Gene regulatory network inference and analysis of multidrug-resistant Pseudomonas aeruginosa

BACKGROUND Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.

Evaluation of multidrug resistance-1 gene C>T polymorphism frequency in patients with asthma

OBJECTIVES:Asthma is a chronic inflammatory lung disease characterized by bronchial hyperresponsiveness and airflow obstruction. Genetic and oxidative stress factors, in addition to pulmonary and systemic inflammatory processes, play a pivotal role in the pathogenesis of asthma. The products of the multidrug resistance-1 gene protect lung tissue from oxidative stress. Here, we aimed to evaluate the association between the multidrug resistance-1 gene C>T polymorphism and asthma with regard to oxidative stress-related parameters of asthmatic patients.METHODS:Forty-five patients with asthma and 27 healthy age-matched controls were included in this study. Blood samples were collected in tubes with ethylenediaminetetraacetic acid. DNA was extracted from the blood samples. The multidrug resistance-1 gene polymorphism was detected by polymerase chain reaction and a subsequent enzyme digestion technique. The serum levels of total oxidant status and total antioxidant status were determined by the colorimetric measurement method.RESULTS:The heterozygous polymorphic genotype was the most frequent in both groups. A significant difference in the multidrug resistance-1 genotype frequencies between groups indicated an association of asthma with the TT genotype. A significant difference between groups was found for wild type homozygous participants and carriers of polymorphic allele participants. The frequency of the T allele was significantly higher in asthmatic patients. The increase in the oxidative stress index parameter was significant in the asthma group compared with the control group.CONCLUSIONS:The multidrug resistance-1 gene C/T polymorphism may be an underlying genetic risk factor for the development of asthma via oxidant-antioxidant imbalance, leading to increased oxidative stress.

Mutaciones asociadas a resistencia a rifampicina e isoniacida en aislamientos clínicos del complejo Mycobacterium tuberculosis de pacientes del Hospital Roosevelt / Mutations associated with resistance to rifampin and isoniazid in clinical isolates of the Mycobacterium tuberculosis complex from patients at Roosevelt Hospital

El aumento de tuberculosis y la multidrogo resistencia de cepas de micobacterias es un problema de los sistemas de salud, en 2009, en el Hospital Roosevelt Gordillo y cols, determinaron la TB-MDR en pacientes con tuberculosis diagnosticada microbiológicamente, la tasa de resistencia fue de 4.3%. Objetivo: Determinar los patrones de resistencia y perfiles genéticos de cepas con monoresistencia y cepas TB-MDR del Complejo M. tuberculosis. Métodos: Se utilizaron dos métodos para evaluar las cepas de M. tuberculosis, un método fenotípico, MGIT, y un método Genotípico, Genotype HAIN LifeScience para determinar el perfil genético de las cepas. Resultados: Se evaluaron 846 cepas de micobacterias de los años 2008 al 2013, encontrándose un 2.2% de TB-MDR. Las cepas evaluadas genotípicamente fueron 761, a las cuales se determinó los genes de resistencia, encontrándose monoresistencia a Isoniacida en 58 cepas, 7.6%, monoresistencia a Rifampicina en 18 cepas, 2.4% y 15 cepas MDR, 2.0%. Las mutaciones más frecuentes en monoresistencia fueron inhA MUT1 y katG MUT1 y la combinación de ambos genes 3.2%, 3.0% y 1.3%, para cepas TB-MDR la combinación rpoB Mutación silenciosa + katG MUT1 + inhA MUT1. Se encontró que en pacientes con cepas MDR el 3.1% son HIV+ y el 1.5% son HIV-...(AU)

Introduction: The increase of tuberculosis and multidrug resistance in mycobacteria strains is a problem for health systems, in 2009, in Hospital Roosevelt, Gordillo and cols, determined the TB-MDR in patients diagnosed with tuberculosis microbiologically, the resistance rate was 4.3%. Objective: To determine the resistance patterns and genetic profiles of monoresistant strains and MDR-TB strains of M. tuberculosis complex. Methods: Two methods for evaluating M. tuberculosis strains were used, a phenotypic method, MGIT, and a genotypic method, Genotype HAIN LifeScience to determine the genetic profile of the strains. Results: 846 strains of mycobacteria of the years 2008 to 2013 were evaluated, finding 2.2% of MDR-TB. The strains genotypically evaluated were 761, of wich, resistance genes were determined, finding isoniazid monoresistance in 58 strains, 7.6%, Rifampicin monoresistance in 18 strains, 2.4% and 15 MDR strains, 2.0%. The most frequent mutations for monoresistant strains were inhA MUT1 and katG MUT1 and the combination of both genes 3.2%, 3.0% and 1.3%, respectively, and the most frequent mutations for TB-MDR strains was the combination rpoB silent mutation + katG MUT1 + inhA MUT1. There was found that in patients with MDR strains 3.1% are HIV+ and 1.5% are HIV-. Conclusions: The percentage of TB-MDR strains was 2.3%, and the most common genes were rpoB silent mutation, inhA MUT1 y katG MUT1. There was found a higher percentage of monoresistance in isoniazid than rifampicin, being the HIV+ patient population the one that presented higher percentages in both monoresistance to RIF and INH and TB-MDR strains.

Multidrug resistance 1 gene polymorphisms may determine Crohn's disease behavior in patients from Rio de Janeiro

OBJECTIVES: Conflicting data from studies on the potential role of multidrug resistance 1 gene polymorphisms in inflammatory bowel disease may result from the analysis of genetically and geographically distinct populations. Here, we investigated whether multidrug resistance 1 gene polymorphisms are associated with inflammatory bowel diseases in patients from Rio de Janeiro. METHODS: We analyzed 123 Crohn's disease patients and 83 ulcerative colitis patients to determine the presence of the multidrug resistance 1 gene polymorphisms C1236T, G2677T and C3435T. In particular, the genotype frequencies of Crohn's disease and ulcerative colitis patients were analyzed. Genotype-phenotype associations with major clinical characteristics were established, and estimated risks were calculated for the mutations. RESULTS: No significant difference was observed in the genotype frequencies of the multidrug resistance 1 G2677T/A and C3435T polymorphisms between Crohn's disease and ulcerative colitis patients. In contrast, the C1236T polymorphism was significantly more common in Crohn's disease than in ulcerative colitis (p = 0.047). A significant association was also found between the multidrug resistance 1 C3435T polymorphism and the stricturing form of Crohn's disease (OR: 4.13; p = 0.009), whereas no association was found with penetrating behavior (OR: 0.33; p = 0.094). In Crohn's disease, a positive association was also found between the C3435T polymorphism and corticosteroid resistance/refractoriness (OR: 4.14; p = 0.010). However, no significant association was found between multidrug resistance 1 gene polymorphisms and UC subphenotypic categories. CONCLUSION: The multidrug resistance 1 gene polymorphism C3435T is associated with the stricturing phenotype and an inappropriate response to therapy in Crohn's disease. This association with Crohn's disease may support additional pathogenic roles ...

Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil / Genes de multirresistência, incluindo blaKPC e blaCTX-M-2, em Klebsiella pneumoniae isoladas em Recife, Brasil

INTRODUCTION: The prevalence of cephalosporins and carbapenem-resistant Klebsiella pneumoniae strains is rising in Brazil, with potential serious consequences in terms of patients' outcomes and general care. METHODS: This study characterized 24 clinical isolates of K. pneumoniae from two hospitals in Recife, Brazil, through the antimicrobial susceptibility profile, analyses of β-lactamase genes (blaTEM, blaSHV,blaCTX-MblaKPC, blaVIM, blaIMP, and blaSPM), plasmidial profile and ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTS: ERIC-PCR and plasmidial analysis grouped the isolates in 17 and 19 patterns, respectively. Six isolates from one hospital presented the same pattern by ERIC-PCR, indicating clonal dissemination. All isolates presented blaSHV, 62.5% presented blaCTX-M-2, 29% blaTEM, and 41.7% blaKPC. Metallo-β-lactamase genes blaand blawere not detected. Eleven isolates were identified carrying at least 3 β-lactamase studied genes, and 2 isolates carried blaSHVblaTEM, blaCTX-M-2 and blaKPC simultaneously. CONCLUSIONS: The accumulation of resistance genes in some strains, observed in this study, imposes limitations in the therapeutic options available for the treatment of infections caused by K. pneumoniae in Recife, Brazil. These results should alert the Brazilian medical authorities to establish rigorous methods for more efficiently control the dissemination of antimicrobial resistance genes in the hospital environment.

INTRODUÇÃO: A prevalência de cepas de Klebsiella pneumoniae resistentes a cefalosporinas e carbapenêmicos está aumentando no Brasil, com sérias consequências em termos de desfechos dos pacientes e cuidados gerais. MÉTODOS: Este estudo caracterizou 24 isolados clínicos de K. pneumoniae provenientes de dois hospitais de Recife, Brasil, através do perfil de susceptibilidade a antimicrobianos, análise de genes de β-lactamase (blaTEM,blaSHV,blaCTX-MblaKPC,blaVIM, blaIMP,and blaSPM), perfil plasmidial e ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTADOS: A análise da ERIC-PCR e do perfil plasmidial agrupou os isolados em 17 e 19 perfis, respectivamente. Seis isolados de um hospital apresentaram o mesmo padrão de ERIC-PCR, indicando disseminação clonal. Todos os isolados apresentaram blaSHV, 62,5% apresentaram blaCTX-M-2, 29% blaTEM e 41,7% blaKPC. Genes de metalo-β-lactamase blaVIM, blaIMP e blaSPM não foram detectados. Onze isolados foram identificados carreando, pelo menos, três dos genes de β-lactamase estudados, dentre estes, dois isolados continham blaSHV,blaTEM, blaCTX-M-2 e blaKPC simultaneamente. CONCLUSÕES: O acúmulo de genes de resistência em algumas cepas, observado nesse estudo, impõem limitações nas opções terapêuticas disponíveis para o tratamento de infecções causadas por K. pneumoniae em Recife, Brasil. Estes resultados devem alertar as autoridades médicas brasileiras para estabelecer rigorosos métodos para controlar eficientemente a disseminação de genes de resistência a antimicrobianos no ambiente hospitalar.

QRDR mutations, efflux system & antimicrobial resistance genes in enterotoxigenic Escherichia coli isolated from an outbreak of diarrhoea in Ahmedabad, India.

Background & objectives: Diverse mechanisms have been identified in enteric bacteria for their adaptation and survival against multiple classes of antimicrobial agents. Resistance of bacteria to the most effective fluoroquinolones have increasingly been reported in many countries. We have identified that most of the enterotoxigenic Escherichia coli (ETEC) were resistant to several antimicrobials in a diarrhoea outbreak at Ahmedabad during 2000. The present study was done to identify several genes responsible for antimicrobial resistance and mobile genetic elements in the ETEC strains. Methods: Seventeen ETEC strains isolated from diarrhoeal patients were included in this study. The antimicrobial resistance was confirmed by conventional disc diffusion method. PCR and DNA sequencing were performed for the identification of mutation in the quinolone resistance-determining regions (QRDRs). Efflux pump was tested by inhibiting the proton-motive force. DNA hybridization assay was made for the detection of integrase genes and the resistance gene cassettes were identified by direct sequencing of the PCR amplicons. Results: Majority of the ETEC had GyrA mutations at codons 83 and 87 and in ParC at codon 80. Six strains had an additional mutation in ParC at codon 108 and two had at position 84. Plasmid-borne qnr gene alleles that encode quinolone resistance were not detected but the newly described aac(6’)-Ib-cr gene encoding a fluoroquinolne-modifying enzyme was detected in 64.7 per cent of the ETEC. Class 1 (intI1) and class 2 (intI2) integrons were detected in six (35.3%) and three (17.6%) strains, respectively. Four strains (23.5%) had both the classes of integrons. Sequence analysis revealed presence of dfrA17, aadA1, aadA5 in class 1, and dfrA1, sat1, aadA1 in class 2 integrons. In addition, the other resistance genes such as tet gene alleles (94.1%), catAI (70.6%), strA (58.8%), blaTEM-1(35.2%), and aphA1-Ia (29.4%) were detected in most of the strains. Interpretation & conclusions: Innate gene mutations and acquisition of multidrug resistance genes through mobile genetic elements might have contributed to the emergence of multidrug resistance (MDR) in ETEC. This study reinforces the necessity of utilizing molecular techniques in the epidemiological studies to understand the nature of resistance responsible for antimicrobial resistance in different species of pathogenic bacteria.

Frequency of the mdr-1 C>T gene polymorphism in patients with COPD

BACKGROUND AND AIM: The multi-drug resistant-1 (MDR-1) gene is located on human chromosome 7 and encodes a glycosylated membrane protein that is a member of the ATP-binding cassette transporters superfamily. The aim of the study was to reveal the role of the C3435T MDR-1 gene polymorphism in chronic obstructive pulmonary disease. METHOD: DNA samples from 41 patients with chronic obstructive pulmonary disease and 50 healthy control participants were used to compare MDR-1 gene profiles. Genotyping assays were performed using the StripAssay technique that is based on reverse-hybridization. RESULTS: The T allele polymorphism in the MDR-1 gene located at position 3435 in exon 26 was shown to correlate with chronic obstructive pulmonary disease. CONCLUSION: These preliminary results suggest that the T allele polymorphism of the MDR-1 gene is associated with chronic obstructive pulmonary disease.

Impact of gene polymorphism on therapeutic drug monitoring of phenytoin and outcome in epileptic patients

This work was performed to explore the effect of polymorphism in multidrug resistant gene [MDR1] on plasma phenytoin levels and patients' outcome to evaluate its involvement in drug resistance and toxicity that is usually associated with antiepileptic drugs. Therefore, we genotyped the MDR1 in 100 patients, suffering from partial or generalized tonic-clonic seizures and receiving phenytoin, and 50 healthy control subjects. Steady state plasma phenytoin levels were also determined in the epileptic patients. Patients were evaluated after three and six months and were classified either as drug resistant patients or responsive patients. Results revealed 37 patients with drug-responsive epilepsy and 63 patients with drug-resistant epilepsy. Genotyping of our patients and control subjects revealed a genotype distribution of [CC, CT, TT: 55.50%, 38.00%, 6.50%] for drug resistant patients, [CC, CT, TT: 13.50%, 46.00%, 40.50%] for drug responsive patients, and [CC, CT, TT: 24.00%, 48.00%, 28.00%] for the control subjects. Patients with drug-resistant epilepsy were more likely to have the CC than the TT genotype compared with either responsive patients [P< 0.0001] or control [P <0.0001]. The C polymorphism was overrepresented among patients with drug-resistant epilepsy as compared with either those with drug-responsive epilepsy [P <0.001] or control [P <0.001]. Out of the total 100 epileptic patients; 13 patients had their plasma phenytoin levels exceeding the maximum safe concentration. These 13 patients were more likely to have TT genotype than the CC genotype compared with the remainder patients who had their plasma phenytoin levels 0.05] or allele frequency [P>0.05]. In Since most of the antiepileptic drugs are MDR1 substrates, thus the MDR1 is an important candidate gene potentially influencing the response to antiepileptic drugs. Our findings suggest that using genotype data may make it possible to safely reduce the time required to reach an effective dose. Therefore, it is a priority to assess the utility of dose adjustment on the basis of genotype for these medicines that are substrates for this gene

High baseline serum total and LDL cholesterol levels are associated with MDR1 haplotypes in Brazilian hypercholesterolemic individuals of European descent

The MDR1 gene encodes the P-glycoprotein, an efflux transporter with broad substrate specificity. P-glycoprotein has raised great interest in pharmacogenetics because it transports a variety of structurally divergent drugs, including lipid-lowering drugs. The synonymous single-nucleotide polymorphism C3435T and the nonsynonymous single-nucleotide polymorphism G2677T/A in MDR1 have been indicated as potential determinants of variability in drug disposition and efficacy. In order to evaluate the effect of G2677T/A and C3435T MDR1 polymorphisms on serum levels of lipids before and after atorvastatin administration, 69 unrelated hypercholesterolemic individuals from São Paulo city, Brazil, were selected and treated with 10 mg atorvastatin orally once daily for four weeks. MDR1 polymorphisms were analyzed by PCR-RFLP. C3435T and G2677T polymorphisms were found to be linked. The allelic frequencies for C3435T polymorphism were 0.536 and 0.464 for the 3435C and 3435T alleles, respectively, while for G2677T/A polymorphism allele frequencies were 0.580 for the 2677G allele, 0.384 for the 2677T allele and 0.036 for the 2677A allele. There was no significant relation between atorvastatin response and MDR1 polymorphisms (repeated measures ANOVA; P > 0.05). However, haplotype analysis revealed an association between T/T carriers and higher basal serum total (TC) and LDL cholesterol levels (TC: 303 ± 56, LDL-C: 216 ± 57 mg/dl, respectively) compared with non-T/T carriers (TC: 278 ± 28, LDL-C: 189 ± 24 mg/dl; repeated measures ANOVA/Tukey test; P < 0.05). These data indicate that MDR1 polymorphism may have an important contribution to the control of basal serum cholesterol levels in Brazilian hypercholesterolemic individuals of European descent.

Polimorfismos del gen de resistencia a múltiples drogas (MDR1) en poblaciones chilenas: mapuche, mestiza y maorí / Polymorphisms of the multiple drug resistance gene (MDR1) in Mapuche, Mestizo and Maori populations in Chile

BACKGROUND: There are significant differences in drug responses among different ethnic groups. The multidrug transporter P-gp, encoded by the MDR1 gene, plays a key role in determining drug bioavailability, and an association between a polymorphism in exon 26 (C3435T) and lower P-gp expression has been found. The co-segregation of this polymorphism with the polymorphism in exon 12 (C1236T) and in exon 21 (G2677T/A) determines several MDR1 haplotypes in humans. AIM: To characterize the polymorphisms of exons 26, 21 and 12 of the MDR1 gene in different Chilean populations. MATERIAL AND METHODS: Using a polymerase chain reaction and restriction fragment length polymorphism technique, we studied the allelic frequencies and the distribution of MDR1 haplotypes in 3 Chilean populations: Mestizo (n=104), Mapuche (n=96, living in the National Reservation of the Huapi Island, Ranico Lake) and Maori (n=52, living in Eastern Island). RESULTS: The frequency of the normal MDR1*1 haplotype, without mutations, was lower in Mapuches than in Mestizos or Maoris (p<0.005) but similar to that reported in Asian population (p=0.739), probably due to the Asian origin of the Amerindian populations. In addition, the MDR1*l haplotype fequency hin Mestizos was similar to the frequency reported in Caucasians (p=0.49), in agreement with the origin of our population, with a strong influence of Caucasian genes from the Spanish conquerors. The MDR1*2 haplotype distribution, with the three polymoyphisms and probably lower multidrug transporter expression, was similar in the three Chilean populations studied (p>0.0.5), but lower than the frequencies reported in Caucasians or Asians (p<0.05). CONCLUSIONS: We found significant differences in the frequencies of genetic polymorphisms of the MDR1 gene in Chilean populations, related to the ethnic origins of our ancestors.

Multiple drug resistance protein (MDR-1), multidrug resistance-related protein (MRP) and lung resistance protein (LRP) gene expression in childhood acute lymphoblastic leukemia

CONTEXTO: Apesar dos avanços nos índices de cura da leucemia linfoblástica aguda (LLA) aproximadamente 25% das crianças sofrem recaídas da doença. A expressão dos genes de resistência múltipla a drogas (MDR-1), genes relacionados à proteína de resistência múltipla a drogas (MRP) e genes da proteína de resistência pulmonar (LRP) podem conferir o fenótipo de resistência ao tratamento das neoplasias. OBJETIVO: Analisar a expressão dos genes de resistência MDR-1, MRP e LRP em crianças diagnosticadas com LLA por meio da técnica da reação em cadeia da polimerase da transcriptase reversa (RT-PCR) semiquantitativa, associando estas expressões à sobrevida livre de eventos (SLE) e a variáveis clínico-laboratoriais. TIPO DE ESTUDO: Estudo clínico retrospectivo. LOCAL: Laboratório de Oncologia Pediátrica do Departamento de Puericultura e Pediatria da Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo, Brasil. MÉTODOS: Amostras de medula óssea de 30 crianças com o diagnóstico de leucemia linfoblástica aguda foram avaliadas quanto à expressão do RNA-mensageiro para os genes MDR-1, MRP e LRP, pela reação em cadeia da RT-PCR semiquantitativa. RESULTADOS: Dos três genes estudados, somente a expressão aumentada de LRP esteve relacionada a uma pior SLE (p = 0.005). A presença do antígeno para leucemia linfoblástica aguda comum (CALLA) se correlacionou à expressão aumentada de LRP (p = 0.009) e a risco aumentado de ocorrência de recaída ou óbito (p = 0.05). O risco relativo de ocorrência de recaída ou óbito é seis vezes maior em crianças com alta expressão de LRP ao diagnóstico (p = 0.05), o que se confirma na análise multivariada dos três genes estudados (p = 0.035). DISCUSSAO: A resistência celular a drogas é um determinante de resposta ao tratamento oncológico e sua avaliação por RT-PCR pode ser de importância. CONCLUSÕES: A avaliação da expressão dos genes de resistência a drogas antineoplásicas na leucemia linfoblástica aguda da criança ao diagnóstico, particularmente do gene LRP, pode ser de relevância clínica e deve ser objeto de estudos prospectivos.