Epidemics of drug-resistant bacteria emerge worldwide, even as resistant strains frequently have reduced fitness compared to their drug-susceptible counterparts. Data from model systems suggest that ...the fitness cost of antimicrobial resistance can be reduced by compensatory mutations; however, there is limited evidence that compensatory evolution has any significant role in the success of drug-resistant bacteria in human populations. Here we describe a set of compensatory mutations in the RNA polymerase genes of rifampicin-resistant M. tuberculosis, the etiologic agent of human tuberculosis (TB). M. tuberculosis strains harboring these compensatory mutations showed a high competitive fitness in vitro. Moreover, these mutations were associated with high fitness in vivo, as determined by examining their relative clinical frequency across patient populations. Of note, in countries with the world's highest incidence of multidrug-resistant (MDR) TB, more than 30% of MDR clinical isolates had this form of mutation. Our findings support a role for compensatory evolution in the global epidemics of MDR TB.
Deciphering physiological changes that mediate transition of Mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an ...individual host for decades. We have combined RNA sequencing (RNA-seq) of 5′ triphosphate-enriched libraries with regular RNA-seq to characterize the architecture and expression of M. tuberculosis promoters. We identified over 4,000 transcriptional start sites (TSSs). Strikingly, for 26% of the genes with a primary TSS, the site of transcriptional initiation overlapped with the annotated start codon, generating leaderless transcripts lacking a 5′ UTR and, hence, the Shine-Dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria. Genes encoding proteins with active growth functions were markedly depleted from the leaderless transcriptome, and there was a significant increase in the overall representation of leaderless mRNAs in a starvation model of growth arrest. The high percentage of leaderless genes may have particular importance in the physiology of nonreplicating M. tuberculosis.
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•A resource for the identification of in vitro active promoters in M. tuberculosis•A quarter of all genes in M. tuberculosis are expressed as leaderless mRNAs•Leaderless mRNAs are differentially associated with toxin-antitoxin modules•Abundance of leaderless mRNAs increases during starvation-induced growth arrest
In this study, Cortes, Young, and colleagues report genome-wide mapping of transcriptional start sites combined with RNA sequencing and shotgun proteomics in the human pathogen Mycobacterium tuberculosis. A striking finding is a high proportion of genes expressed in the form of leaderless transcripts lacking the Shine-Dalgarno sequence conventionally required for translation. The distribution of functional gene classes between leaderless and Shine-Dalgarno transcriptomes suggests that changes in the specificity of translation may play a role in bacterial adaptation during infection.
Nonalcoholic fatty liver disease (NAFLD) is increasingly recognized among overweight adolescents. Metabolic syndrome often coexists with NAFLD in adults. This study aimed to determine whether, in US ...adolescents, metabolic syndrome increases the odds of alanine aminotransferase (ALT)>40 U/L, a surrogate for NAFLD.
A cross-sectional study of the association between metabolic syndrome and ALT>40 U/L was undertaken in a sample of 12- to 19-year-olds from the National Health and Nutrition Examination Survey, 1999 to 2002, representative of the US adolescent population. Subjects were excluded for incomplete data, pregnancy, steroid or hepatotoxic drug use, cholestasis or viral hepatitis, and alcohol intake. The metabolic syndrome was defined by adult criteria adapted for pediatric body size and blood pressure. The association of metabolic syndrome with ALT>40 U/L, effect modifiers, and confounders were analyzed using National Health and Nutrition Examination Survey sampling weights.
Of 4902 adolescents, the 1323 included were similar to the excluded ones, except for the important variable sex (55% vs 49% male, P=0.03, respectively). The metabolic syndrome was associated with ALT>40 U/L (odds ratio OR 16.7, confidence interval CI 6.2-45.1, P<0.001) with significant interaction by sex: OR 20.4 (CI 6.2-66.7, P<0.001) for males versus 3.1 (CI 0.4-25.1, P=0.3) for females. Further stratification of males suggested interaction by ethnicity: OR 5.0 (CI 1.3-19.0, P=0.02) for Hispanics versus 34.3 (CI 7.61-55.4, P<0.001) for non-Hispanics. Among Hispanic males, adjustment for body mass index z score explained the association (OR 0.6; CI 0.1-2.9, P=0.5), whereas among non-Hispanic males, the association remained after adjustment for BMIz (OR 11.1; CI 3.2-38.0, P<0.001).
Metabolic syndrome is strongly associated with ALT>40 U/L in US male adolescents. Body mass index z score explained this association among Hispanics, but not among non-Hispanic males. Significant sex and ethnic differences exist in the association of pediatric metabolic syndrome with elevated ALT.
RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5' and 3' untranslated regions, antisense ...transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses.
Clostridium difficile has rapidly emerged as the leading cause of antibiotic-associated diarrheal disease, with the transcontinental spread of various PCR ribotypes, including 001, 017, 027 and 078. ...However, the genetic basis for the emergence of C. difficile as a human pathogen is unclear. Whole genome sequencing was used to analyze genetic variation and virulence of a diverse collection of thirty C. difficile isolates, to determine both macro and microevolution of the species. Horizontal gene transfer and large-scale recombination of core genes has shaped the C. difficile genome over both short and long time scales. Phylogenetic analysis demonstrates C. difficile is a genetically diverse species, which has evolved within the last 1.1–85 million years. By contrast, the disease-causing isolates have arisen from multiple lineages, suggesting that virulence evolved independently in the highly epidemic lineages.
Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases ...with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data.
We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation.
The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.
Inhaled corticosteroids (ICS) are the mainstay of asthma treatment, but evidence suggests a link between ICS usage and increased rates of respiratory infections. We assessed the composition of the ...asthmatic airways microbiome in asthma patients taking low and high dose ICS and the stability of the microbiome over a 2 week period.
We prospectively recruited 55 individuals with asthma. Of these, 22 were on low-dose ICS and 33 on high-dose ICS (16 on budesonide, 17 on fluticasone propionate). Sputum from each subject underwent DNA extraction, amplification and 16S rRNA gene sequencing of the bacterial component of the microbiome. 19 subjects returned for further sputum induction after 24 h and 2 weeks.
A total of 5,615,037 sequencing reads revealed 167 bacterial taxa in the asthmatic airway samples, with the most abundant being Streptococcus spp. No significant differences in sputum bacterial load or overall community composition were seen between the low- and high-dose ICS groups. However, Streptococcus spp. showed significantly higher relative abundance in subjects taking low-dose ICS (p = 0.002). Haemophilus parainfluenzae was significantly more abundant in subjects on high-dose fluticasone propionate than those on high-dose budesonide (p = 0.047). There were no statistically significant changes in microbiota composition over a 2-week period.
Whilst no significant differences were observed between the low- and high-dose ICS groups, increased abundance of the potential pathogen H. parainfluenzae was observed in patients taking high-dose fluticasone propionate compared to those taking high-dose budesonide. The microbiota were stable over fourteen days, providing novel evidence of the established community of bacteria in the asthmatic airways.
ClinicalTrials.gov NCT02671773.
Current tools available to study the molecular epidemiology of tuberculosis do not provide information about the directionality and sequence of transmission for tuberculosis cases occurring over a ...short period of time, such as during an outbreak. Recently, whole genome sequencing has been used to study molecular epidemiology of Mycobacterium tuberculosis over short time periods.
To describe the microevolution of M. tuberculosis during an outbreak caused by one drug-susceptible strain. METHOD AND MEASUREMENTS: We included 9 patients with tuberculosis diagnosed during a period of 22 months, from a population-based study of the molecular epidemiology in San Francisco. Whole genome sequencing was performed using Illumina's sequencing by synthesis technology. A custom program written in Python was used to determine single nucleotide polymorphisms which were confirmed by PCR product Sanger sequencing.
We obtained an average of 95.7% (94.1-96.9%) coverage for each isolate and an average fold read depth of 73 (1 to 250). We found 7 single nucleotide polymorphisms among the 9 isolates. The single nucleotide polymorphisms data confirmed all except one known epidemiological link. The outbreak strain resulted in 5 bacterial variants originating from the index case A1 with 0-2 mutations per transmission event that resulted in a secondary case.
Whole genome sequencing analysis from a recent outbreak of tuberculosis enabled us to identify microevolutionary events observable during transmission, to determine 0-2 single nucleotide polymorphisms per transmission event that resulted in a secondary case, and to identify new epidemiologic links in the chain of transmission.
Enhanced transcription of the Rv2660c locus in response to starvation of Mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation. ...Using strand-specific RNA sequencing, we show that the up-regulated transcript is in fact a small RNA encoded on the opposite strand to the annotated Rv2660c. The transcript originates within a prophage and is expressed only in strains that carry PhiRv2. The small RNA contains both host and phage sequences and provides a useful biomarker to monitor bacterial starvation during infection and/or non-replicating persistence. Using different approaches we do not find any evidence of Rv2660c at the level of mRNA or protein. Further efforts to understand the mechanism by which Rv2660c improves efficacy of the H56 vaccine are likely to provide insights into the pathology and immunology of tuberculosis.
Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using ...shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected.
Dominant clinically important species identified within the microbiomes included
,
and members of the
and
genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of
and high levels of
within some infants. We were able to demonstrate that in a third of the infants the
identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples.
We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.
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