms in Mycobacterium tuberculosis gidB gene

ABSTRACT Mutations related to streptomycin resistance in the rpsL and rrs genes are well known and can explain about 70% of this phenotypic resistance. Recently, the gidB gene was found to be associated with low-level streptomycin resistance in Mycobacterium tuberculosis. Mutations in gidB have been reported with high frequency, and this gene appears to be very polymorphic, with frameshift and point mutations occurring in streptomycin-susceptible and streptomycin-resistant strains. In this study, mutations in gidB appeared in 27% of streptomycin-resistant strains that contained no mutations in the rpsL or rrs genes, and they were associated with low-level streptomycin resistance. However, the association of certain mutations in gidB with streptomycin resistance needs to be further investigated, as we also found mutations in gidB in streptomycin-susceptible strains. This occurred only when the strain was resistant to rifampin and isoniazid. Two specific mutations appeared very frequently in this and other studies of streptomycin-susceptible and -resistant strains; these mutations were not considered related to streptomycin resistance, but as a polymorphism. We stratified the strains according to the different phylogenetic lineages and showed that the gidB 16 polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype, while the gidB 92 polymorphism (92C allele) was associated with the Beijing lineage in another population. In the sample studied, the two characterized single-nucleotide polymorphisms could distinguish LAM and Beijing lineages from the other lineages.

Synthesis and evaluation of rifabutin analogs against Mycobacterium avium and H37Rv, MDR and NRP Mycobacterium tuberculosis

Clinical utility of rifabutin 1 (RBT), a potent antibiotic used in multidrug regimens for tuberculosis (TB) as well as for infections caused by Mycobacterium avium complex (MAC), has been hampered due to dose-limiting toxicity. RBT analogs 2-11 were synthesized and evaluated against M. avium 1581 and Mycobacterium tuberculosis susceptible and resistant strains in vitro. A selection of candidates were also assayed against non-replicating persistent (NRP) M. tuberculosis. Subsequent in vivo studies with the best preclinical candidate drugs 5 and 8, in a model of progressive pulmonary tuberculosis of Balb/C mice infected either with H(37)Rv drug-sensible strain or with multidrug resistant (MDR) clinical isolates, resistant to all primary antibiotics including rifampicin, were performed. The results disclosed here suggest that 5 and 8 have potential for clinical application.

Biological cost in Mycobacterium tuberculosis with mutations in the rpsL, rrs, rpoB, and katG genes

When bacteria develop drug-resistant mutations, there is often an associated biological cost; however, some strains can exhibit low- or no-cost mutations. In the present study, a quantitative resazurin reduction assay was used to measure the biological cost of Mycobacterium tuberculosis isolates that contained different mutations in the rpsL, rrs, rpoB, and katG genes, and showed different resistance profiles. Biological costs were determined by comparing the growth curves of drug-resistant isolates with drug-susceptible strains. Some strains, such as those with rpoB mutations other than S531L and strains with mutations in all of the studied genes, grew more slowly than did drug-susceptible strains. However, some strains grew more quickly than drug-susceptible strains, such as those that had only the rpsL K43R mutation. Strains with the mutation katG S315T presented heterogeneous biological costs. When analyzed individually, strains with the mutations rpsL43/katG315, rpoB531, and rpoB531/katG315 grew faster than drug-susceptible strains. The results suggest that some strains with the most common mutations correlated to a high resistance toward streptomycin, isoniazid and rifampicin can grow as well as or better than susceptible strains.

Performance of the GenoType MTBDRplus Assay Directly on Sputum Specimens from Brazilian Patients with Tuberculosis Treatment Failure or Relapse

ABSTRACT Rapid identification of drug resistance in clinical isolates of Mycobacterium tuberculosis is important in determining treatment for tuberculosis. The aim of this work was evaluate the performance of the GenoType MDRTBplus assay directly on sputum of patients who had treatment failure or relapse in a routine outpatient setting in southern Brazil.

Mycobacterium tuberculosis of the RDRio Genotype Is the Predominant Cause of Tuberculosis and Associated with Multidrug Resistance in Porto Alegre City, South Brazil

ABSTRACT Spoligotyping has shown Mycobacterium tuberculosis strains to be composed of different lineages, and some of them are not just geographically restricted but also affect specific ethnic populations and are associated with outbreaks and drug resistance. We recently described a particular subtype within the Latin American-Mediterranean (LAM) family, called RDRio, widespread in Brazil. Moreover, recent data also indicate that RDRio is present in many countries on all continents and is associated with cavitary disease and multidrug resistance (MDR). To further explore the relationship between RDRio and MDR, we conducted a study in a tuberculosis (TB) reference center responsible for the care of MDR patients in Rio Grande do Sul, the southernmost Brazilian state. From a collection of 237 clinical isolates, RDRio alone was responsible for one-half of all MDR cases, including one large group composed of strains with identical IS6110-restriction fragment length polymorphism (RFLP) and having the LAM5 signature. We additionally had complete data records for 96 patients and could make comparisons between the presence and absence of RDRio. No difference in clinical, radiological or laboratory features was observed, but a significantly greater number of cases with MDR were described in patients infected with an RDRio strain (P = 0.0015). Altogether, RDRio was responsible for 38% of all TB cases. These data support and confirmed previous findings that RDRio is the main agent responsible for TB in Brazil and is associated with drug resistance. Considering that RDRio is a globally distributed genotype, such findings raise concern about the increase in MDR in certain human populations.

Clonal diversity of M. tuberculosis isolated in a sea port city in Brazil

Genotyping tools have been widely used to study the occurrence of outbreaks and to identify the patterns of transmission of Mycobacterium tuberculosis strains. The clonal diversity of 65 clinical isolates of M. tuberculosis was determined by PCR methods. The Double Repeat Element method (DRE-PCR) and spoligotyping identified 45 and 26 distinct patterns respectively. Among these, LAM (38%) was the most frequent lineage, followed by Haarlem (31%) and T (20%). Five orphan patterns were not present in the SITVIT database. Mycobacterial interspersed repetitive units (MIRU) using 12 loci revealed 46 distinct patterns. MIRU loci 10, 23, 26 and 40 had the highest discriminatory power. The high genetic diversity found among M. tuberculosis isolates in this study suggests a high level of recent TB transmission, indicating an endemic mode of TB transmission and a putative importation of new TB genotypes. In addition, the high diversity among the isolates could indicate early detection of the infection in patients and an efficient rate of cure.

Tuberculosis recurrence in a high incidence setting for HIV and tuberculosis in Brazil

BackgroundTo compare epidemiological data between recurrent cases after cure (RC), distinguishing relapse from reinfection, after dropout (RD) and new cases (NC) in an ambulatory setting in a TB-endemic country.MethodsRecords of patients who started treatment for pulmonary TB between 2004 and 2010 in a TB clinic were reviewed. Epidemiological data were analyzed. Spoligotyping and MIRU patterns were used to determine relapse or reinfection in 13 RC available.ResultsOf the eligible group (1449), 1060 were NC (73.2%), among the recurrent cases, 203 (14%) were RC and 186 (12.8%) were RD. Of RC, 171 (84.2%) occurred later than 6 months after a previous episode, 13 had available DNA, in 4 (30.7%) the disease was attributed to reinfection and in 9 (69.3%), to relapse. Comparing RC to NC, HIV (p < 0.0001) was independent risk factor for RC. When RC and RD were compared, alcohol abuse (p = 0.001) and treatment noncompliance (p = 0.006) were more frequent in RD.ConclusionsHIV is the sole more important associated factor for RC. This finding points the need to improve the approach to manage TB in order to decrease the chance for exposure especially in vulnerable people with increased risk of developing disease and to improve DOTS strategy to deal with factors associated to treatment noncompliance.