This study aimed to reconstruct the evolutionary history of Beijing strains of Mycobacterium tuberculosis and to test the hypothesis that evolution has influenced the ability of the Beijing strains ...within the different Beijing sublineages to spread and cause disease. A PCR-based method was used to analyze the genome structure of 40 different loci in 325 Beijing isolates collected from new and retreatment tuberculosis patients from an urban setting and 270 Beijing isolates collected from high-risk tuberculosis patients from a rural setting in the Western Cape, South Africa. The resulting data were subjected to phylogenetic analysis using the neighbor joining algorithm. Phylogenetic reconstructions were highly congruent with the "gold standard" phylogenetic tree based on synonymous single-nucleotide polymorphisms, thereby allowing a prediction of the order in which the evolutionary events had occurred. A total of seven independently evolving Beijing sublineages were identified. Analysis of epidemiological data in relation to the Beijing sublineage suggested an association between recent evolutionary change and frequency of occurrence in an urban population (P < 0.001) as well as in the rural population (P < 0.001). This concept was further supported by an association between more recently evolved Beijing strains and an increased ability to transmit and to cause disease (odds ratio, 5.82; 95% confidence interval, 3.13 to 10.82 P < 0.001). An association between Beijing sublineage and demographic and clinical parameters and drug resistance could not be demonstrated. From these data, we suggest that the pathogenic characteristics of Beijing strains are not conserved but rather that strains within individual lineages have evolved unique pathogenic characteristics.
Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during ...the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis.
Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline.
Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30% to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event.
Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.
Bacterial pathogens cause significant morbidity and mortality annually to both humans and animals. With the rampant spread of drug resistance and the diminishing effectiveness of current antibiotics, ...there is a pressing need for effective diagnostics for detection of bacterial pathogens and their drug resistances. Bacteriophages offer several unique opportunities for bacterial detection. This review highlights the means by which bacteriophages have been utilized to achieve and facilitate specific bacterial detection.
Summary The diverse clinico- and histopathological features, frequency of transmission and treatment outcome of Mycobacterium tuberculosis have been associated with several environmental, host and ...bacterial factors. Many Mycobacterium tuberculosis genotypes have been studied in an attempt to understand the genetic variations among the different genotypes and to clarify their contribution to phenotypic differences. Strains of the Beijing genotype have been extensively investigated due to their increased ability to spread and cause disease. Here we review the evidence of hypervirulence of the Beijing genotype as well as other Beijing-associated phenotypic characteristics such as alternate host immune modulation, clinical and pathological features, drug resistance, resistance to BCG vaccination and other epidemiological features to enhance our understanding of the contribution of pathogenic factors. From the data collected it is clear that the genetic background of Mycobacterium tuberculosis may influence the differential induction of the immune response, drug resistance patterns and clinical, epidemiological and pathogenic features which define disease progression following infection. This highlights the importance of ongoing research into the genetic mechanisms underlying the phenotypic and genotypic characteristics of different Mycobacterium tuberculosis genotype strains. Furthermore, these findings could help to direct future drug, vaccine and diagnostic test development towards targeting critical virulence factors and to identify persons at risk for developing active disease thereby limiting transmission and the perpetuation of the tuberculosis epidemic.
Differentiation of members of the Mycobacterium tuberculosis complex by conventional mycobacteriological methods is time consuming, making surveillance of species-specific disease difficult. A ...two-step, multiplex polymerase chain reaction (PCR) method based on genomic regions of difference (RD1, RD1(mic), RD2(seal), RD4, RD9 and RD12) was developed for the differentiation of M. canettii, M. tuberculosis, M. africanum, M. microti, M. pinnipedii, M. caprae, M. bovis and M. bovis BCG. The size of the respective multiplex PCR amplification products corresponded to the presence of the different M. tuberculosis complex members. This method allows for rapid differentiation, making it suitable for routine laboratories and surveillance purposes.
Background. Bacille Calmette-Guérin (BCG)—a live, attenuated vaccine—is routinely given to neonates in settings where tuberculosis is endemic, irrespective of human immunodeficiency virus (HIV) ...exposure. HIV-infected infants and other immunodeficient infants are at risk of BCG-related complications. We report the presentation, treatment, and mortality of children who develop BCG disease, with emphasis on HIV-infected children. In addition, we present a revised classification of BCG disease in children and propose standard diagnostic and management guidelines. Methods. This retrospective, hospital-based study was conducted in the Western Cape Province, South Africa. Mycobacterium tuberculosis complex isolates recovered from children aged <13 years during the period of August 2002 through January 2005 were speciated by polymerase chain reaction to confirm Mycobacterium bovis BCG. Clinical data were collected through medical file review. BCG disease was classified according to standard and revised disease classifications. Mortality was assessed at the end of the study period. Results. BCG disease was diagnosed in 25 children; 22 (88%) had local disease, and 8 (32%) had distant or disseminated disease; 5 children (20%) had both local and distant or disseminated disease. Seventeen children were HIV infected; 2 children had other immunodeficiencies. All 8 children with distant or disseminated disease were immunodeficient; 6 were HIV infected. The mortality rate was 75% for children with distant or disseminated disease. Conclusions. BCG vaccination poses a risk to infants perinatally infected with HIV and to other primary immunodeficient children. The proposed pediatric BCG disease classification reflects clinically relevant disease categories in HIV-infected children. The suggested diagnostic and treatment guidelines should improve existing case management and surveillance. Prospective evaluation of management strategies for BCG disease in HIV-infected and HIV-uninfected children is essential.
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