Andrea von Groll

Fluoroquinolone Resistance in Mycobacterium tuberculosis and Mutations in gyrA and gyrB

ABSTRACT This study evaluated cross-resistance of Mycobacterium tuberculosis strains to ofloxacin, moxifloxacin, and gatifloxacin and investigated the presence of mutations in gyrA and gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosis strains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three fluoroquinolones. However, one strain, with an Asn-533 → Thr mutation in gyrB, was susceptible to ofloxacin but resistant to moxifloxacin and gatifloxacin.

Efflux as a mechanism for drug resistance in Mycobacterium tuberculosis.

Tuberculosis remains an important global public health problem, with an estimated prevalence of 14 million individuals with tuberculosis worldwide in 2007. Because antibiotic treatment is one of the main tools for tuberculosis control, knowledge of Mycobacterium tuberculosis drug resistance is an important component for the disease control strategy. Although several gene mutations in specific loci of the M. tuberculosis genome have been reported as the basis for drug resistance, additional resistance mechanisms are now believed to exist. Efflux is a ubiquitous mechanism responsible for intrinsic and acquired drug resistance in prokaryotic and eukaryotic cells. Mycobacterium tuberculosis presents one of the largest numbers of putative drug efflux pumps compared with its genome size. Bioinformatics as well as direct and indirect evidence have established relationships among drug efflux with intrinsic or acquired resistance in M. tuberculosis. This minireview describes the current knowledge on drug efflux in M. tuberculosis.

Rapid culture-based methods for drug-resistance detection in Mycobacterium tuberculosis

Tuberculosis still represents a major public health problem, especially in low-resource countries where the burden of the disease is more important. Multidrug-resistant and extensively drug drug-resistant tuberculosis constitute serious problems for the efficient control of the disease stressing the need to investigate resistance to first- and second-line drugs. Conventional methods for detecting drug-resistance in Mycobacterium tuberculosis are slow and cumbersome. The most commonly used proportion method on Löwenstein-Jensen medium or Middlebrook agar requires a minimum of 3-4 weeks to produce results. Several new approaches have been proposed in the last years for the rapid and timely detection of drug-resistance in tuberculosis. This review will address phenotypic culture-based methods for rapid drug susceptibility testing in M. tuberculosis.

Linear DNA Low Efficiency Transfection by Liposome Can Be Improved by the Use of Cationic Lipid as Charge Neutralizer

A plasmid expressing the β‐galactosidase enzyme was used to transfect Vero cells in order to evaluate the efficiency of a liposome‐mediated transfection by circular and linear DNA. The results obtained showed a low rate of transfection by linear DNA:liposome complexes. To explore whether the structure of the complexes was interfering with the transfection, atomic force microscopy (AFM) was used. It has confirmed the difference between the linear and circular condensates: whereas the circular DNA:liposome complexes presented compact spherical or cylindrical structures of about 100–800 nm, the linear DNA showed pearl necklace‐like structures, with pearls varying from 250 to 400 nm. On the basis of the theory proposed by Kuhn et al. (1999), low concentrations of cationic amphihile were used to neutralize or reverse the DNA charge in order to improve the transfection efficiency of the linearDNA. Using this method, we were able to obtain the expression of the transgene without an associated toxicity observed with the linear DNA liposome delivery.

Fitness study of the RDRio lineage and Latin American-Mediterranean family of Mycobacterium tuberculosis in the city of Rio Grande, Brazil

RD(Rio) is a novel Mycobacterium tuberculosis lineage of the Latin American-Mediterranean (LAM) family. LAM has been found worldwide but is more predominant in South America. The aim of this study was to assess the presence of the RD(Rio) lineage and LAM family in the city of Rio Grande, Brazil, and to investigate the fitness of these strains based on determination of their growth rate. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different patterns were found by spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats. The predominant genotypes belonged to the LAM family (54% of the strains) followed by clade T (22%) and Haarlem (16%). The RD(Rio) lineage represented 38% of the total strains and 70.4% of the LAM strains found in this study. Strains belonging to the LAM family showed a fitness advantage when comparing their rate of growth with that of non-LAM strains, but a significant difference between RD(Rio) and non-RD(Rio) strains was not confirmed.

Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

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.

Determinants of HIV-1 mother-to-child transmission in Southern Brazil

Different human immunodeficiency virus type 1 (HIV-1) subtypes may have distinct biological, immunological and pathogenic properties. Efficiency of mother-to-child transmission (MTCT) may be among those properties, but few and controversial results have been described so far. In this study, 102 children born from HIV-1-infected mothers between 1998 and 2004 in the city of Rio Grande, Brazil were analyzed for potential risk factors associated with MTCT. That geographic region is characterized by a high proportion of subtype C-infected subjects, and it allowed comparison between subtypes B and C and their influence on MTCT. The analysis also included clinical, obstetric and immunological parameters. Multivariate regression analyses were conducted to evaluate the influence of the parameters on MTCT, and prevalence ratios (PR) and 95% confidence intervals (CI95) were also calculated. A surprisingly high prevalence of subtype C of over 70% was found. Only the HIV viral load and the use of ACTG 076 protocol were predictive of MTCT. HIV subtype and CD4 T-cell counts were not associated with increased risk of transmission. Although a clear expansion of subtype C is evident in southern Brazil, it does not seem to correlate with increased risk of vertical transmission.

Rapid Detection of Mycobacterium tuberculosis Resistance to Second-Line Drugs by Use of the Manual Mycobacterium Growth Indicator Tube System

ABSTRACT The objective of this study was to evaluate the manual mycobacterium growth indicator tube (MGIT) system for the testing of Mycobacterium tuberculosis susceptibility to second-line drugs compared to the proportion method. One hundred eighty-eight M. tuberculosis isolates were tested for susceptibility to ofloxacin, kanamycin, ethionamide, and capreomycin by the manual MGIT, and results were compared to those obtained with the proportion method on 7H11 agar, considered a reference method. Results for ofloxacin and capreomycin were excellent, with 100% accuracy, and a result of 99.4% accuracy was achieved for kanamycin. For ethionamide, accuracy was lower, with a result of 86.7% compared to that of the proportion method. We proposed the following critical concentrations for the drugs: for ofloxacin, 2.0 μg/ml; for kanamycin, 2.5 μg/ml; for ethionamide, 5 μg/ml; and for capreomycin, 2.5 μg/ml. The time required to obtain results was an average of 8 days by the manual MGIT and 3 weeks by the reference method. Our results show that the manual MGIT is an accurate method for the rapid susceptibility testing of M. tuberculosis to second-line drugs. There is no need for a machine when using the manual MGIT, and results can be read with a simple UV lamp or with a semiquantitative reader, which considerably reduces the cost of the method.

Comparative evaluation of the Nitrate Reductase Assay and the Resazurin Microtitre Assay for drug susceptibility testing of Mycobacterium tuberculosis against first line anti-tuberculosis drugs

Tuberculosis remains as a serious infection disease of worldwide distribution, with high morbidity and mortality, mainly in low socio-economic condition countries. The state of emergency of tuberculosis caused by the resistant and multidrug-resistant (MDR) strains, became the main threat to the tuberculosis treatment and control programs. A fast detection method for the resistant strains will allow the implementation of an adequate treatment and contribute for controlling the dissemination of these resistant strains. This study evaluated the performance of the nitrate reductase assay in solid (NRA-LJ) and liquid (NRA-7H9) media, to determine the susceptibility to first line anti-tuberculosis drugs: isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and streptomycin (SMR). Both methods NRA-LJ and NRA-7H9 were evaluated among 18 strains with a known susceptibility profile. The resazurin microtiter assay (REMA) was performed as a reference method. One hundred percent of accordance was observed between NRA-7H9 and REMA for the four tested drugs. When the NRA-LJ method was compared to REMA, the sensitivity and the specificity to INH, RMP, EMB and SMR were 100%, 100 %, 85.7%, 76.9% and 80%, 100%, 75% and 80%, respectively. From the 57 clinical isolates of M. tuberculosis evaluated by NRA-7H9 and REMA, 56 (98.2%) were sensitive to all antibiotics tested (INH, RMP, EMB and SMR) by the NRA-7H9 method, while three of these strains were resistant to INH by REMA. One strain showed resistance to INH and RMP for both methods, and MIC of 1.0 μg/ml to INH for both methods, while MIC of 1.0 and 2.0 μg/ml to RMP for REMA and NRA-7H9, respectively. The three assays showed a high level of agreement for rapid detection of rifampicin and isoniazid resistance. Regarding rapidness, the detection of color change in the NRA method is within instants as compared to the overnight incubation required for the REMA test. NRA might represent an inexpensive and alternative assay for rapid detection of resistance in low-income countries.