Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass α-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heatshock response and is related to a major antigen induced in response to anaerobic stress.

Antimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a ‘one-stop’ test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams (‘participants’) were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories.

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysidqtotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenferiae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tubernlosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium awium and Mycobacterium bowis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium waccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His,-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bowis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitm.

Background Mass drug administration (MDA) with azithromycin is the primary strategy for global trachoma control efforts. Numerous studies have reported secondary effects of MDA with azithromycin, including reductions in childhood mortality, diarrhoeal disease and malaria. Most recently, the MORDOR clinical trial demonstrated that MDA led to an overall reduction in all-cause childhood mortality in targeted communities. There is however concern about the potential of increased antimicrobial resistance in treated communities. This study evaluated the impact of azithromycin MDA on the prevalence of gastrointestinal carriage of macrolide-resistant bacteria in communities within the MORDOR Malawi study, additionally profiling changes in the gut microbiome after treatment. For faecal metagenomics, 60 children were sampled prior to treatment and 122 children after four rounds of MDA, half receiving azithromycin and half placebo. Results The proportion of bacteria carrying macrolide resistance increased after azithromycin treatment. Diversity and global community structure of the gut was minimally impacted by treatment, however abundance of several species was altered by treatment. Notably, the putative human enteropathogen Escherichia albertii was more abundant after treatment. Conclusions MDA with azithromycin increased carriage of macrolide-resistant bacteria, but had limited impact on clinically relevant bacteria. However, increased abundance of enteropathogenic Escherichia species after treatment requires further, higher resolution investigation. Future studies should focus on the number of treatments and administration schedule to ensure clinical benefits continue to outweigh costs in antimicrobial resistance carriage. Trial registration ClinicalTrial.gov, NCT02047981. Registered January 29th 2014, https://clinicaltrials.gov/ct2/show/NCT02047981

Objectives: People living with HIV (PLWH) are at increased risk of infections with resistant organisms due to more frequent healthcare utilization. Our objective was to investigate the association between HIV and antimicrobial resistance (AMR). Methods: We searched MEDLINE, EMBASE, Web of Science, LILACS and African Journals Online. Studies were eligible if they reported on AMR for colonization or infection with bacterial pathogens (excluding mycobacteria and bacteria causing sexually transmitted infections) and were stratified by HIV status, species and antimicrobials tested. Pooled odds ratios were used to evaluate the association between HIV and resistance. Results: In total, 92 studies published between 1995 and 2020 were identified. The studies included the following organisms: Staphylococcus aureus (n ¼ 47), Streptococcus pneumoniae (n ¼ 28), Escherichia coli (n ¼ 6) and other Gram-negative bacteria. PLWH had a 2.12 (95%CI 1.36e3.30) higher odds for colonization and 1.90 (95%CI 1.45e2.48) higher odds for infection with methicillin-resistant S. aureus, a 2.28 (95%CI 1.75e2.97) higher odds of infection with S. pneumoniae with decreased penicillin susceptibility, and a 1.59 (95%CI 0.83-3.05) higher odds of resistance to third-generation cephalosporins in E. coli and Klebsiella pneumoniae. Conclusion: This review shows an increased risk of AMR in PLWH across a range of bacterial pathogens and multiple drug classes. The lack of laboratory capacity for identifying AMR, and limited access to alternative treatment options in countries with the highest burden of HIV, highlight the need for more research on AMR in PLWH. Overall, the quality of studies was moderate or low, which may impact the findings of this review.