Silke Feuerriegel

Bacterial factors favoring the unprecedented multidrug-resistant tuberculosis (MDR-TB) epidemic in the former Soviet Union remain unclear. We utilized whole genome sequencing and Bayesian statistics to analyze the evolutionary history, temporal emergence of resistance and transmission networks of MDR Mycobacterium tuberculosis complex isolates from Karakalpakstan, Uzbekistan (2001–2006). One clade (termed Central Asian outbreak, CAO) dating back to 1974 (95% HPD 1969–1982) subsequently acquired resistance mediating mutations to eight anti-TB drugs. Introduction of standardized WHO-endorsed directly observed treatment, short-course in Karakalpakstan in 1998 likely selected for CAO-strains, comprising 75% of sampled MDR-TB isolates in 2005/2006. CAO-isolates were also identified in a published cohort from Russia (2008–2010). Similarly, the presence of mutations supposed to compensate bacterial fitness deficits was associated with transmission success and higher drug resistance rates. The genetic make-up of these MDR-strains threatens the success of both empirical and standardized MDR-TB therapies, including the newly WHO-endorsed short MDR-TB regimen in Uzbekistan.

Bacterial factors favoring the unprecedented multidrug-resistant tuberculosis (MDR-TB) epidemic in the former Soviet Union remain unclear. We utilized whole genome sequencing and Bayesian statistics to analyze the evolutionary history, temporal emergence of resistance and transmission networks of MDR-MTBC strains from Karakalpakstan, Uzbekistan (2001-2006). One MTBC-clone (termed Central Asian outbreak, CAO) with resistance mediating mutations to eight anti-TB drugs existed prior the worldwide introduction of standardized WHO-endorsed directly observed treatment, short-course (DOTS). DOTS implementation in Karakalpakstan in 1998 likely selected for these CAO-strains, comprising 75% of sampled MDR-TB strains in 2005/2006. CAO-strains were also identified in a previously published cohort from Samara, Russia (2008-2010). Similarly, transmission success and resistance development was linked to mutations compensating fitness deficits associated with rifampicin resistance. The genetic make-up of these outbreak clades threatens the success of both empirical and standardized guideline driven MDR-TB therapies, including the newly WHO-endorsed short MDR-TB regimen in Uzbekistan.

Rapid and accurate detection of antibiotic resistance in pathogens is an urgent need, affecting both patient care and population-scale control. Microbial genome sequencing promises much, but many barriers exist to its routine deployment. Here, we address these challenges, using a de Bruijn graph comparison of clinical isolate and curated knowledge-base to identify species and predict resistance profile, including minor populations. This is implemented in a package, Mykrobe predictor, for S. aureus and M. tuberculosis, running in under three minutes on a laptop from raw data. For S. aureus, we train and validate in 495/471 samples respectively, finding error rates comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.3%/99.5% across 12 drugs. For M. tuberculosis, we identify species and predict resistance with specificity of 98.5% (training/validating on 1920/1609 samples). Sensitivity of 82.6% is limited by current understanding of genetic mechanisms. We also show that analysis of minor populations increases power to detect phenotypic resistance in second-line drugs without appreciable loss of specificity. Finally, we demonstrate feasibility of an emerging single-molecule sequencing technique.

Sequence analysis of known antibiotic resistance genes of the Mycobacterium tuberculosis complex (MTBC) is increasingly being used to infer phenotypic resistance to a variety of antibiotics. However, a clear understanding of the genotype–phenotype relationship is required to interpret genotypic susceptibility results accurately. In this context, it is particularly important to distinguish phylogenetically informative neutral polymorphisms from true resistance-conferring mutations. Using a collection of 71 strains that encompasses all major MTBC genotypes, we mapped the genetic diversity in 18 genes that are known to be involved or were previously implicated in antibiotic resistance to eight current as well as two novel antibiotics. This included bedaquiline, capreomycin, ethambutol, fluoroquinolones, isoniazid, PA-824, para-aminosalicylic acid, prothionamide, rifampicin and streptomycin. Moreover, we included data from one of our prior studies that focused on two of the three known pyrazinamide resistance genes. We found 58 phylogenetic polymorphisms that were markers for the genotypes M. tuberculosis Beijing, Haarlem, Latin American–Mediterranean (LAM), East African Indian (EAI), Delhi/Central Asian (CAS), Ghana, Turkey (Tur), Uganda I and II, Ural and X-type, as well as for Mycobacterium africanum genotypes West African I (WA I) and II (WA II), Mycobacterium bovis, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium microti and Mycobacterium canettii. This study represents one of the most extensive overviews of phylogenetically informative polymorphisms in known resistance genes to date, and will serve as a resource for the design and interpretation of genotypic susceptibility assays.

The ribosomal L3 protein was identified as a novel target in linezolid (LZD)-resistant Mycobacterium tuberculosis strains. Next-generation sequencing confirmed rplC T460C as the sole mutation in an LZD-resistant M. tuberculosis H37Rv strain selected in vitro . Sequencing analysis revealed the rplC T460C mutation in eight further LZD-resistant isolates (three in vitro -selected mutants and five patient isolates, including isolates from three different patients that developed LZD resistance during treatment) but in none of the susceptible control strains ( n = 84).

Drug resistance displays a problem for the therapy of Mycobacterium tuberculosis infections. For molecular resistance testing, it is essential to have precise knowledge on genomic variations involved in resistance development. However, data from high-incidence settings are only sparely available. Therefore we performed a systematic approach and analyzed a total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone for mutations in katG, rpoB, rrs, rpsL, gidB, embB, pncA and where applicable in inhA and ahpC. Of the strains investigated 50 were either mono- or poly-resistant to isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide or MDR and 47 fully susceptible strains served as controls.

Mechanisms of resistance to ethambutol in Mycobacterium tuberculosis remain inadequately described. Although there is mounting evidence that mutations of codon 306 in embB play a key role, a significant number of phenotypically ethambutol-resistant strains do not carry mutations in this codon. Here, other mutations in the embCAB operon are suggested to be involved in resistance development. The entire embCAB operon (∼10 kb) was analysed in 34 phenotypically ethambutol-resistant M. tuberculosis strains without mutations in embB306 and in 12 ethambutol-susceptible strains. Furthermore, 106 control strains were investigated for the presence of particular mutations only. Overall, 18 non-synonymous mutations in 15 distinct codons of the embCAB operon were identified in ethambutol-resistant strains but not in ethambutol-susceptible isolates. The majority occurred in the embB gene (10 distinct codons), in a 570 bp region also encompassing embB306. Mutations in embC and embA were found rarely and in most cases in combination with polymorphisms in embB. One synonymous mutation (embA 228 bp) and two non-synonymous mutations (embCVal981Leu and embCArg738Gln) were found in ethambutol-susceptible strains as well as resistant strains and were confirmed to represent phylogenetic markers for strains of the Beijing, Haarlem and Delhi/CAS genotypes, respectively. Besides mutations in embB306, mutations in embB406 and embB497 were confirmed as hot spots for genomic variation in ethambutol-resistant clinical isolates. Of all resistant strains 70.6% carry a mutation in a relatively short region in embB, which therefore represents a promising target for inclusion in molecular assays for rapid detection of ethambutol resistance.

The rapid detection of Mycobacterium tuberculosis isolates resistant to second-line drugs is crucial for the institution of appropriate treatment regimens as early as possible. Although molecular methods have successfully been used for the rapid detection of resistance to first-line drugs, there are limited data on mutations that confer resistance to second-line drugs. To address this question, we analyzed Mycobacterium tuberculosis strains resistant to ofloxacin ( n = 26) and to capreomycin and/or amikacin ( n = 48) from Uzbekistan for variations in target genes ( gyrA , gyrB , rrs , and tlyA ). Strains susceptible to ofloxacin ( n = 49) and capreomycin and/or amikacin ( n = 39) were included as controls. Mutations in gyrA or gyrB were found in 96% (25/26 strains) of the ofloxacin-resistant strains, while none of the susceptible strains displayed mutations in those two genes. The most common mutation occurred in gyrA at codon 94 (17/26 strains [65.4%]), followed by mutations at codons 90 and 91. Two strains showed a mutation in gyrB , at codons 485 and 543, respectively; both mutations have not been reported previously. The most frequent mutation in strains resistant to both amikacin and capreomycin was A1401G in rrs (34/40 strains [85.0%]). Three strains had mutations in tlyA , of which two (at codons 18 and 118) were associated with resistance to capreomycin alone. Overall, none of the 10 resistant strains (5 amikacin-resistant and capreomycin-susceptible strains) and none of the 39 susceptible control strains had mutations in the genes investigated. Our results clearly demonstrate the potential of sequence analyses of short regions of relatively few target genes for the rapid detection of resistance to second-line drugs among strains isolated from patients undergoing treatment for multidrug-resistant tuberculosis. The mechanisms that confer amikacin resistance in this setting remain unclear.

The genetic basis for multidrug resistance (mdr) due to overexpression of mdr efflux pumps in Escherichia coli usually includes alterations in genes encoding global regulators, like MarA, SoxS, and Rob. In Salmonella , in addition to these regulators, Rma, for which no homolog exists in E. coli , seems to play a role in the regulation of efflux pumps. However, the exact mechanisms remain unclear. Therefore, this study aimed at investigating the influence of the regulator Rma on the expression of mdr efflux pumps in Salmonella Hadar by overexpression of the potential activator. Overexpression of rma (about 80-fold) from a plasmid led to an increase in the minimum inhibitory concentrations (MICs) of (fluoro)quinolones (2–4-fold), chloramphenicol (3-fold), tetracycline (4-fold), and novobiocin (3-fold), which is associated with an elevated expression of the mdr efflux pumps AcrAB-TolC, AcrEF-TolC, and MdtABC (formerly known as YegMNO)-TolC (all about 15-fold) as has been demonstrated by quantitative real-time PCR experiments. To assess a putative general relevance of Rma for fluoroquinolone resistance, clinical isolates of Salmonella Typhimurium DT104 and DT204c and Salmonella Hadar, which showed elevated MICs, were investigated concerning rma and acr A gene expression levels. All clinical isolates showed an elevated expression of rma (up to 16-fold) and acr A (up to 3-fold) compared to the respective wild-type strains. Thus, the global regulator Rma is important in Salmonella concerning elevated expression of several mdr efflux pumps and hence resulting increase in antibiotic resistance.