Genetic heterogeneity of Escherichia coli isolated from pasteurized milk in State of Paraná, Brazil

A contaminação de alimentos por patógenos entéricos é uma das principais causas de doenças diarréicas em todo o mundo, resultando em altas taxas de morbidade e mortalidade e perdas econômicas significativas. As bactérias são importantes agentes de doenças de origem alimentar, particularmente Escherichia coli diarreiogênicas. O presente estudo teve como objetivo avaliar a diversidade genética e a resistência a antimicrobianos de E. coli isoladas de leite pasteurizado, processados em 21 laticínios na região noroeste do Paraná - Brasil. Os 95 isolados de E. coli foram submetidos a testes de suscetibilidade aos antimicrobianos de acordo com as recomendações do Clinical and Laboratory Standards Institute e avaliados genotipicamente por ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus - Polymerase Chain Reaction). O principal perfil de resistência encontrado entre os isolados foi resistência à cefalotina (55,78%). ERIC-PCR revelou alta diversidade genética, agrupando os 95 isolados bacterianos em 90 diferentes perfis genotípicos. Estes resultados mostraram uma população heterogênea de E. coli em amostras de leite produzido na região noroeste do Paraná e a necessidade de boas práticas na manipulação de todo o processamento de leite pasteurizado, a fim de reduzir o risco de doenças transmitidas por alimentos.Food contamination caused by enteric pathogens is a major cause of diarrheal disease worldwide, resulting in high morbidity and mortality and significant economic losses. Bacteria are important agents of foodborne diseases, particularly diarrheagenic Escherichia coli. The present study assessed the genetic diversity and antimicrobial resistance of E. coli isolates from pasteurized milk processed in 21 dairies in northwestern State of Parana, Brazil. The 95 E. coli isolates were subjected to antimicrobial susceptibility testing according to the recommendations of the Clinical and Laboratory Standards Institute and assessed genotypically by Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR). The highest rate of resistance was observed for cephalothin (55.78%). ERIC-PCR revealed high genetic diversity, clustering the 95 bacterial isolates into 90 different genotypic patterns. These results showed a heterogeneous population of E. coli in milk samples produced in the northwestern region of Paraná and the need for good manufacturing practices throughout the processing of pasteurized milk to reduce the risk of foodborne illnesses

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

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 (TM) MGIT (TM) Mycobacterial Detection System). DNA polymorphisms were also determined by PCR-SSCP in isolates resistant to INH and RIF. BACTEC (TM) MGIT (TM) 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 (TM) MGIT (TM) 960 system. ROC curve showed cut-off values of 0.0625 mu g/mL and 0.125 mu g/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 (TM) MGIT (TM) 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. (C) 2011 Elsevier Masson SAS. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Genes differentially expressed by Mycobacterium tuberculosis after exposure to ruthenium phosphinic compound and isoniazid

Background- The evaluation of the effects of new compounds and nonconventional anti-tuberculous drugs have grown and become increas-ingly more popular in recent years. Studies have shown anti-tuberculous activity for Ruthenium complexes, including organometallic com-pounds containing phosphine ligands such as picolinic acid generating great expectations and hopes. Methods- The Representational Difference Analysis (RDA) was applied in order to gain insight about differences in expression of Mycobacte-rium tuberculosis H37Rv exposed to [Ru(dppb)(pic)(bypy)] PF6 (SCAR1) and isoniazid (INH). Total RNA was extracted from the bacillus not exposed and exposed to SCAR1 and INH separately at concentration of MIC for 12 hours at 35°C. RDA was carried out and differentially expressed products were sequenced. Results- RDA-sequencing identified, for both compounds, orthologs that encode hypothetical and predict proteins. One related cell wall syn-thesis gene, identified by RDA, and genes related to INH target as inhA, katG and ahpC had their expression confirmed and quantified by real-time PCR. The gene encoding the cell wall associated hydrolase was induced 4.627 and 1.189, inhA 0.983 and 1.027, katG 1.111 and 1.345 and ahpC 1.063 and 1.039 fold after exposure to SCAR1 and INH respectively, compared to not exposed growth. Conclusion- The RDA brings, for the first time, directions to study related genes with metabolic pathways of SCAR1. RDA and Real-Time PCR highlight the idea that one of the SCAR1 interaction, in M tuberculosis may be in the cell wall biosynthesis considering the differential expression of a cell wall hydrolase and warrants further investigation