Is the efflux pump inhibitor Verapamil a potential booster for isoniazid against Mycobacterium tuberculosis?

The membrane-based efflux pump systems are recognized to have an important role in pathogenicity and drug resistance in Mycobacterium tuberculosis by the extrusion of toxic substrates and drugs from the inner bacillus. This study aimed to investigate the in vitro interaction of Verapamil (VP), an efflux pump inhibitor, with the classical first-line anti-tuberculosis drug isoniazid (INH) in resistant and susceptible M. tuberculosis clinical isolates. Seven multidrug-resistant (MDR), three INH monoresistant and four susceptible M. tuberculosis clinical isolates were tested for the INH and VP combination by modified Resazurin Microtiter Assay Plate (REMA). Fractional Inhibitory Concentration (FIC) and Modulation Factor (MF) were determined. The INH plus VP combination showed no significant change in the Minimum inhibitory concentration (MIC) values of INH (FIC≥ 0.5; MF=1 or 2).The use of VP in tuberculosis therapy should be managed carefully, considering the resistance caused by specific mutation in katG and inhA genes, in which the use of these EPIs may have no success. The use of EPIs as an adjunctive drug in the anti-tuberculosis therapy should be further investigated on a larger number of M. tuberculosis clinical isolates with different resistant profile.

Nanotechnology-Based Drug Delivery Systems for Treatment of Tuberculosis--A Review.

Tuberculosis (TB) is an infectious and transmissible disease that is caused by Mycobacterium tuberculosis and primarily affects the lungs, although it can affect other organs and systems. The pulmonary presentation of TB, in addition to being more frequent, is also the most relevant to public health because it is primarily responsible for the transmission of the disease. The to their low World Health Organization (WHO) recommends a combined therapeutic regimen of several drugs, such as rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (ETB). These drugs have low plasma levels after oral administration, due to their low water solubility, poor permeability and ability to be rapidly metabolized by the liver and at high concentrations. Furthermore, they have short t1/2 (only 1-4 hours) indicating a short residence in the plasma and the need for multiple high doses, which can result in neurotoxicity and hepatotoxicity. Nanotechnology drug delivery systems have considerable potential for the treatment of TB. The systems can also be designed to allow for the sustained release of drugs from the matrix and drug delivery to a specific target. These properties of the systems enable the improvement of the bioavailability of drugs, can reduce the dosage and frequency of administration, and may solve the problem of non-adherence to prescribed therapy, which is a major obstacle to the control of TB. The purpose of this study was to systematically review nanotechnology-based drug delivery systems for the treatment of TB.

Anti-Mycobacterium tuberculosis activity of antituberculosis drugs and amoxicillin/clavulanate combination.

We report the in vitro drugs interaction by the resazurin drugs combination microtiter assay (REDCA) of amoxicillin (AMO)/clavulanate (CLAV) with isoniazid (INH), ethambutol (EMB), and rifampicin (RIF) against susceptible and resistant Mycobacterium tuberculosis isolates. The addition of AMO/CLAV to classical antituberculosis drugs should be explored as a promising alternative for the treatment of resistant tuberculosis (TB).

Opportunistic pathogens and elements of the resistome that are common in bottled mineral water support the need for continuous surveillance.

Several differences concerning bacterial species, opportunistic pathogens, elements of the resistome as well as variations concerning the CFU/mL counts were identified in some of the five most marketed bottled mineral water from Araraquara city, São Paulo, Brazil. Two out of five brands tested were confirmed as potential source of opportunistic pathogens, including Mycobacterium gordonae, Ralstonia picketti and Burkholderia cepacia complex (Bcc). A total of one hundred and six isolates were recovered from four of these bottled mineral water brands. Betaproteobacteria was predominant followed by Alphaproteobacteria, Gammaproteobacteria and Firmicutes. Ninety percent of the bacteria isolated demonstrated resistance to seventeen of the nineteen antimicrobials tested. These antimicrobials included eight different classes, including 3rd and 4th generation cephalosporins, carbapenems and fluoroquinolones. Multidrug resistant bacteria were detected for fifty-nine percent of isolates in three water brands at counts up to 103 CFU/ml. Of major concern, the two bottled mineral water harboring opportunistic pathogens were also source of elements of the resistome that could be directly transferred to humans. All these differences found among brands highlight the need for continuous bacteriological surveillance of bottled mineral water.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil.

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Synthesis and evaluation of a pyrazinoic acid prodrug in Mycobacterium tuberculosis.

Tuberculosis (TB) is a disease caused mainly by infection of Mycobacterium tuberculosis affecting more than ten million people around the world. Despite TB can be treated, the rise of MDR-TB and XDR-TB cases put the disease in a worrying status. As pyrazinamide-resistant strains exhibit low or none pyrazinamidase activity, it is proposed that the active form of pyrazinamide (PZA) is pyrazinoic acid (POA), although this acid has poor penetration in mycobacteria. In this work, we present a convenient one-pot synthesis of 2-chloroethyl pyrazinoate, and its activity in M. tuberculosis H37Rv (ATCC27294) in MIC assay using the MABA technique. The obtained MIC of the compound was 3.96 g/mL, and discussion about the activity profile of some previously evaluated pyrazinoates is also performed.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Nanostructured lipid systems as a strategy to improve the in vitro cytotoxicity of ruthenium(II) compounds.

Tuberculosis is an ancient disease that is still present as a global public health problem. Our group has been investigating new molecules with anti-TB activity. In this context, inorganic chemistry has been a quite promising source of such molecules, with excellent results seen with ruthenium compounds. Nanostructured lipid systems may potentiate the action of drugs by reducing the required dosage and side effects and improving the antimicrobial effects. The aim of this study was to develop a nanostructured lipid system and then characterize and apply these encapsulated compounds (SCARs 1, 2 and 4) with the goal of improving their activity by decreasing the Minimum Inhibitory Concentration (MIC90) and reducing the cytotoxicity (IC50). The nanostructured system was composed of 10% phase oil (cholesterol), 10% surfactant (soy oleate, soy phosphatidylcholine and Eumulgin®) and 80% aqueous phase (phosphate buffer pH = 7.4). Good activity against Mycobacterium tuberculosis was maintained after the incorporation of the compounds into the nanostructured lipid system, while the cytotoxicity decreased dramatically, in some cases up to 20 times less toxic than the unencapsulated drug.

Characterization and Quantification of Compounds in the Hydroalcoholic Extract of the Leaves from Terminalia catappa Linn. (Combretaceae) and Their Mutagenic Activity.

Terminalia is a genus of Combretaceous plants widely distributed in tropical and subtropical regions. Thus, the aim of this study was to quantify the majority compounds of the hydroalcoholic extract (7 : 3, v/v) of the leaves from T. catappa by HPLC-PDA, chemically characterize by hyphenated techniques (HPLC-ESI-IT-MS(n)) and NMR, and evaluate its mutagenic activity by the Salmonella/microsome assay on S. typhimurium strains TA98, TA97a, TA100, and TA102. The quantification of analytes was performed using an external calibration standard. Punicalagin is the most abundant polyphenol found in the leaves. The presence of this compound as a mixture of anomers was confirmed using HPLC-PDA and (1)H and (13)C NMR. Mutagenic activity was observed in strains TA100 and TA97a. As the extract is a complex mixture of punicalagin, its derivatives, and several other compounds, the observed mutagenicity may be explained in part by possible synergistic interaction between the compounds present in the extract. These studies show that mutagenic activity of T. catappa in the Ames test can only be observed when measured at high concentrations. However, considering the mutagenic effects observed for T. catappa, this plant should be used cautiously for medicinal purposes.

Esteroide e Triterpenos de espécies de Qualea - Bioatividade sobre Mycobacterium tuberculosis / Sterol and Triterpenes from Qualea species: Bioactivity on Mycobacterium tuberculosis

O extrato clorofórmico das cascas de Qualea parviflora (Vochysiaceae) foi fracionado em cromatografia em coluna usando sílica gel para fornecer triterpenos (lupeol, lupenona, betulina, ácido epibetulínico, e friedelina) e um esteroide (β-sitosterol). Os compostos β- sitosterol, lupenona e lupeol foram identificados também em Q. grandiflora e Q. multiflora, enquanto friedelina foi detectada apenas em Q. multiflorautilizando a cromatografia gasosa acoplada à espectrometria de massas. A atividade anti Mycobacterium tuberculosis do extrato clorofórmico e dos compostos isolados foi determinada por MABA e o valor da CIM variou de 250,0 a 31,2 μg/mL. Esta investigação constitui o primeiro relato do estudo químico e antitubercular de compostos apolares das espécies de Qualea.

The chloroform extract of bark of the tropical tree Qualea parviflora (Vochysiaceae) was fractionated by column chromatography on silica gel, yielding triterpenes (lupeol, lupenone, betulin, epibetulinic acid and friedelin) and a steroid (β-sitosterol). β-sitosterol, lupenone and lupeol were also identified in Q. grandiflora and Q. multiflora, while friedelin was detected only in Q. Multiflora, by means of gas chromatography-mass spectrometry. The anti-Mycobacterium tuberculosis activity of the chloroform extract and isolated compounds was assayed by MABA and MIC values ranged from 250.0 to 31.2 μg/mL. This study is the first to investigate the chemistry and antitubercular activity of apolar compounds from Qualeaspecies.

A photoelectrocatalytic process that disinfects water contaminated with Mycobacterium kansasii and Mycobacterium avium.

Nontuberculous mycobacteria are resistant to conventional water treatment; indeed, they have been recovered from a wide variety of environmental sources. Here, we applied the photoelectrocatalytic technique using a Ti/TiO2-Ag photoanode to inactivate mycobacteria. For a mycobacteria population of 5 × 10(8) CFU mL(-1), we achieved 99.9 and 99.8% inactivation of Mycobacterium kansasii and Mycobacterium avium with rate constant of 6.2 × 10(-3) and 4.2 × 10(-3) min(-1), respectively, after 240 min. We compared the proposed method with the photolytic and photocatalytic methods. Using a mycobacteria population of 7.5 × 10(4) CFU mL(-1), the proposed Ti/TiO2-Ag photoanode elicited total mycobacteria inactivation within 3 min of treatment; the presence of Ag nanoparticles in the electrode provided 1.5 larger degradation rate constant as compared with the Ti/TiO2 anode (1.75 × 10(-2) for M. kansassi and 1.98 × 10(-2) for M. avium). We monitored the degradation of the metabolites released during cellular lysis by TOC removal, sugar release, chromatography, and mass spectrometry measurements; photoelectrocatalysis and Ti/TiO2-Ag photoanodes furnished the best results.

Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs.

We assessed the performance of REMA in comparison with BACTEC MGIT 960 in the susceptibility testing of 80 Mycobacterium tuberculosis clinical isolates from Clemente Ferreira Institute against four drugs. REMA proved to be a rapid and accurate method, providing excellent correlation with BACTEC MGIT 960, with the exception of results for the ethambutol drug.

Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil.

BACKGROUND: Mycobacterium spp. is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk and related dairy products. To address this concern, the Brazilian control and eradication program focusing on bovine tuberculosis, was established in 2001. However, bovine tuberculosis continues to afflict approximately 1,3 percent of the cattle in Brazil. In the present study, 300 samples of milk from bovine herds, obtained from both individual and collective bulk tanks and informal points of sale, were cultured on Löwenstein-Jensen and Stonebrink media. Polymerase chain reaction (PCR)-based tests and restriction-enzyme pattern analysis were then performed on the colonies exhibiting phenotypes suggestive of Mycobacterium spp., which were characterized as acid-fast bacilli. RESULTS: Of the 300 bovine milk samples that were processed, 24 were positively identified as Mycobacterium spp.Molecular identification detected 15 unique mycobacterial species: Mycobacterium bovis, M. gordonae, M. fortuitum, M. intracellulare, M. flavescens, M. duvalii, M. haemophilum, M. immunogenum, M. lentiflavum, M. mucogenicum, M. novocastrense, M. parafortuitum, M. smegmatis, M. terrae and M. vaccae. The isolation of bacteria from the various locations occurred in the following proportions: 9 percent of the individual bulk-tank samples, 7 percent of the collective bulk-tank samples and 8 percent of the informal-trade samples. No statistically significant difference was observed between the presence of Mycobacterium spp. in the three types of samples collected, the milk production profiles, the presence of veterinary assistance and the reported concerns about bovine tuberculosis prevention in the herds. CONCLUSION: The microbiological cultures associated with PCR-based identification tests are possible tools for the investigation of the presence of Mycobacterium spp. in milk samples. Using these methods, we found that the Brazilian population may be regularly exposed to mycobacteria by consuming raw bovine milk and related dairy products. These evidences reinforces the need to optimize quality programs of dairy products, to intensify the sanitary inspection of these products and the necessity of further studies on the presence of Mycobacterium spp. in milk and milk-based products.

Isolation and identification of environmental mycobacteria in the waters of a hemodialysis center.

The use of poorly treated water during hemodialysis may lead to contamination with nontuberculous mycobacteria (NTM). This study aimed to isolate and identify NTM species in the water of a Brazilian hemodialysis center. We collected 210 samples of water from the hydric system of the unit (post-osmosis system, hemodialysis rooms, reuse system, and hemodialysis equipment) and from the municipal supply network; we isolated the NTM by a classic microbiological technique and identified them by the PCR restriction enzyme pattern of the hsp65 gene (PRA). Fifty-one (24.3 %) of the collected samples tested positive for NTM; both the municipal supply network (2 samples, 3.2 %) and the hydric system of the hemodialysis center (49 samples, 96.1 %) contained NTM. We isolated and identified potentially pathogenic bacteria such as Mycobacterium lentiflavum (59.0 %) and M. kansasii (5.0 %), as well as rarely pathogenic bacteria like M. gordonae (24.0 %), M. gastri (8.0 %), and M. szulgai (4.0 %). The ability of NTM to cause diseases is well documented in the literature. Therefore, the identification of NTM in the water of a Brazilian hemodialysis center calls for more effective water disinfection procedures in this unit.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting.

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Comparison of three molecular typing methods to assess genetic diversity for Mycobacterium tuberculosis.

This study describes the comparison of three methods for genotyping of Mycobacterium tuberculosis, namely MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats), spoligotyping and, for the first time, MLST (Multilocus Sequence Typing). In order to evaluate the discriminatory power of these methods, a total of 44 M. tuberculosis isolates obtained from sputum specimens of patients from Brazil were genotyped. Among the three methods, MLST showed the lowest discriminatory power compared to the other two techniques. MIRU-VNTR showed better discriminatory power when compared to spoligotyping, however, the combination of both methods provides the greatest level of discrimination and therefore this combination is the most useful genotyping tool to be applied to M. tuberculosis isolates.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Disseminated Mycobacterium tuberculosis infection in a dog.

An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs.

Mycobacterium tuberculosis population structure shift in a 5-year molecular epidemiology surveillance follow-up study in a low endemic agro-industrial setting in São Paulo, Brazil.

Starting with 257 outpatients attending the specialized health service for tuberculosis (TB) between 2002 and 2006 in Araraquara, an agro-industrial area with low tuberculosis (TB) incidence in São Paulo state, Brazil, positive mycobacterial cultures were obtained in 130 cases, of which 121 were confirmed as Mycobacterium tuberculosis complex. This report assesses the genetic diversity observed on 69.42% (n=84) of the clinical isolates, for which both spoligotyping and 12-loci MIRU typing data were fully interpretable. In order to monitor changes in the population dynamics of circulating M. tuberculosis strains over time, spoligotypes were compared from this study (n=84) with an earlier study from 1998 to 2001 (n=70 strains); and these two datasets from low-incidence Araraquara area were also compared with a 2-year cohort in the nearby higher-incidence São Paulo city area from 2006 to 2008 (n=93). The results obtained showed that with 58.3% (49/84) of the strains, the Latin-American-Mediterranean (LAM) was the predominant lineage in the present follow-up study; major patterns being SIT42/LAM9 11.9% (10/84), and SIT20/LAM1 10.7% (9/84). As compared with the 1998-2001 period when 40% (28/70) of the isolates belonged to the ill-defined T family, it was replaced by LAM strains between 2002 and 2006 with a visible shift to a population structure characteristic of the metropolitan São Paulo city. Further typing of the follow-up isolates from 2002 to 2006 using 12 loci MIRUs in conjunction with conventional epidemiology did not link this population structure shift to an increase in ongoing transmission or drug-resistance. Instead, it is most probably linked to movements of the important migrant community of Araraquara to higher TB incidence metropolitan areas such as São Paulo city. This is of particular concern owing to the increment in the global burden of LAM strains and the recent association of certain LAM sublineages with multidrug- and extensively drug-resistant TB. These observations suggest the need for further molecular monitoring of the TB population structure and the evaluation of transmission trends amongst migrant workers and other risk groups, such as persons in homeless shelters, in correctional facilities, drug users, and those with HIV infection, etc.

Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs

We assessed the performance of REMA in comparison with BACTEC MGIT 960 in the susceptibility testing of 80 Mycobacterium tuberculosis clinical isolates from Clemente Ferreira Institute against four drugs. REMA proved to be a rapid and accurate method, providing excellent correlation with BACTEC MGIT 960, with the exception of results for the ethambutol drug.