Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are ...threatening the control of MDR tuberculosis (MDR-TB) in Eswatini.
We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009-2010; (2) MDR strains from the Nhlangano region, 2014-2017).
MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987-1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2.
The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.
Bovine tuberculosis (bTB) caused by
is a transmissible disease notifiable to the World Organization for Animal Health and to the European Union, with ongoing efforts of surveillance and eradication ...in every EU member state. In Germany, a country which has been declared officially free from bovine tuberculosis since 1997 by the EU,
infections still occur sporadically in cattle and other mammals, including humans. Here, the transmission routes of a bTB outbreak in a wildlife park in Germany affecting different cervid species, bison, lynx, and pot-bellied pigs were followed by employing whole-genome sequencing (WGS) combined with spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. One single
strain persisted from 2002 to 2015, and transmission between the park and a distantly located captive cervid farm was verified. The spoligotyping patterns remained identical, while MIRU-VNTR typing of 24 loci of the standardized panel and locus 2163a as an additional locus revealed one change at locus 2165 in a strain from a fallow deer and one at locus 2461 in isolates from red deer over the whole time period. WGS analysis confirmed the close relatedness of the isolates, with a maximum of 12 single nucleotide polymorphisms (SNPs) detected between any two sequenced isolates. In conclusion, our data confirm a longitudinal outbreak of
in a German wildlife park and provide the first insights into the dynamics of different genotyping markers in
.
Today, next-generation whole-genome sequencing (WGS) is increasingly used to determine the genetic relationships of bacteria on a nearly whole-genome level for infection control purposes and ...molecular surveillance. Here, we conducted a multicenter ring trial comprising five laboratories to determine the reproducibility and accuracy of WGS-based typing. The participating laboratories sequenced 20 blind-coded
DNA samples using 250-bp paired-end chemistry for library preparation in a single sequencing run on an Illumina MiSeq sequencer. The run acceptance criteria were sequencing outputs >5.6 Gb and Q30 read quality scores of >75%. Subsequently, spa typing, multilocus sequence typing (MLST), ribosomal MLST, and core genome MLST (cgMLST) were performed by the participants. Moreover, discrepancies in cgMLST target sequences in comparisons with the included and also published sequence of the quality control strain ATCC 25923 were resolved using Sanger sequencing. All five laboratories fulfilled the run acceptance criteria in a single sequencing run without any repetition. Of the 400 total possible typing results, 394 of the reported spa types, sequence types (STs), ribosomal STs (rSTs), and cgMLST cluster types were correct and identical among all laboratories; only six typing results were missing. An analysis of cgMLST allelic profiles corroborated this high reproducibility; only 3 of 183,927 (0.0016%) cgMLST allele calls were wrong. Sanger sequencing confirmed all 12 discrepancies of the ring trial results in comparison with the published sequence of ATCC 25923. In summary, this ring trial demonstrated the high reproducibility and accuracy of current next-generation sequencing-based bacterial typing for molecular surveillance when done with nearly completely locked-down methods.
We applied whole-genome sequencing to reconstruct the spatial and temporal dynamics underpinning the expansion of Clostridium difficile ribotype 027 in Germany. Based on re-sequencing of genomes from ...57 clinical C. difficile isolates, which had been collected from hospitalized patients at 36 locations throughout Germany between 1990 and 2012, we demonstrate that C. difficile genomes have accumulated sequence variation sufficiently fast to document the pathogen's spread at a regional scale. We detected both previously described lineages of fluoroquinolone-resistant C. difficile ribotype 027, FQR1 and FQR2. Using Bayesian phylogeographic analyses, we show that fluoroquinolone-resistant C. difficile 027 was imported into Germany at least four times, that it had been widely disseminated across multiple federal states even before the first outbreak was noted in 2007, and that it has continued to spread since.
•Solute transport modeling of the Soultz-sous-Forêts EGS•Joint analysis of different hydraulic and geophysical experiments•Hydraulic characterization of the pathways between the wells within the ...reservoir•Reevaluation of the fracture-controlled flow field in the EGS
The Enhanced Geothermal System in Soultz-sous-Forêts, located in the geothermal favorable Upper Rhine Graben, is a fracture-controlled reservoir that was highly investigated in the last decades generating a huge geoscientific database. Numerical reservoir models use this database to simulate the operation of the subsurface heat exchanger, yet suffer from simplifications regarding the transfer of experimental into model data, dimensional extension, and computational power and efficiency. The new extensive transient 3D simulations, based on geophysical, geological and hydraulic data, highlight the hydraulic and transport feedback of the Soultz EGS due to convective and advective fluid flow. Developed with the goal of simulating the vast tracer test data during the reservoir-testing phase in 2005, the finite element model is focusing on the main fractured zones, which connect the wells in the deep reservoir. It comprises 13 major hydraulically active faults and fractures in a 13 × 11 x 5 km extending model domain, as well as open-hole sections of the wells GPK1 to GPK4 and their casing leakages. The simulation of the tracer experiment confirms the strong heterogeneity of the reservoir and highlights the importance of a potential fractured zone, hydraulically separating the reservoir in a northern (GPK1 to 3) and southern section (GPK4). This zone tends to connect the reservoir to the main fault system by hydraulically separating GPK4 from the other wells. The calibration and sensitivity analyses provide a unique, broad understanding of the reservoir flow zones providing information on the extension of the Soultz reservoir in the future and on the fluid pathways in the deep subsurface of the Upper Rhine Graben.
Since 2013, over 100 cases of Mycobacterium chimaera prosthetic valve endocarditis and disseminated disease were notified in Europe and the USA, linked to contaminated heater–cooler units (HCUs) used ...during cardiac surgery. We did a molecular epidemiological investigation to establish the source of these patients' disease.
We included 24 M chimaera isolates from 21 cardiac surgery-related patients in Switzerland, Germany, the Netherlands, and the UK, 218 M chimaera isolates from various types of HCUs in hospitals, from LivaNova (formerly Sorin; London, UK) and Maquet (Rastatt, Germany) brand HCU production sites, and unrelated environmental sources and patients, as well as eight Mycobacterium intracellulare isolates. Isolates were analysed by next-generation whole-genome sequencing using Illumina and Pacific Biosciences technologies, and compared with published M chimaera genomes.
Phylogenetic analysis based on whole-genome sequencing of 250 isolates revealed two major M chimaera groups. Cardiac surgery-related patient isolates were all classified into group 1, in which all, except one, formed a distinct subgroup. This subgroup also comprised isolates from 11 cardiac surgery-related patients reported from the USA, most isolates from LivaNova HCUs, and one from their production site. Isolates from other HCUs and unrelated patients were more widely distributed in the phylogenetic tree.
HCU contamination with M chimaera at the LivaNova factory seems a likely source for cardiothoracic surgery-related severe M chimaera infections diagnosed in Switzerland, Germany, the Netherlands, the UK, the USA, and Australia. Protective measures and heightened clinician awareness are essential to guarantee patient safety.
Partly funded by the EU Horizon 2020 programme, its FP7 programme, the German Center for Infection Research (DZIF), the Swiss National Science Foundation, the Swiss Federal Office of Public Health, and National Institute of Health Research Oxford Health Protection Research Units on Healthcare Associated Infection and Antimicrobial Resistance.
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).
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