RESUMEN

Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses. KEY POINTS: • vB_SauM-UFV_DC4 is a new viral species belonging to a new genus within the class Caudoviricetes. • vB_SauM-UFV_DC4 carries a set of RNA polymerase subunits and an RNA-directed DNA polymerase. • vB_SauM-UFV_DC4 and closely related jumbo phages showed a similar set of lytic proteins.

Asunto(s)

Bacteriófagos , Fagos de Staphylococcus , Animales , Bovinos , Femenino , Humanos , Fagos de Staphylococcus/genética , Staphylococcus aureus/genética , Proteómica , Genoma Viral , Genómica , Bacteriófagos/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN de Transferencia

RESUMEN

Proteobacteria comprising species of Pseudomonas syringae group cause diseases of many plants around the world. The phytopathogen has a complex taxonomic structure, which is constantly being revised due to the emergence of new molecular and biochemical diagnostic methods. Here for the first time, we describe the genetic and phenotypic diversity of 57 strains of Pseudomonas syringae isolated from affected soybeans, cereals, sunflowers, and other plants in the Russian Federation from 1950 to 2019. Genetic diversity was assessed by Multi Locus Sequence Analysis (MLSA) using fragments of the genes of glyceraldehyde-3-phosphate dehydrogenase (gapdh), the DNA-directed RNA polymerase subunit D (rpoD), gyrase (topoisomerase) B subunit (gyrB), and citrate synthase I (gltA). The synthesis of syringomycin and coronatine by bacteria was assessed by the reaction of susceptible yeast culture, seedlings of barley, tomato, and sunflower, and by presence of toxin genes confirmed by PCR test. The pathogenicity of the strains was confirmed on seedlings of dicotyledonous and monocotyledonous plants of peas, soybean, sunflowers, barley and wheat, as the most affected crops. The sensitivity of bacteria to 10 antibiotics of the main mechanisms of activity and two bactericidal commercial products was tested by standard disc method. The obtained results showed a high genetic homogeneity of the Russian population of P. syringae, which infects various agricultural crops, and an increase in the proportion of antibiotic-resistant strains over the years.

Asunto(s)

Enfermedades de las Plantas , Pseudomonas syringae , Antibacterianos , Citrato (si)-Sintasa , ARN Polimerasas Dirigidas por ADN/genética , Pseudomonas syringae/genética , Glycine max , Federación de Rusia

RESUMEN

Promoter identification is a fundamental step in understanding bacterial gene regulation mechanisms. However, accurate and fast classification of bacterial promoters continues to be challenging. New methods based on deep convolutional networks have been applied to identify and classify bacterial promoters recognized by sigma (σ) factors and RNA polymerase subunits which increase affinity to specific DNA sequences to modulate transcription and respond to nutritional or environmental changes. This work presents a new multiclass promoter prediction model by using convolutional neural networks (CNNs), denoted as PromoterLCNN, which classifies Escherichia coli promoters into subclasses σ70, σ24, σ32, σ38, σ28, and σ54. We present a light, fast, and simple two-stage multiclass CNN architecture for promoter identification and classification. Training and testing were performed on a benchmark dataset, part of RegulonDB. Comparative performance of PromoterLCNN against other CNN-based classifiers using four parameters (Acc, Sn, Sp, MCC) resulted in similar or better performance than those that commonly use cascade architecture, reducing time by approximately 30-90% for training, prediction, and hyperparameter optimization without compromising classification quality.

Asunto(s)

ARN Polimerasas Dirigidas por ADN , Factor sigma , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Factor sigma/genética , Factor sigma/metabolismo

RESUMEN

In the past few years, our understanding of the RNA virosphere has changed dramatically due to the growth and spurt of metagenomics, exponentially increasing the number of RNA viral sequences, and providing a better understanding of their range of potential hosts. As of today, the only conserved protein among RNA viruses appears to be the monomeric RNA-dependent RNA polymerase. This enzyme belongs to the right-hand DNA-and RNA polymerases, which also includes reverse transcriptases and eukaryotic replicative DNA polymerases. The ubiquity of this protein in RNA viruses makes it a unique evolutionary marker and an appealing broad-spectrum antiviral target. In this work pairwise structural comparisons of viral RdRps and RTs were performed, including tertiary structures that have been obtained in the last few years. The resulting phylogenetic tree shows that the RdRps from (+)ss- and dsRNA viruses might have been recruited several times throughout the evolution of mobile genetic elements. RTs also display multiple evolutionary routes. We have identified a structural core comprising the entire palm, a large moiety of the fingers and the N-terminal helices of the thumb domain, comprising over 300 conserved residues, including two regions that we have named the "knuckles" and the "hypothenar eminence". The conservation of an helix bundle in the region preceding the polymerase domain confirms that (-)ss and dsRNA Reoviruses' polymerases share a recent ancestor. Finally, the inclusion of DNA polymerases into our structural analyses suggests that monomeric RNA-dependent polymerases might have diverged from B-family polymerases.

Asunto(s)

ARN Polimerasas Dirigidas por ADN , Evolución Molecular , Secuencia de Aminoácidos , ADN Polimerasa Dirigida por ADN , ARN Polimerasas Dirigidas por ADN/genética , Filogenia , ARN/genética

RESUMEN

Genomic-based surveillance on the occurrence of drug resistance and its transmission dynamics has emerged as a powerful tool for the control of tuberculosis (TB). A whole-genome sequencing approach, phenotypic testing and clinical-epidemiological investigation were used to undertake a retrospective population-based study on drug-resistant (DR)-TB in Rio Grande do Sul, the largest state in Southern Brazil. The analysis included 305 resistant Mycobacterium tuberculosis strains sampled statewide from 2011 to 2014, and covered 75.7% of all DR-TB cases identified in this period. Lineage 4 was found to be predominant (99.3%), with high sublineage-level diversity composed mainly of 4.3.4.2 [Latin American and Mediterranean (LAM)/RD174], 4.3.3 (LAM/RD115) and 4.1.2.1 (Haarlem/RD182) sublineages. Genomic diversity was also reflected in resistance of the variants to first-line drugs. A large number of distinct resistance-conferring mutations, including variants that have not been reported previously in any other setting worldwide, and 22 isoniazid-monoresistant strains with mutations described as disputed in the rpoB gene but causing rifampicin resistance generally missed by automated phenotypic tests as BACTEC MGIT. Using a cut-off of five single nucleotide polymorphisms, the estimated recent transmission rate was 55.1%, with 168 strains grouped into 28 genomic clusters. The most worrying fact concerns multi-drug-resistant (MDR) strains, of which 73.4% were clustered. Different resistance profiles and acquisition of novel mutations intraclusters revealed important amplification of resistance in the region. This study described the diversity of M. tuberculosis strains, the basis of drug resistance, and ongoing transmission dynamics across the largest state in Southern Brazil, stressing the urgent need for MDR-TB transmission control state-wide.

Asunto(s)

Antibióticos Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/genética , Tuberculosis Resistente a Múltiples Medicamentos/transmisión , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antituberculosos/uso terapéutico , Brasil/epidemiología , Perfilación de la Expresión Génica , Genoma Bacteriano/genética , Humanos , Isoniazida/uso terapéutico , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple/genética , Estudios Retrospectivos , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Secuenciación Completa del Genoma , Adulto Joven

RESUMEN

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.

Asunto(s)

Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Brasil , Catalasa/genética , Catalasa/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/aislamiento & purificación , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Estudios Prospectivos

RESUMEN

BACKGROUND: Since the implementation of the Xpert MTB/RIF in Sao Paulo, Brazil, numerous Mycobacterium tuberculosis isolates presenting "rifampicin-resistant genotype with rifampicin-susceptible phenotype" were observed. OBJECTIVE: To evaluate the prevalence, rpoB mutations and transmission of M. tuberculosis resistant to rifampicin on Xpert MTB/RIF but susceptible on BACTEC MGIT system, in Sao Paulo state. METHODS: Patients' isolates with this pattern of rifampicin discordance, collected from 2014 to 2017, had their rpoB predominant rifampicin-resistance-determining region sequenced and were genotyped by IS6110 restriction fragment-length polymorphism. FINDINGS: The prevalence of rifampicin-discordant M. tuberculosis with genotypic resistance was 55.1% (156/283). Among the sequenced and genotyped isolates, 75.5% (111/147) were in clusters, largely associated with the type of rpoB mutation. Most isolates (98.6%; 72/73) harbouring the predominant mutation, His445Asn, were pooled into the two largest clusters, SP2ga (42/72; 58.3%) and SP5o (12/72; 16.7%). Ranking second, isolates carrying the silent mutation Phe433Phe were mostly (92.3%; 24/26) gathered into four groups of the family SP25. CONCLUSION: These findings suggest that this unusual high rifampicin discrepancy proportion was greatly influenced by few actively circulating clusters. Further studies on many of the rpoB mutations identified in our setting are needed to elucidate their association with phenotypic rifampicin resistance.

Asunto(s)

Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Epidemias/estadística & datos numéricos , Mutación , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/transmisión , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antibióticos Antituberculosos/farmacología , Brasil/epidemiología , Estudios Transversales , ARN Polimerasas Dirigidas por ADN/genética , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Fenotipo , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Adulto Joven

RESUMEN

Tuberculosis (TB) is an ongoing public health care, with the state of affairs exacerbated by the growth of anti-TB drug-resistant forms in South Africa. Not much attention is given to zoonotic TB. Thus, this study aimed to determine the presence of rpoB mutations among Mycobacterium tuberculosis complex (MTBC) isolates of lymph nodes from slaughtered cattle. A count of 14,950 carcasses from selected abattoirs were examined for nodular lesions and enlarged lymph nodes; 376 lymph nodes were cultured for MTBC. Positive isolates were tested for drug sensitivity against three anti-TB drugs, rifampicin, isoniazid, and ethambutol, using the Lowenstein-Jensen proportion method. Rifampicin-resistant isolates were sequenced, and spoligotyping was performed for lineage classification. A total of 162 isolates were confirmed as MTBC and 42 isolates were resistant to rifampicin. All rifampicin-resistant isolates carried the H526D rpoB mutation, and almost all of them carried an additional nonsynonymous nucleotide substitution in the hot spot region, in three other codons (510, 516 and 522). In total, 5 different mutations at four codons are reported, including one isolate showing 3 of them which has never been reported in South Africa. In addition, we report 4 different spoligo patterns, with 34 isolates known and 8 unknown spoligotype international types. From the known clades, 5 (11.9%) isolates were identified as Bov_4 caprae lineage, 29 (69%) Beijing, and 8 (19.1%) remaining unknown clades. The detection of MTBC-resistant patterns from cattle lymph nodes (Eastern Cape, South Africa) necessitates the investigation of other possible routes of MTBC transmission.

Asunto(s)

Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/veterinaria , Mataderos , Animales , Antituberculosos/farmacología , Bovinos , Genotipo , Ganglios Linfáticos/microbiología , Pruebas de Sensibilidad Microbiana , Mutación , Sudáfrica , Tuberculosis Resistente a Múltiples Medicamentos/microbiología

RESUMEN

Background: Rifampicin (RIF) resistance in Mycobacterium tuberculosis is frequently caused by mutations in the rpoB gene. These mutations are associated with a fitness cost, which can be overcome by compensatory mutations in other genes, among which rpoC may be the most important. We analyzed 469 Peruvian M. tuberculosis clinical isolates to identify compensatory mutations in rpoC/rpoA associated with RIF resistance. Methods: The M. tuberculosis isolates were collected and tested for RIF susceptibility and spoligotyping. Samples were sequenced and aligned to the reference genome to identify mutations. By analyzing the sequences and the metadata, we identified a list of rpoC mutations exclusively associated with RIF resistance and mutations in rpoB. We then evaluated the distribution of these mutations along the protein sequence and tridimensional structure. Results: One hundred and twenty-five strains were RIF susceptible and 346 were resistant. We identified 35 potential new compensatory mutations, some of which were distributed on the interface surface between rpoB and rpoC, arising in clusters and suggesting the presence of hotspots for compensatory mutations. Conclusion: This study identifies 35 putative novel compensatory mutations in the ß' subunit of M. tuberculosis RNApol. Six of these (S428T, L507V, A734V, I997V, and V1252LM) are considered most likely to have a compensatory role, as they fall in the interaction zone of the two subunits and the mutation did not lead to any change in the protein's physical-chemical properties.

Asunto(s)

Antituberculosos/farmacología , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Perú/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología

RESUMEN

OBJECTIVE: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. METHODS: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. RESULTS: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. CONCLUSIONS: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

Asunto(s)

Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Mycobacterium avium/genética , Micobacterias no Tuberculosas/aislamiento & purificación , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Brasil , Chaperonina 60/genética , ARN Polimerasas Dirigidas por ADN/genética , Variación Genética , Humanos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium avium/aislamiento & purificación

RESUMEN

Zymomonas mobilis is a bacterium of industrial interest due to its high ethanol productivity and high tolerance to stresses. Although the physiological parameters of fermentation are well characterized, there are few studies on the molecular mechanisms that regulate the response to fermentative stress. Z. mobilis ZM4 presents five different sigma factors identified in the genome annotation, but the absence of sigma 38 leads to the questioning of which sigma factors are responsible for its mechanism of fermentative stress response. Thus, in this study, factors sigma 32 and sigma 24, traditionally related to heat shock, were tested for their influence on the response to osmotic and ethanol stress. The overexpression of these sigma factors in Z. mobilis ZM4 strain confirmed that both are associated with heat shock response, as described in other bacteria. Moreover, sigma 32 has also a role in the adaptation to osmotic stress, increasing both growth rate and glucose influx rate. The same strain that overexpresses sigma 32 also showed a decrease in ethanol tolerance, suggesting an antagonism between these two mechanisms. It was not possible to conclude if sigma 24 really affects ethanol tolerance in Z. mobilis, but the overexpression of this sigma factor led to a decrease in ethanol productivity.

Asunto(s)

Fermentación , Presión Osmótica , Factor sigma/genética , Estrés Fisiológico/genética , Zymomonas/genética , Zymomonas/fisiología , ARN Polimerasas Dirigidas por ADN/genética , Etanol/farmacología , Glucosa/metabolismo , Proteínas de Choque Térmico/genética , Zymomonas/efectos de los fármacos

RESUMEN

ABSTRACT Objective: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. Methods: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. Results: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. Conclusions: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

---

RESUMO Objetivo: As micobactérias não tuberculosas (MNT) são um grupo heterogêneo de bactérias amplamente distribuídas na natureza e relacionadas com infecções oportunistas em seres humanos. Os objetivos deste estudo foram identificar MNT em pacientes com suspeita de tuberculose e culturas positivas e avaliar a diversidade genética de cepas identificadas como Mycobacterium avium. Métodos: Foram estudadas amostras pulmonares e extrapulmonares provenientes de 1.248 pacientes. As amostras que apresentaram resultado positivo em cultura e negativo para o complexo M. tuberculosis na identificação molecular foram avaliadas por meio da detecção dos genes hsp65 e rpoB e de sequenciamento de fragmentos conservados desses genes. Todas as cepas identificadas como M. avium foram genotipadas pelo método mycobacterial interspersed repetitive unit-variable-number tandem-repeat com oito loci. Resultados: Das 332 micobactérias isoladas, 25 (7,5%) eram MNT. Dessas 25, 18 (72%) eram M. avium, 5 (20%) eram M. abscessus, 1 (4%) era M. gastri e 1 (4%) era M. kansasii. As 18 cepas de M. avium apresentaram alta diversidade, e apenas duas eram geneticamente relacionadas. Conclusões: Esses resultados mostram a necessidade de considerar a investigação de MNT em pacientes com suspeita de tuberculose ativa e culturas positivas e de avaliar a diversidade genética de cepas de M. avium.

Asunto(s)

Humanos , Micobacterias no Tuberculosas/aislamiento & purificación , Mycobacterium avium/genética , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Proteínas Bacterianas/genética , Variación Genética , Brasil , ARN Polimerasas Dirigidas por ADN/genética , Técnicas de Tipificación Bacteriana , Chaperonina 60/genética , Mycobacterium avium/aislamiento & purificación , Infecciones por Mycobacterium no Tuberculosas/microbiología

RESUMEN

BACKGROUND: Molecular tests can allow the rapid detection of tuberculosis (TB) and multidrug-resistant TB (MDR-TB). TB-SPRINT 59-Plex Beamedex® is a microbead-based assay developed for the simultaneous spoligotyping and detection of MDR-TB. The accuracy and cost evaluation of new assays and technologies are of great importance for their routine use in clinics and in research laboratories. The aim of this study was to evaluate the performance of TB-SPRINT at three laboratory research centers in Brazil and calculate its mean cost (MC) and activity-based costing (ABC). METHODS: TB-SPRINT data were compared with the phenotypic and genotypic profiles obtained using Bactec™ MGIT™ 960 system and Genotype® MTBDRplus, respectively. RESULTS: Compared with MGIT, the accuracies of TB-SPRINT for the detection of rifampicin and isoniazid resistance ranged from 81 to 92% and 91.3 to 93.9%, respectively. Compared with MTBDRplus, the accuracies of TB-SPRINT for rifampicin and isoniazid were 99 and 94.2%, respectively. Moreover, the MC and ABC of TB-SPRINT were USD 127.78 and USD 109.94, respectively. CONCLUSION: TB-SPRINT showed good results for isoniazid and rifampicin resistance detection, but still needs improvement to achieve In Vitro Diagnostics standards.

Asunto(s)

Farmacorresistencia Bacteriana , Citometría de Flujo/métodos , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , Costos y Análisis de Costo , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Citometría de Flujo/economía , Genotipo , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Regiones Promotoras Genéticas , Juego de Reactivos para Diagnóstico , Rifampin , Sensibilidad y Especificidad , Tuberculosis/economía

RESUMEN

Tuberculosis (TB) is the leading cause of death among infectious diseases worldwide. Among the estimated cases of drug-resistant TB, approximately 60% occur in the BRICS countries (Brazil, Russia, India, China and South Africa). Among Brazilian states, primary and acquired multidrug-resistant TB (MDR-TB) rates were the highest in Rio Grande do Sul (RS). This study aimed to perform molecular characterisation of MDR-TB in the State of RS, a high-burden Brazilian state. We performed molecular characterisation of MDR-TB cases in RS, defined by drug susceptibility testing, using 131 Mycobacterium tuberculosis (M.tb) DNA samples from the Central Laboratory. We carried out MIRU-VNTR 24loci, spoligotyping, sequencing of the katG, inhA and rpoB genes and RDRio sublineage identification. The most frequent families found were LAM (65.6%) and Haarlem (22.1%). RDRio deletion was observed in 42 (32%) of the M.tb isolates. Among MDR-TB cases, eight (6.1%) did not present mutations in the studied genes. In 116 (88.5%) M.tb isolates, we found mutations associated with rifampicin (RIF) resistance in rpoB gene, and in 112 isolates (85.5%), we observed mutations related to isoniazid resistance in katG and inhA genes. An insertion of 12 nucleotides (CCAGAACAACCC) at the 516 codon in the rpoB gene, possibly responsible for a decreased interaction of RIF and RNA polymerase, was found in 19/131 of the isolates, belonging mostly to LAM and Haarlem families. These results enable a better understanding of the dynamics of transmission and evolution of MDR-TB in the region.

Asunto(s)

Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/genética , Adolescente , Adulto , Distribución por Edad , Antituberculosos/uso terapéutico , Brasil/epidemiología , Costo de Enfermedad , ARN Polimerasas Dirigidas por ADN/genética , Bases de Datos Factuales , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Femenino , Genotipo , Humanos , Incidencia , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Repeticiones de Minisatélite/genética , Mycobacterium tuberculosis/aislamiento & purificación , Estudios Retrospectivos , Medición de Riesgo , Distribución por Sexo , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Adulto Joven

RESUMEN

OBJECTIVE: To evaluate the use of a sequencing procedure for the entire rpoB gene of Mycobacterium tuberculosis to identify mutations pre-rifampicin resistance determining region (RRDR), within RRDR, and post-RRDR in isolates circulating in a region affected by tuberculosis (TB). METHODS: Five primers were designed, with which five DNA fragments of rpoB were obtained, sequenced by Sanger, and analysed in silico in order to identify mutations over the entire rpoB gene in rifampicin-sensitive and rifampicin-resistant TB. RESULTS: It was possible to analyse the entire rpoB gene in five rifampicin-sensitive and 15 rifampicin-resistant isolates. Thirty-six mutations were identified. Two mutations were found pre-RRDR, nine within-RRDR and 25 post-RRDR. The most frequent mutations within RRDR were S531L (53%), followed by S512T (20%), all of which were found in rifampicin-resistant isolates. Of the 25 mutations found post-RRDR, 14 were only in resistant isolates, and the most frequent was D853N, which was present in 85% of isolates. Mutations E818K, D836N and T882P were observed in 80% of the rifampicin-resistant and rifampicin-sensitive isolates. CONCLUSIONS: The proposed sequencing method allowed identification of mutations in the entire rpoB gene. This procedure represents a useful tool for diagnosing rifampicin resistance. The number of mutations that were found raises new questions about the diversity of mutations in the rpoB gene and their role in rifampicin resistance in regions where TB is endemic.

Asunto(s)

Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Análisis de Secuencia de ADN/métodos , Tuberculosis/microbiología , Farmacorresistencia Bacteriana , Enfermedades Endémicas , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Rifampin/uso terapéutico , Tuberculosis/tratamiento farmacológico

RESUMEN

The prevalence of Helicobacter pylori resistance to levofloxacin and metronidazole was high in the Dominican Republic. We used two-fold agar dilution method to determine the minimum inhibitory concentration of five alternative antibiotics in 63 Dominican strains. We also assessed the genetic mutations associated with the antibiotic resistance using next-generation sequencing. We revealed that all 63 strains were sensitive towards sitafloxacin, furazolidone, and rifabutin. In contrast, the prevalence of rifaximin and garenoxacin resistance were high (82.5% and 34.9%, respectively). Patients more than or equal to 60 years old had the highest risk of double-antibiotic resistance (7/9, 77.8%, OR = 31.5, P = 0.009) and garenoxacin resistances (8/9, 88.9%, OR = 45.33, P = 0.002) with an increasing risk simultaneously by age (P = 0.004, r = 0.357). Almost all rifaximin resistant strains possessed multiple mutations with more than three mutations within rpoB including the most frequent novel mutations of S352L, I2726L, and V2465A. There was a significant association between vacA genotype and rifaximin resistance (P = 0.042). Among 23 levofloxacin-resistant strains, 82.6% (19/23, P <0.001) were also resistant to garenoxacin, and 39.1% (9/23) had a high minimal inhibitory concentration ≥8 µg/mL with positive trend correlation (P = <0.001, r = 0.84). Among 19 garenoxacin resistant strains, 16 (84.2%) contained mutations at D91 and N87 of gyrA. In conclusion, sitafloxacin, rifabutin, and furazolidone might be considered as alternative antibiotics to be included in H. pylori eradication regimen in regions with high prevalence of levofloxacin and metronidazole resistance, such as the Dominican Republic.

Asunto(s)

Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Helicobacter pylori/fisiología , Levofloxacino/farmacología , Metronidazol/farmacología , Adulto , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , República Dominicana/epidemiología , Farmacorresistencia Bacteriana Múltiple/genética , Femenino , Infecciones por Helicobacter/diagnóstico , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/epidemiología , Helicobacter pylori/efectos de los fármacos , Humanos , Levofloxacino/uso terapéutico , Masculino , Metronidazol/uso terapéutico , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación , Prevalencia , Virulencia/genética

RESUMEN

Cowpea (Vigna unguiculata L.) is a legume species that considerably benefits from inoculation with nitrogen fixing bacteria of the genus Bradyrhizobium. One of the strains recommended for inoculation in cowpea in Brazil is UFLA03-84 (Bradyrhizobium sp.). The aim of our study was to define the taxonomic position of the UFLA03-84 strain and of two other strains of Bradyrhizobium (UFLA03-144 and INPA237B), all belonging to the same phylogenetic group and isolated from soils of the Brazilian Amazon. Multilocus sequence analysis (MLSA) of the housekeeping genes atpD, gyrB, recA, and rpoB grouped (with similarity higher than 99%) the three strains with Bradyrhizobium viridifuturi SEMIA 690T. The analyses of average nucleotide identity and digital DNA-DNA hybridization supported classification of the group as Bradyrhizobium viridifuturi. The three strains exhibited similar behavior in relation to the most of the phenotypic characteristics evaluated. However, some characteristics exhibited variation, indicating phenotypic diversity within the species. Phylogenetic analysis of the nodC and nifH genes showed that the three strains are members of the same symbiovar (tropici) that contains type strains of Bradyrhizobium species coming from tropical soils (SEMIA 690TB. viridifuturi, CNPSo 1112TB. tropiciagri, CNPSo 2833TB. embrapense, and B. brasilense UFLA03-321T).

Asunto(s)

Bradyrhizobium/clasificación , Bradyrhizobium/genética , Genes Esenciales/genética , Nódulos de las Raíces de las Plantas/microbiología , Vigna/microbiología , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Bradyrhizobium/aislamiento & purificación , Brasil , Girasa de ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Genoma Bacteriano/genética , Proteínas de la Membrana/genética , Tipificación de Secuencias Multilocus , N-Acetilglucosaminiltransferasas/genética , Fijación del Nitrógeno/genética , Oxidorreductasas/genética , ARN Ribosómico 16S/genética , Rec A Recombinasas/genética , Microbiología del Suelo

RESUMEN

AIM: To evaluate modulatory effect of verapamil (VP) in rifampicin (RIF) activity and its effect in efflux pumps (EPs) transcript levels in Mycobacterium tuberculosis. MATERIALS & METHODS: RIF and VP minimal inhibitory concentration, combinatory effect and detection of mutations were determined in 16 isolates. EPs transcript levels were determined in four isolates by real-time PCR after exposure to drugs. RESULTS: VP showed good combinatory effect among RIF-resistant isolates. This effect was also observed in the relative transcript levels of EPs, mainly after 72 h of exposure, depending on the EP gene, genotype and the resistance profile of the isolate. CONCLUSION: Additional regulatory mechanisms in the EP activities, as well as, interactions with other drug-specific resistance mechanisms need further investigation in M. tuberculosis.

Asunto(s)

Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Verapamilo/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Brasil , Catalasa/genética , ARN Polimerasas Dirigidas por ADN/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Regulación Bacteriana de la Expresión Génica , Genotipo , Humanos , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Oxidorreductasas/genética , Factores de Tiempo , Tuberculosis Resistente a Múltiples Medicamentos/microbiología

RESUMEN

INTRODUCTION: Previous knowledge of molecular mechanisms related with multi-drug resistances in tuberculosis is important if molecular diagnostic procedures want to be used in specific geographical regions. For that reason, the aim of this study was to investigate the mutations at rpoB, katG and inhA in multi-drug resistant tuberculosis isolates from Southeast Mexico. METHODS: Isolates of tuberculosis with a confirmed resistance against rifampicin and isoniazid were collected and sequencing analysis was performed of the rpoB rifampicin resistance-determining region, the katG and the encoding region of inhA. RESULT: Of 74 isolates with multidrug resistance, 34 (46%) presented six mutations in katG; the most abundant was katG315 in 29 (39%) isolates. At inhA, nine (11%) isolates presented three mutations; the most frequent was inhA21, located in five (6%) strains. Eleven polymorphisms were observed at rpoB in 61 (82%) isolates, prevailing rpoB531 and rpoB 526 in 48 (64%) and ten (12%) isolates, respectively. Eleven double combinations were observed in 39 (52%) isolates, the most common of which was rpoB531+katG315, found in 22 (29%) strains. CONCLUSION: This study provides valuable information on the diversity of polymorphisms in genes related to multidrug-resistant tuberculosis, as well as the presence of new mutations not previously described; this information should be considered in the implementation of molecular diagnostic tests.

Asunto(s)

Proteínas Bacterianas/genética , Catalasa/genética , ARN Polimerasas Dirigidas por ADN/genética , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Oxidorreductasas/genética , Adulto , Femenino , Humanos , Masculino , México , Persona de Mediana Edad , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/microbiología

RESUMEN

Promoters are DNA sequences located upstream of the transcription start site of genes. In bacteria, the RNA polymerase enzyme requires additional subunits, called sigma factors (σ) to begin specific gene transcription in distinct environmental conditions. Currently, promoter prediction still poses many challenges due to the characteristics of these sequences. In this paper, the nucleotide content of Escherichia coli promoter sequences, related to five alternative σ factors, was analyzed by a machine learning technique in order to provide profiles according to the σ factor which recognizes them. For this, the clustering technique was applied since it is a viable method for finding hidden patterns on a data set. As a result, 20 groups of sequences were formed, and, aided by the Weblogo tool, it was possible to determine sequence profiles. These found patterns should be considered for implementing computational prediction tools. In addition, evidence was found of an overlap between the functions of the genes regulated by different σ factors, suggesting that DNA structural properties are also essential parameters for further studies.

Asunto(s)

Escherichia coli/enzimología , Escherichia coli/genética , Regiones Promotoras Genéticas , Factor sigma/genética , Algoritmos , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Nucleótidos/análisis , Factor sigma/metabolismo , Transcripción Genética