RESUMEN

BACKGROUND: Mycobacterium abscessus complex (MABC), an opportunistic nontuberculous mycobacteria (NTM), can lead to poor clinical outcomes in pulmonary infections. Conflicting data exist on person-to-person transmission of MABC within and across healthcare facilities. To investigate further, a comprehensive retrospective study across five healthcare institutions on the Island of Montréal was undertaken. METHODS: We analyzed the genomes of 221 MABC isolates obtained from 115 individuals (2010-2018) to identify possible links. Genetic similarity, defined as ≤25 single-nucleotide polymorphisms (SNPs), was investigated through a blinded epidemiological inquiry. RESULTS: Bioinformatics analyses identified 28 sequence types (STs), including globally observed dominant circulating clones (DCCs). Further analysis revealed 210 isolate pairs within the SNP threshold. Among these pairs, there was one possible lab contamination where isolates from different patients processed in the same lab differed by only 2 SNPs. There were 37 isolate pairs from patients who had provided specimens from the same hospital; however, epidemiological analysis found no evidence of healthcare-associated person-to-person transmission between these patients. Additionally, pan-genome analysis showed higher discriminatory power than core genome analysis for examining genomic similarity. CONCLUSIONS: Genomics alone is insufficient to establish MABC transmission, particularly considering the genetic similarity and wide distribution of DCCs, although pan-genome analysis has the potential to add further insight. Our findings indicate that MABC infections in Montréal are unlikely attributable to healthcare-associated person-to-person transmission.

RESUMEN

In Mycobacterium tuberculosis, molecular predictions of ethambutol resistance rely primarily on the detection of mutations within embB. However, discordance between embB406 mutations and gold standard phenotypic drug sensitivity testing (DST) questions the significance of embB406 mutations used in molecular DST. This study tabulates embB mutations found in Canadian M. tuberculosis isolates and evaluates the impact of specific mutations on ethambutol resistance. The National Reference Centre for Mycobacteriology culture collection (n = 2796) was screened for isolates with embB mutations. Phenotypic DST was performed on the BACTEC™ MGIT™ 960 at ethambutol concentrations of 2-5 µg/mL. Whole genome sequencing was used for drug resistance predictions, phylogenomics and single nucleotide polymorphism analysis. Detection of resistance-associated embB mutations corresponded to a positive predictive value of 64.3%, negative predictive value of 99.2%, 98.7% specificity, and 73.3% sensitivity compared to phenotypic DST. Two embB406 mutation subtypes (Gly406Asp, Gly406Ala) were found among 16 isolates, of which 12 were sensitive at 5 µg/mL ethambutol with variable resistance between 2-4 µg/mL. A novel frameshift mutation in regulator embR (Gln258fs) was found in nine isolates. Mutations in embB406 were associated with low-level ethambutol resistance undetectable at the recommended critical concentration (5 µg/mL). These novel mutations may exacerbate variability in ethambutol resistance.

RESUMEN

Phenotypic susceptibility testing of the Mycobacterium tuberculosis complex (MTBC) isolate requires culture growth, which can delay rapid detection of resistant cases. Whole genome sequencing (WGS) and data analysis pipelines can assist in predicting resistance to antimicrobials used in the treatment of tuberculosis (TB). This study compared phenotypic susceptibility testing results and WGS-based predictions of antimicrobial resistance (AMR) to four first-line antimicrobials-isoniazid, rifampin, ethambutol, and pyrazinamide-for MTBC isolates tested between the years 2018-2022. For this 5-year retrospective analysis, the WGS sensitivity for predicting resistance for isoniazid, rifampin, ethambutol, and pyrazinamide using Mykrobe was 86.7%, 100.0%, 100.0%, and 47.8%, respectively, and the specificity was 99.4%, 99.5%, 98.7%, and 99.9%, respectively. The predictive values improved slightly using Mykrobe corrections applied using TB Profiler, i.e., the WGS sensitivity for isoniazid, rifampin, ethambutol, and pyrazinamide was 92.31%, 100%, 100%, and 57.78%, respectively, and the specificity was 99.63%. 99.45%, 98.93%, and 99.93%, respectively. The utilization of WGS-based testing addresses concerns regarding test turnaround time and enables analysis for MTBC member identification, antimicrobial resistance prediction, detection of mixed cultures, and strain genotyping, all through a single laboratory test. WGS enables rapid resistance detection compared to traditional phenotypic susceptibility testing methods using the WHO TB mutation catalog, providing an insight into lesser-known mutations, which should be added to prediction databases as high-confidence mutations are recognized. The WGS-based methods can support TB elimination efforts in Canada and globally by ensuring the early start of appropriate treatment, rapidly limiting the spread of TB outbreaks.

Asunto(s)

Antituberculosos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Secuenciación Completa del Genoma , Secuenciación Completa del Genoma/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/farmacología , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Estudios Retrospectivos , Farmacorresistencia Bacteriana/genética , Genoma Bacteriano , Etambutol/farmacología , Isoniazida/farmacología , Pirazinamida/farmacología , Tuberculosis/microbiología , Tuberculosis/tratamiento farmacológico , Rifampin/farmacología

RESUMEN

Epidemiologic research on zoonotic tuberculosis historically used Mycobacterium bovis as a surrogate measure, however, increased reports of human tuberculosis caused by other animal-associated Mycobacterium tuberculosis complex members like Mycobacterium orygis necessitates their inclusion. We performed a retrospective cohort study including persons infected with any animal-lineage M. tuberculosis complex species in Alberta, Canada, from January 1995 to July 2021, identifying 42 patients (20 M. bovis, 21 M. orygis, one M. caprae). Demographic, epidemiologic and clinical characteristics were compared against persons with culture-confirmed M. tuberculosis infection. The proportion of culture-positive infections caused by M. orygis increased continuously from 2016-2020. Significantly more females at a higher median age were impacted by M. orygis, with all patients originating from South Asia. M. bovis caused significantly more extra-pulmonary disease, and disproportionately impacted young females, particularly those pregnant or post-partum. All infections were acquired abroad. These findings can aid in developing targeted public health interventions.

RESUMEN

A recently described member of the Mycobacterium tuberculosis complex (MTBC) is Mycobacterium orygis, which can cause disease primarily in animals but also in humans. Although M. orygis has been reported from different geographic regions around the world, due to a lack of proper identification techniques, the contribution of this emerging pathogen to the global burden of zoonotic tuberculosis is not fully understood. In the present work, we report single nucleotide polymorphism (SNP) analysis using whole genome sequencing (WGS) that can accurately identify M. orygis and differentiate it from other members of the MTBC species. WGS-based SNP analysis was performed for 61 isolates from different provinces in Canada that were identified as M. orygis. A total of 56 M. orygis sequences from the public databases were also included in the analysis. Several unique SNPs in the gyrB, PPE55, Rv2042c, leuS, mmpL6, and mmpS6 genes were used to determine their effectiveness as genetic markers for the identification of M. orygis. To the best of our knowledge, five of these SNPs, viz., gyrB 277 (A→G), gyrB 1478 (T→C), leuS 1064 (A→T), mmpL6 486 (T→C), and mmpS6 334 (C→G), are reported for the first time in this study. Our results also revealed several SNPs specific to other species within MTBC. The phylogenetic analysis shows that the studied genomes were genetically diverse and clustered with M. orygis sequences of human and animal origin reported from different geographic locations. Therefore, the present study provides a new insight into the high-confidence identification of M. orygis from MTBC species based on WGS data, which can be useful for reference and diagnostic laboratories.

Asunto(s)

Mycobacterium tuberculosis , Mycobacterium , Tuberculosis , Animales , Humanos , Filogenia , Tuberculosis/diagnóstico , Tuberculosis/microbiología , Secuenciación Completa del Genoma , Polimorfismo de Nucleótido Simple , Mycobacterium tuberculosis/genética

RESUMEN

Tuberculosis is a significant cause of morbidity worldwide and is a priority at the provincial and federal levels in Canada. It is known that tuberculosis transmission networks are complex and span many years as well as different jurisdictions and countries. MIRU-VNTR is a universal tuberculosis genotyping method that utilizes a 24-loci pattern and it has shown promise in identifying inter and intrajurisdictional clusters within Canada. MIRU-VNTR data collected over 10 years from the National Reference Centre for Mycobacteriology (NRCM) were analyzed in this study. Some clusters were unique to a single province/territory, while others spanned multiple provinces and/or territories in Canada. The use of a universal laboratory test can enhance contact tracing, provide geographical information on circulating genotypes, and hence, aid in tuberculosis investigation by public health. The housing of all data on one platform, technical ease of the method, easy exchange of data between jurisdictions, and strong collaboration with laboratories and surveillance units at the provincial and federal levels have the potential to identify possible outbreaks in real time.

RESUMEN

Real-time genome monitoring of the SARS-CoV-2 pandemic outbreak is of utmost importance for designing diagnostic tools, guiding antiviral treatment and vaccination strategies. In this study, we present an accurate method for temporal and geographical comparison of mutational events based on GISAID database genome sequencing. Among 42523 SARS-CoV-2 genomes analyzed, we found 23202 variants compared to the reference genome. The Ti/Tv (transition/transversion) ratio was used to filter out possible false-positive errors. Transition mutations generally occurred more frequently than transversions. Our clustering analysis revealed remarkable hotspot mutation patterns for SARS-CoV-2. Mutations were clustered based on how their frequencies changed over time according to each geographical location. We observed some clusters showing a clear variation in mutation frequency and continuously evolving in the world. However, many mutations appeared in specific periods without a clear pattern over time. Various important nonsynonymous mutations were observed, mainly in Oceania and Asia. More than half of these mutations were observed only once. Four hotspot mutations were found in all geographical locations at least once: T265I (NSP2), P314L (NSP12), D614G (S), and Q57H (ORF3a). The current analysis of SARS-CoV-2 genomes provides valuable information on the geographical and temporal mutational evolution of SARS-CoV-2.

Asunto(s)

COVID-19 , Bases de Datos de Ácidos Nucleicos , Evolución Molecular , Genoma Viral , Mutación , Pandemias , Filogenia , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/genética , Humanos

RESUMEN

BACKGROUND: Mycobacterium abscessus is a rapidly growing mycobacteria involved in severe infections of the lung, skin, or soft tissue. Macrolides such as clarithromycin are the recommended first line drugs for treatment of M. abscessus infections. However, M. abscessus has dual mechanisms of resistance to macrolides, making treatment by macrolides difficult. A functional erm(41) gene confers for inducible resistance while acquired mutations on the 23S rRNA rrl gene confer for constitutive resistance. METHODS: We have developed a real-time PCR assay to detect both inducible and acquired resistance to clarithromycin, and compared the results to traditional erm(41) and rrl sequencing and phenotypic susceptibility testing using Sensititre™ plates. RESULTS: Of the total 126 M. abscessus isolates tested, truncated erm(41) was found in 23/126 (18.3%) of the samples, 27/126 (21.4%) had a T28C mutation in erm(41), and 2/126 (1.6%) had an acquired A2058C mutation in rrl. The phenotypic results correlated with the expected sequencing results in 121/126 samples (96%). Phenotypic testing compared to real-time PCR resolved 2 of these discrepancies by showing the existence of both erm(41) alleles in the isolates that sequencing missed. One culture was found to be mixed with two M. abscessus subsp. as per hsp65 sequencing and 2 isolates had discordance between molecular and phenotypic results. It was presumed that 3 isolates showed discrepancy between sequencing and real-time PCR, but one culture was mixed and other 2 detected both alleles by real-time PCR leading to 100% concordance when compared to sequencing. CONCLUSION: In conclusion, real-time PCR is more accurate for detection of both acquired and induced clarithromycin resistance, specifically when mixed genic profiles are present in a sample.

Asunto(s)

Antibacterianos/farmacología , Claritromicina/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Genes Bacterianos , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , ARN Ribosómico 23S/genética

RESUMEN

Background: Mycobacterium abscessus is a rapid growing nontuberculous mycobacteria (NTM) and a clinically significant pathogen capable of causing variable infections in humans that are difficult to treat and may require months of therapy/surgical interventions. Like other NTMs, M. abscessus can be associated with outbreaks leading to complex investigations and treatment of affected cases. Typing schemes for bacterial pathogens provide numerous applications; including identifying chain of transmission and tracking genomic evolution, are lacking or limited for many NTMs including M. abscessus. Methods: We extended the existing scheme from PubMLST using whole-genome data for M. abscessus by extracting data for 15 genetic regions within the M. abscessus genome. A total of 168 whole genomes and 11 gene sequences were used to build this scheme (MAB-multilocus sequence typing [MLST]). Results: All seven genes from the PubMLST scheme, namely argH, cya, gnd, murC, pta, purH, and rpoB, were expanded by 10, 14, 13, 10, 13, 10, and 9 alleles, respectively. Another eight novel genes were added including hsp 65, erm(41), arr, rrs, rrl, gyrA, gyrB, and recA with 16, 16, 25, 7, 32, 35, 29, and 15 alleles, respectively, with 85 unique sequence types identified among all isolates. Conclusion: MAB-MLST can provide identification of M. abscessus complex to the subspecies level based on three genes and can provide antimicrobial resistance susceptibility prediction based on results from seven genes. MAB-MLST generated a total of 85 STs, resulting in subtyping of 90 additional isolates that could not be genotyped using PubMLST and yielding results comparable to whole-genome sequencing (WGS). Implementation of a Galaxy-based data analysis tool, MAB-MLST, that simplifies the WGS data and yet maintains a high discriminatory index that can aid in deciphering an outbreak has vast applicability for routine diagnostics.

Asunto(s)

Tipificación de Secuencias Multilocus , Mycobacterium abscessus/clasificación , Secuenciación Completa del Genoma , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , ADN Bacteriano , Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano , Genotipo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/genética , Filogenia , Análisis de Secuencia de ADN

RESUMEN

Mycobacterium africanum is an important cause of human tuberculosis and is found almost exclusively in West Africa. We identified a cluster of patients in Montreal, Canada, with M africanum disease that share identical genotypic signatures by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and a putative epidemiological link, thus providing evidence of possible local transmission of M africanum in Montreal over a 10-year period.

RESUMEN

INTRODUCTION: Mycobacterium xenopi is a rare opportunistic pathogen mainly causing infections in immunocompromised human patients or those with underlying chronic structural lung disease. Cases of disease in veterinary medicine remain scarce. Few animal species, including birds, are suspected of being vectors of the disease and there has not yet been a report of clinical disease in birds. We report the first case, to our knowledge, of systemic infection in a domestic bird. CASE PRESENTATION: A female fiery-shouldered conure was submitted after death for necropsy following episodes of heavy breathing. The necropsy revealed multiple granulomatous lesions within the liver, air sacs and kidneys. Ziehl-Neelsen stains demonstrated the presence of numerous intralesional acid-fast bacilli. PCR assays and culture confirmed the presence of M. xenopi. CONCLUSION: Through this case we hope to describe the characteristics of M. xenopi disease in birds and the possible close relationship between animal and human infections.

RESUMEN

BACKGROUND: Intravesical Bacillus Calmette-Guérin (BCG) instillation has become one of the mainstays of adjunctive therapy in the treatment of superficial bladder cancer. Ophthalmologic complications are rare, but few cases are reported in the literature. METHODS: Retrospective observational case report. RESULTS: The authors report a case of unilateral Mycobacterium bovis BCG endophthalmitis after intravesical BCG instillations. Despite appropriate systemic antituberculous and corticosteroid therapy, the patient almost completely lost sight in the affected eye. This is the fourth case in the literature of proven M. bovis endophthalmitis suggesting a direct choroidal mycobacterial infection and not only a hypersensitivity immunologic reaction as previously suggested. CONCLUSION: This case highlights the direct choroidal mycobacterial infection of the disease after BCG instillations for bladder cancer and failure of treatment despite culture-proven drug sensitivity, thus suggesting the need to revaluate adequate treatment to avoid loss of vision.

Asunto(s)

Vacuna BCG/efectos adversos , Endoftalmitis/microbiología , Infecciones Bacterianas del Ojo/microbiología , Inmunoterapia/efectos adversos , Infecciones por Mycobacterium/microbiología , Administración Intravesical , Anciano de 80 o más Años , Endoftalmitis/etiología , Humanos , Masculino , Mycobacterium bovis , Estudios Retrospectivos

RESUMEN

BACKGROUND: BCG vaccine, introduced almost 100years ago, is the only option to prevent TB disease. It effectively protects newborns from meningeal TB but fails to prevent adult pulmonary TB. TB kills 1.3million people annually in areas where BCG vaccination is widely practiced. Thus, more effective TB vaccines are urgently needed. Others and we have shown that BCG mimics features of virulent M. tuberculosis, in particular attenuation of essential macrophage functions such as phagosome maturation and antigen presentation. One of these studies revealed that defect in antigen presentation is largely due to down-regulation of the cysteine protease Cathepsin S (CatS), which prevents MHC II molecule maturation and proper antigen peptide loading. Recent studies also suggested a potential role for cysteine proteases in the regulation of apoptosis, a key cellular process used by the macrophage to (i) contain and process ingested bacteria and (ii) facilitate cross-talk antigen presentation between the macrophage and dendritic cells. METHOD: To reverse the phenotype of vaccine-mediated macrophage attenuation, we engineered a novel BCG strain that expresses and secretes active CatS (rBCG-CatS) to examine its pro-apoptotic properties in vitro, and subsequently, immunogenicity in mice. RESULTS: Transcriptomic profiling of macrophages infected with rBCG-CatS, but not BCG, revealed upregulation of key pro-apoptotic genes and downregulation of anti-apoptotic genes, which were further confirmed by RT-qPCR analyses of expression of selected genes. Macrophages infected with rBCG-CatS undergo apoptosis as indicated by increased levels of annexin V staining and intracellular caspase-3 cleavage. Consistent with these findings, mice vaccinated with rBCG-CatS showed increased antigen-specific CD4+ T-cell responses, as well as enhanced cytokine production and proliferation in CD4+ upon ex vivo re-stimulation. CONCLUSION: Collectively, this study shows that a pro-apoptotic BCG strain alleviates adverse traits of the wild-type strain, resulting in a highly immunogenic TB vaccine.

Asunto(s)

Presentación de Antígeno , Apoptosis , Vacuna BCG/inmunología , Catepsinas/biosíntesis , Expresión Génica , Mycobacterium bovis/inmunología , Tuberculosis/prevención & control , Animales , Vacuna BCG/administración & dosificación , Vacuna BCG/genética , Catepsinas/genética , Femenino , Macrófagos/inmunología , Macrófagos/fisiología , Ratones Endogámicos C57BL , Mycobacterium bovis/genética , Mycobacterium tuberculosis , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

The whole genome sequencing of extensively drug-resistant Shewanella xiamenensis T17 isolated from hospital effluents in Algeria revealed the presence of a novel 268.4 kb plasmid designated pSx1, which carries several antibiotic-resistance genes in the novel Tn1696 derivative (Tn6297), in addition to the chromosomal blaOXA-48-like gene (blaOXA-416). The presence of the plasmid was confirmed by nuclease S1-PFGE analysis and transformation by electroporation into Escherichia coli DH10B. Tn6297 contains an In27 class 1 integron harboring the dfrA12-orfF-aadA2 array, msr(E) and mph(E) associated with IS26; a new efflux pump multidrug resistance composite transposon delimited by two ISEc29s; Tn-tet harboring tetR and tetA(C); a class 1 integron with the qacG gene cassette; qnrVC6 and dfrA23 associated with ISCR1; and a complex class 1 integron In4-like containing aacC1, aadA1, blaVEB-16, catA2, sul1Δ, cmlA9, tetR, tetA(G), aac(6')-II, and blaPSE-1. Its mer operon carries merB, but lacks merC, in contrast to Tn1696 and Tn21. This study represents the first characterization of a multidrug-resistant transposon and multidrug resistance plasmid in Shewanella and is the first report of blaOXA-416 in Algeria, providing evidence that Shewanella spp. could be an important reservoir and vehicle for drug resistance genes.

Asunto(s)

Elementos Transponibles de ADN , Farmacorresistencia Bacteriana Múltiple/genética , Plásmidos/metabolismo , Shewanella/genética , beta-Lactamasas/genética , Argelia/epidemiología , Antibacterianos/farmacología , Clonación Molecular , Electroporación , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/epidemiología , Infecciones por Bacterias Gramnegativas/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento , Hospitales , Humanos , Integrones , Pruebas de Sensibilidad Microbiana , Plásmidos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Shewanella/efectos de los fármacos , Shewanella/aislamiento & purificación , Shewanella/metabolismo , Transformación Bacteriana , beta-Lactamasas/metabolismo

RESUMEN

BACKGROUND: The increasing emergence of drug-resistant tuberculosis presents a threat to the effective control of tuberculosis (TB). Rapid detection of drug-resistance is more important than ever to address this scourge. The purpose of this study was to genotypically characterize the first-line antitubercular drug-resistant isolates collected over 11 years in Quebec. RESULTS: The main mutations found in our resistant strains collection (n = 225) include: the S315T substitution in katG (50.2 %), the -15 C/T mutation in the inhA promoter (29 %); the S531L substitution in rpoB (43 %); the deletion 8 bp 446 / + R140S in pncA (72.9 %); the M306I (35.7 %) and M306V (21.4 %) substitutions in embB. Ten of the mutations in katG and 4 mutations identified in pncA were previously undescribed. CONCLUSION: Screening of mutations conferring resistance to first-line antituberculous drugs using DNA-sequencing approach seems to be feasible and would drastically shorten the time to determine the resistance profile compared to the proportion method.

Asunto(s)

Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Amidohidrolasas/genética , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/genética , Genoma Bacteriano , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Oxidorreductasas/genética , Pentosiltransferasa/genética , Quebec , Análisis de Secuencia de ADN , Eliminación de Secuencia , Tuberculosis/microbiología

RESUMEN

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer's instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.

Asunto(s)

Servicios de Laboratorio Clínico , Técnicas de Laboratorio Clínico/métodos , Salud Pública , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Actinobacteria/clasificación , Actinobacteria/aislamiento & purificación , Técnicas de Tipificación Bacteriana/métodos , Hongos/clasificación , Hongos/aislamiento & purificación , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/aislamiento & purificación , Humanos , Mycobacterium/clasificación , Mycobacterium/aislamiento & purificación , Técnicas de Tipificación Micológica/métodos , Quebec , Reproducibilidad de los Resultados , Levaduras/clasificación , Levaduras/aislamiento & purificación

RESUMEN

Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain.

Asunto(s)

Mycobacterium tuberculosis/genética , Genes Bacterianos , Genética de Población , Humanos , Inuk , Polimorfismo de Nucleótido Simple , Quebec/epidemiología , Selección Genética , Tuberculosis/epidemiología , Tuberculosis/transmisión

RESUMEN

BACKGROUND: Between November 2011 and November 2012, a Canadian village of 933 persons had 50 culture-positive cases of tuberculosis, with 49 sharing the same genotype. METHODS: We performed Illumina-based whole-genome sequencing on Mycobacterium tuberculosis isolates from this village, during and before the outbreak. Phylogenetic trees were generated using the maximum likelihood method. RESULTS: Three distinct genotypes were identified. Strain I (n = 7) was isolated in 1991-1996. Strain II (n = 8) was isolated in 1996-2004. Strain III (n = 62) first appeared in 2007 and did not arise from strain I or II. Within strain III, there were 3 related but distinct clusters: IIIA, IIIB, and IIIC. Between 2007 and 2010, cluster IIIA predominated (11 of 22 vs 2 of 40; P < .001), whereas in 2011-2012 clusters IIIB (n = 18) and IIIC (n = 20) predominated over cluster IIIA (n = 11). Combined evolutionary and epidemiologic analysis of strain III cases revealed that the outbreak in 2011-2012 was the result of ≥6 temporally staggered events, spanning from 1 reactivation case to a point-source outbreak of 20 cases. CONCLUSIONS: After the disappearance of 2 strains of M. tuberculosis in this village, its reemergence in 2007 was followed by an epidemiologic amplification, affecting >5% of the population.

Asunto(s)

Enfermedades Transmisibles Emergentes/epidemiología , Brotes de Enfermedades , Tuberculosis/epidemiología , Adolescente , Adulto , Regiones Árticas , Canadá/epidemiología , Enfermedades Transmisibles Emergentes/microbiología , Femenino , Genoma Bacteriano , Genotipo , Humanos , Masculino , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Filogenia , Análisis de Secuencia de ADN , Tuberculosis/microbiología , Adulto Joven

RESUMEN

Mycobacterium xenopi is an opportunistic mycobacterial pathogen of increasing clinical importance. Surveillance of M. xenopi is hampered by the absence of tools for genotyping and molecular epidemiology. In this study, we describe the development and evaluation of an effective multilocus sequence typing strategy for M. xenopi.

Asunto(s)

Tipificación de Secuencias Multilocus/métodos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium xenopi/clasificación , Mycobacterium xenopi/genética , Humanos , Epidemiología Molecular/métodos , Infecciones por Mycobacterium no Tuberculosas/epidemiología