New antibacterial agents: Hybrid bioisoster derivatives as potential E. coli FabH inhibitors.

The development of resistance to antibiotics by microorganisms is a major problem for the treatment of bacterial infections worldwide, and therefore, it is imperative to study new scaffolds that are potentially useful in the development of new antibiotics. In this regard, we propose the design, synthesis and biological evaluation of hybrid sulfonylhydrazone bioisosters/furoxans with potential antibacterial (Escherichia coli) activity. The most active compound of the series, (E)-3-methyl-4-((2-tosylhydrazono)methyl)-1,2,5-oxadiazole 2-oxide, with a MIC=0.36µM, was not cytotoxic when tested on Vero cells (IC50>100µM). To complement the in vitro screening, we also studied the interaction of the test compounds with ß-ketoacyl acyl carrier protein synthase (FabH), the target for the parent compounds, and we observed three important hydrogen-bonding interactions with two important active site residues in the catalytic site of the enzyme, providing complementary evidence to support the target of the new hybrid molecules.

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.

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 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.

Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping.

We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB), for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping.

A promising technology based on photoelectrocatalysis against Mycobacterium tuberculosis in water disinfection.

Mycobacterium tuberculosis is highly infectious, persistent and has been detected in more than one quarter of the world's population. It is notoriously resistant to sterilization and disinfection procedures, largely due to an unusual hydrophobic cell wall and effective defense mechanisms against oxidative stress. This work shows an effective method to reduce M. tuberculosis quantity in water by using Ti/TiO2 nanotubes electrodes bare and coated with Ag nanoparticles by using photoelectrocatalytic oxidation process. The results have indicated 99.999% of inactivation of a solution spiked with standard and resistant strains of 1×104 CFU mL-1 M. tuberculosis after 5 min of treatment at Ti/TiO2 photoanode in 0.05 mol L-1 Na2SO4 (pH 6) under applied potential of + 1.5 V versus Ag/AgCl and UV irradiation. The mycobacteria degradation was monitored by dissolved total organic carbon (TOC) removal, carbohydrate release, chromatography coupled to mass spectroscopy measurements and it is slightly superior to photocatalysis and photolysis processes. All the results corroborated with the complete inactivation and degradation of the byproducts generated during cell lysis.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis.

Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil. The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.

Genotyping and rifampicin and isoniazid resistance in Mycobacterium bovis strains isolated from the lymph nodes of slaughtered cattle.

In developing nations, 10-20% of the human cases of tuberculosis are caused by Mycobacterium bovis. However, this percentage may be underestimated because most laboratories in developing countries do not routinely perform mycobacterial cultures, and only a few have the systems in place to identify M. bovis. There are few studies investigating genotypic diversity and drug resistance in M. bovis from animal and/or human infections. The genotypic diversity of M. bovis strains obtained from bovine lymph nodes were investigated by spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing (MIRU-VNTR). The phenotypic resistance to isoniazid and rifampicin and MIC values of the isolates were determined using the resazurin microtiter assay plate method (REMA). The evaluation of the possible genetic basis for such resistance was performed with GenoType MTBDRplus. Sixty-seven isolates were obtained, of which 11 (16%) were MDR-TB, 8 (12%) were isoniazid-resistant, and 2 (3%) were rifampicin-resistant. Mutations associated with drug resistance were not found. Genotyping techniques enabled the grouping of the strains into 12 clusters and 21 isolates with unique profiles. The high frequency of M. bovis reinforces the impact of the pathogen as a major causal agent of bovine tuberculosis in the study area. The resistance of the strains to drugs used for first-line treatment of human tuberculosis raises public health concerns. Further studies are required to elucidate the basis of drug resistance and genotypic diversity in M. bovis.

Antimycobacterial activity of pyrazinoate prodrugs in replicating and non-replicating Mycobacterium tuberculosis.

Tuberculosis (TB) is an important infectious disease caused by Mycobacterium tuberculosis (Mtb) and responsible for thousands of deaths every year. Although there are antimycobacterial drugs available in therapeutics, just few new chemical entities have reached clinical trials, and in fact, since introduction of rifampin only two important drugs had reached the market. Pyrazinoic acid (POA), the active agent of pyrazinamide, has been explored through prodrug approach to achieve novel molecules with anti-Mtb activity, however, there is no activity evaluation of these molecules against non-replicating Mtb until the present. Additionally, pharmacokinetic must be preliminary evaluated to avoid future problems during clinical trials. In this paper, we have presented six POA esters as prodrugs in order to evaluate their anti-Mtb activity in replicating and non-replicating Mtb, and these showed activity highly influenced by medium composition (especially by albumin). Lipophilicity seems to play the main role in the activity, possibly due to controlling membrane passage. Novel duplicated prodrugs of POA were also described, presenting interesting activity. Cytotoxicity of these prodrugs set was also evaluated, and these showed no important cytotoxic profile.

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.

Drug resistance in Mycobacterium tuberculosis clinical isolates from Brazil: phenotypic and genotypic methods.

We determined the susceptibility profile of 80 Mycobacterium tuberculosis (MTB) clinical isolates from Brazil against isoniazid (INH) and rifampicin (RIF) drugs by two phenotypic methods (Resazurin Microtiter Assay - REMA and BACTEC™ MGIT™ Mycobacterial Detection System). DNA polymorphisms were also determined by PCR-SSCP in isolates resistant to INH and RIF. BACTEC™ MGIT™ 960 detected 22 susceptible isolates to INH and RIF, 48 MDR isolates (resistant at least to INH and RIF) and nine mono-resistant isolates (eight to INH and one to RIF). REMA performance was determined by Receiver Operating Characteristic curve, whose assay was validated utilizing as reference the BACTEC™ MGIT™ 960 system. ROC curve showed cut-off values of 0.0625µg/mL and 0.125µg/mL, for INH and RIF, respectively. REMA-INH demonstrated sensitivity and specificity of 100% while REMA-RIF showed sensitivity of 97.2% and specificity of 100%. PCR-SSCP detected DNA polymorphisms in 87.5% and 75.5% of isolates classified as INH-resistant and RIF-resistant, respectively. One discordant sample found to RIF (resistant by BACTEC™ MGIT™ 960 and susceptible by REMA) showed no mutation by PCR-SSCP. In conclusion, our studies demonstrated that the combination of phenotypic method REMA, which allowed rapid detection of MDR-MTB with higher levels of sensitivity and specificity, with the genotypic method PCR-SSCP, which demonstrated high accuracy in the search of polymorphisms in the resistance genes, proved to be a useful strategy to study MDR-MTB clinical isolates from national reference center located in São Paulo city.

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.

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.

Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping

We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB), for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis

Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil. The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.

Quantitative trait loci (QTL) mapping for growth traits on bovine chromosome 14

Quantitative trait loci (QTL) mapping in livestock allows the identification of genes that determine the genetic variation affecting traits of economic interest. We analyzed the birth weight and weight at 60 days QTL segregating on bovine chromosome BTA14 in a F2 resource population using genotypes produced from seven microsatellite markers. Phenotypes were derived from 346 F2 progeny produced from crossing Bos indicus Gyr x Holstein Bos taurus F1 parents. Interval analysis to detect QTL for birth weight revealed the presence of a QTL (p < 0.05) at 1 centimorgan (cM) from the centromere with an additive effect of 1.210 ± 0.438 kg. Interval analysis for weight at 60 days revealed the presence of a QTL (p < 0.05) at 0 cM from the centromere with an additive effect of 2.122 ± 0.735 kg. The region to which the QTL were assigned is described in the literature as responsible for some growth traits, milk yield, milk composition, fat deposition and has also been related to reproductive traits such as daughter pregnancy rate and ovulation rate. The effects of the QTL described on other traits were not investigated.

Quantitative trait locus affecting birth weight on bovine chromosome 5 in a F2 Gyr x Holstein population

Segregation between a genetic marker and a locus influencing a quantitative trait in a well delineated population is the basis for success in mapping quantitative trait loci (QTL). To detect bovine chromosome 5 (BTA5) birth weight QTL we genotyped 294 F2 Gyr (Bos indicus) x Holstein (Bos taurus) crossbreed cattle for five microsatellite markers. A linkage map was constructed for the markers and an interval analysis for the presence of QTL was performed. The linkage map indicated differences in the order of two markers relative to the reference map (http://www.marc.usda.gov). Interval analysis detected a QTL controlling birth weight (p < 0.01) at 69 centimorgans (cM) from the most centromeric marker with an effect of 0.32 phenotypic standard-error. These results support other studies with crossbred Bos taurus x Bos indicus populations