Insights into transition metal homeostasis of Cupriavidus metallidurans have relied heavily on the construction and characterization of deletion mutants. To reveal the genetic consequences of these deletions, the genomes of C. metallidurans strain CH34 wild type and 34 of its mutants were analyzed. The genome of C. metallidurans wild type remained stable when the strain was strictly kept under the appropriate conditions. Omission of selection pressure or construction of mutants, however, resulted in three different kinds of mutations. Large deletions affected genomic islands or regions in the vicinity of transposon-associated genes. Some of these large deletions could be assigned to groups of overlapping deletions associated with groups of mutants, for instance, those with deletions in metal-efflux systems. Second, single-nucleotide polymorphisms (SNPs), such as point mutations, small insertions, or deletions, with a high variant frequency, were candidates for suppressor mutations, for example, in the corA1 or yidC genes. The third and novel kind of mutations comprised hypervariable regions, groups of SNPs with low variant frequencies in a small region of up to 70 base pairs that were located upstream or within genes. In the well-studied example involving the central zinc uptake regulator Zur, the hypervariable regions in the zur gene enabled the production of a GTP cyclohydrolase, restoring folate biosynthesis in double mutants. These data demonstrate that mutations de-stabilize the genome of C. metallidurans, accumulating changes that result in adaptation to the absence of the respective deleted gene, in some cases via hypervariable regions.IMPORTANCETo determine how bacteria thrive, they are often isolated from their natural environment and maintained in a laboratory. Mutations are introduced, and these derivatives are characterized phenotypically. Understanding how bacteria and their derivatives adapt to the effects of site-directed mutations, curing of plasmids, or just the laboratory environment is important. We show here that changes in the genomes of Cupriavidus metallidurans mutants occurred in all instances except when the wild type was maintained under selection conditions. The secondary mutations identified may be neutral, but some may affect the outcome of subsequent experiments performed to analyze the phenotypes. Our findings indicate that all generated mutations should undergo complete genomic sequencing. The information gained may deepen our understanding of bacterial life processes.

BACKGROUND : Until recently, multidrug-resistant TB (MDR-TB) was treated with lengthy and toxic regimens. New three-drug anti-TB regimens raise the question of whether they are sufficiently active for MDR-TB in Central Asia, an MDR-TB hotspot region. M E T H O D S : In a cohort of rifampicin-resistant (RR) and MDR-TB patients in the Kyrgyz Republic, we investigated the impact of the number of drugs that were tested susceptible by whole-genome sequencing (WGS) and conventional drug susceptibility testing (DST) and used for treatment on the treatment response, defined as 'matches'. Logistic regressions were performed to assess the effect of having >= 4 susceptible drugs in a regimen at baseline and at Month 2 on the treatment response. RESULTS: The study included 227 participants with RR/MDR-TB (30.8% female; median age 30.4 years). The age- and sex-adjusted analysis showed an association between a regimen with >= 4 WGS matches at baseline (adjusted odds ratio [aOR] 2.10, 95% CI 1.00-4.41). No association was found when using conventional DST to define matches. CON C L U SIO N : Our study confirms that the inclusion of four efficacious anti-TB drugs in an MDR-TB regimen increases the chances of a positive treatment response. Susceptibility of at least four drugs in WGS-DST predicts a positive treatment response.

Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.

AbstractBackgroundTheMycobacterium aviumcomplex (MAC) comprises the most frequent non-tuberculous mycobacteria (NTM) in Central Europe and currently includes twelve species.M. avium(MAV),M. intracellularesubsp.intracellulare(MINT), andM. intracellularesubsp.chimaera(MCH) are clinically most relevant. However, the population structure and genomic landscape of MAC linked with potential pathobiological differences remain little investigated.MethodsWhole genome sequencing (WGS) was performed on a multi-national set of MAC isolates from Germany, France, and Switzerland. Phylogenetic analysis was conducted, as well as plasmids, resistance, and virulence genes predicted from WGS data. Data was set into a global context with publicly available sequences. Finally, detailed clinical characteristics were associated with genomic data in a subset of the cohort.ResultsOverall, 610 isolates from 465 patients were included. The majority could be assigned to MAV (n = 386), MCH (n = 111), and MINT (n = 77). We demonstrate clustering with less than 12 SNPs distance of isolates obtained from different patients in all major MAC species and the identification of trans-European or even trans-continental clusters when set into relation with 1307 public sequences. However, none of our MCH isolates clustered closely with the heater-cooler unit outbreak strain Zuerich-1. Known plasmids were detected in MAV (325/1076, 30.2%), MINT (62/327, 19.0%), and almost all MCH-isolates (457/463, 98.7%). Predicted resistance to aminoglycosides or macrolides was rare. Overall, there was no direct link between phylogenomic grouping and clinical manifestations, but MCH and MINT were rarely found in patients with extra-pulmonary disease (OR 0.12 95% CI 0.04–0.28,p < 0.001 and OR 0.11 95% CI 0.02–0.4,p = 0.004, respectively) and MCH was negatively associated with fulfillment of the ATS criteria when isolated from respiratory samples (OR 0.28 95% CI 0.09-0.7, p = 0.011). With 14 out of 43 patients with available serial isolates, co-infections or co-colonizations with different strains or even species of the MAC were frequent (32.6%).ConclusionsThis study demonstrates clustering and the presence of plasmids in a large proportion of MAC isolates in Europe and in a global context. Future studies need to urgently define potential ways of transmission of MAC isolates and the potential involvement of plasmids in virulence.

The European Reference Laboratory Network for tuberculosis (TB) (ERLTB-Net), comprising TB reference laboratories from 30 European Union/European Economic Area countries, was created in 2010 to support European Centre for Disease Prevention and Control (ECDC) and countries in TB diagnostic services, alerts and response to outbreaks. One of the core activities is to build capacity on standardized methods for mycobacterial diagnosis and antimicrobial susceptibility testing (AST) (https://www.ecdc.europa.eu/en/about-us/partnerships-and-networks/disease-and-laboratory-networks/erltb-net). ERLTB-Net recently identified weaknesses in capacity building of diagnosis and AST for non-tuberculous mycobacteria (NTM) [ [1] Nikolayevskyy V. Maurer F.P. Holicka Y. Taylor L. Liddy H. Kranzer K. Novel external quality assurance scheme for drug susceptibility testing of non-tuberculous mycobacteria: a multicentre pilot study. J Antimicrob Chemother. 2019; 74: 1288-1294https://doi.org/10.1093/jac/dkz027 Crossref PubMed Scopus (8) Google Scholar ] and decided to draft guidance on best practices as well as to seek for research areas.

Objectives Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). Methods Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. Results The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. Conclusion The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

ObjectivesHeteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. InMycobacterium tuberculosis(M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the ‘gold standard’ phenotypic assay: the agar proportion method (APM).MethodsUsing two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistantM. tb, we determined the limit of detection (LOD) of known resistance associated mutations.ResultsThe LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data.ConclusionThe gold standard APM detects minority resistant populations at a lower proportion than molecular tests.Mycobacterium bovisBCG strains with defined resistance and extracted DNA fromM. tbprovided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

Heteroresistant infections - defined as infections in which minority drug-resistant (DR) populations are present - are a challenge in infectious disease control. In Mycobacterium tuberculosis , heteroresistance poses challenges in diagnosis and has been linked with poor treatment outcomes. We compared the analytic sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared to the ‘gold standard’ phenotypic assay: the agar proportion method (APM). Using defined mono-resisitant BCG strains we determined the limit of detection (LOD) of rifampin-R (RIF-R) detection was 1% using APM, 60% using Xpert MTB/RIF and 10% using Xpert MTB/RIF Ultra. To evaluate clinical WGS pipelines, a blinded panel of BCG mixtures was sent to 3 clinical labs. These were composed of either a) RIF-R plus isoniazid-R (INH-R) BCG or b) fluoroquinolone-R (FQ-R) plus clofazimine-R/bedaquiline-R (CLZ/BDQ-R) BCG. No labs called resistance at 1%; all labs called RIF-R at 10% or greater and two out of three labs reported FQ-R at 10%. Two labs were able to detect the majority population (either INH-R or CLZ/BDQ-R) at 50%. Importantly, where labs did not report resistance in the majority population, the mutations were present in the raw data but excluded from the final analysis. In conclusion, the gold standard APM more reliably detects minority resistant populations than molecular tests. Further research is required to determine whether the higher LOD of molecular tests is associated with deleterious patient outcomes and the potential effects on transmission of resistance at the population level.

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

The Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak of Mycobacterium tuberculosis complex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods.

Objectives: Since 2013, heater-cooler unit (HCU) associated Mycobacterium chimaera infections linked to a global outbreak have been described. These infections were characterised by high morbidity and mortality due to delayed diagnosis, as well as challenges in antimycobacterial and surgical therapy. This study aimed to investigate the clinical characteristics and outcome of published cases of HCU-associated M. chimaera infections.Methods: We searched PubMed and the Web of Science until 15 June 2022 for case reports, case series, and cohort studies, without language restriction, on patients with M. chimaera infection and a prior history of cardiac surgery. In this systematic review of case reports, no risk of bias assessment could be performed. Clinical, microbiological, and radiological features were recorded. Logistic regression and time-to-event analyses were performed to identify the potential factors associated with better survival.Results: One hundred eighty patients from 54 publications were included. Most patients underwent surgical aortic valve (67.0%; 118/176 of patients with available data) or combined aortic valve and root replacement (15.3%; 27/176). The median period between the time point of surgery and the first symptoms was 17 months (interquartile range 13-26 months). The overall case fatality rate was 45.5% (80/176), with a median survival of 24 months after the initiation of antimycobacterial therapy or diagnosis. A reoperation (including the removal or exchange of foreign material) was associated with better survival in multivariate logistic regression (OR 0.32 for lethal events; 95% CI 0.12-0.79; p 0.015) and in time-to-event analysis (p 0.0094).Discussion: This systematic review and meta-analysis confirm the high overall mortality of HCU -asso-ciated disseminated M. chimaera infections after cardiac surgery. A reoperation seems to be associated with better survival. Physicians have to stay aware of this infection, as patients might still be present today due to the long latency period. Nils Wetzstein, Clin Microbiol Infect 2023;29:1008 & COPY; 2023 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Background The recently published 2023 Duke-ISCVID Criteria for Infective Endocarditis for the first time consider mycobacteria (esp. Mycobacterium chimaera) as 'typical' microorganisms for prosthetic valve endocarditis (major criteria). This reflects the ongoing worldwide outbreak of M. chimaera prosthetic valve endocarditis.Case summary Our case series demonstrates a diagnostic pathway for mycobacterial endocarditis. Symptoms are unspecific, and standard microbiological testing does not result in identification of the causative agent (see Graphical Abstract); therefore patients require special microbiological and imaging diagnostics. One patient with early diagnosis and stringent antibiotic and surgical therapy survived. Two patients with disseminated infection at the time point of diagnosis had fatal outcomes.Discussion The diagnostic approach in our small retrospective case series is in line with the new modified Duke criteria and underlines the diagnostic gap in the previous definitions. Outcome of M. chimaera prosthetic valve endocarditis is related to timely diagnosis and anti-mycobacterial as well as surgical treatment. Non-tuberculous mycobacteria should be given more attention in future endocarditis guidelines.

OBJECTIVES:To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. METHODS:The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. RESULTS:We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. CONCLUSIONS:In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.

Transmission-driven multi-/extensively drug resistant (M/XDR) tuberculosis (TB) is the largest single contributor to human mortality due to antimicrobial resistance. A few major clades of the Mycobacterium tuberculosis complex belonging to lineage 2, responsible for high prevalence of MDR-TB in Eurasia, show outstanding transnational distributions. Here, we determined factors underlying the emergence and epidemic spread of the W148 clade by genome sequencing and Bayesian demogenetic analyses of 720 isolates from 23 countries. We dated a common ancestor around 1963 and identified two successive epidemic expansions in the late 1980s and late 1990s, coinciding with major socio-economic changes in the post-Soviet Era. These population expansions favored accumulation of resistance mutations to up to 11 anti-TB drugs, with MDR evolving toward additional resistances to fluoroquinolones and second-line injectable drugs within 20 years on average. Timescaled haplotypic density analysis revealed that widespread acquisition of compensatory mutations was associated with transmission success of XDR strains. Virtually all W148 strains harbored a hypervirulence-associated ppe38 gene locus, and incipient recurrent emergence of prpR mutation-mediated drug tolerance was detected. The outstanding genetic arsenal of this geographically widespread M/XDR strain clade represents a “perfect storm” that jeopardizes the successful introduction of new anti-M/XDR-TB antibiotic regimens.

Für die Auswertung der Daten, die durch die Hochdurchsatzsequenzierung von Bakterien oder Viren gewonnen werden, benötigt man spezifische bioinformatische Softwarelösungen, kompetentes Personal, eine passende IT-Infrastruktur und ein Konzept für die Datenspeicherung und -sicherheit. Am Forschungszentrum Borstel werden Next-Generation-Sequencing-Verfahren für die hochauflösende Charakterisierung von Erregergenomen und Transkriptomen aus dem Mycobacterium tuberculosis Komplex eingesetzt.

Mycobacterium tuberculosis complex (MTBC) Lineage 3 (L3) strains are abundant in world regions with the highest tuberculosis burden. To investigate the population structure and the global diversity of this major lineage, we analyzed a dataset comprising 2682 L3 strains from 38 countries over 5 continents, by employing 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping (MIRU-VNTR) and drug susceptibility testing. We further combined whole-genome sequencing (WGS) and phylogeographic analysis for 373 strains representing the global L3 genetic diversity. Ancestral state reconstruction confirmed that the origin of L3 strains is located in Southern Asia and further revealed multiple independent introduction events into North-East and East Africa. This study provides a systematic understanding of the global diversity of L3 strains and reports phylogenetic variations that could inform clinical trials which evaluate the effectivity of new drugs/regimens or vaccine candidates.

Infections due to Mycobacterium abscessus are a major cause of mortality and morbidity in cystic fibrosis (CF) patients. Furthermore, M. abscessus has been suspected to be involved in person-to-person transmissions. In 2016, dominant global clonal complexes (DCCs) that occur worldwide among CF patients have been described. To elucidate the epidemiological situation of M. abscessus among CF patients in Germany and to put these data into a global context, we performed whole-genome sequencing of a set of 154 M. abscessus isolates from 123 German patients treated in 14 CF centers. We used MTBseq pipeline to identify clusters of closely related isolates and correlate those with global findings. Genotypic drug susceptibility for macrolides and aminoglycosides was assessed by characterization of the erm(41), rrl, and rrs genes. By this approach, we could identify representatives of all major DCCs (Absc 1, Absc 2, and Mass 1) in our cohort. Intrapersonal isolates showed higher genetic relatedness than interpersonal isolates (median 3 SNPs versus 16 SNPs; P < 0.001). We further identified four clusters with German patients from same centers clustering with less than 25 SNPs distance (range 3 to 18 SNPs) but did not find any hint for in-hospital person-to-person transmission. This is the largest study investigating phylogenetic relations of M. abscessus isolates in Germany. We identified representatives of all reported DCCs but evidence for nosocomial transmission remained inconclusive. Thus, the occurrence of genetically closely related isolates of M. abscessus has to be interpreted with care, as a direct interhuman transmission cannot be directly deduced. IMPORTANCE Mycobacterium abscessus is a major respiratory pathogen in cystic fibrosis (CF) patients. Recently it has been shown that dominant global clonal complexes (DCCs) have spread worldwide among CF patients. This study investigated the epidemiological situation of M. abscessus among CF patients in Germany by performing whole-genome sequencing (WGS) of a set of 154 M. abscessus from 123 German patients treated in 14 CF centers. This is the largest study investigating the phylogenetic relationship of M. abscessus CF isolates in Germany.

Mycobacterium abscessus is an emerging multidrug-resistant non-tuberculous mycobacterium that causes a wide spectrum of infections and has caused several local outbreaks worldwide. To facilitate standardized prospective molecular surveillance, we established a novel core genome multilocus sequence typing (cgMLST) scheme. Whole genome sequencing data of 1991 isolates were employed to validate the scheme, re-analyze global population structure and set genetic distance thresholds for cluster detection and taxonomic identification. We confirmed and amended the nomenclature of the main dominant circulating clones and found that these also correlate well with traditional 7-loci MLST. Dominant circulating clones could be linked to a corresponding reference genome with less than 250 alleles while 99% of pairwise comparisons between epidemiologically linked isolates were below 25 alleles and 90% below 10 alleles. These thresholds can be used to guide further epidemiological investigations. Overall, the scheme will help to unravel the apparent global spread of certain clonal complexes and as yet undiscovered transmission routes.

Background Whole-genome sequencing (WGS) of Mycobacterium tuberculosis complex has become an important tool in diagnosis and management of drug-resistant tuberculosis. However, data correlating resistance genotype with quantitative phenotypic antimicrobial susceptibility testing (AST) are scarce. Methods In a prospective multicentre observational study, 900 clinical M tuberculosis complex isolates were collected from adults with drug-resistant tuberculosis in five high-endemic tuberculosis settings around the world (Georgia, Moldova, Peru, South Africa, and Viet Nam) between Dec 5, 2014, and Dec 12, 2017. Minimum inhibitory concentrations (MICs) and resulting binary phenotypic AST results for up to nine antituberculosis drugs were determined and correlated with resistance-conferring mutations identified by WGS. Findings Considering WHO-endorsed critical concentrations as reference, WGS had high accuracy for prediction of resistance to isoniazid (sensitivity 98.8% [95% CI 98.5-99.0]; specificity 96.6% [95% CI 95.2-97.9]), levofloxacin (sensitivity 94.8% [93.3-97.6]; specificity 97.1% [96.7-97.6]), kanamycin (sensitivity 96.1% [95.4-96.8]; specificity 95.0% [94.4-95.7]), amikacin (sensitivity 97.2% [96.4-98.1]; specificity 98.6% [98.3-98.9]), and capreomycin (sensitivity 93.1% [90.0-96.3]; specificity 98.3% [98.0-98.7]). For rifampicin, pyrazinamide, and ethambutol, the specificity of resistance prediction was suboptimal (64.0% [61.0-67.1], 83.8% [81.0-86.5], and 40.1% [37.4-42.9], respectively). Specificity for rifampicin increased to 83.9% when borderline mutations with MICs overlapping with the critical concentration were excluded. Consequently, we highlighted mutations in M tuberculosis complex isolates that are often falsely identified as susceptible by phenotypic AST, and we identified potential novel resistanceconferring mutations. Interpretation The combined analysis of mutations and quantitative phenotypes shows the potential of WGS to produce a refined interpretation of resistance, which is needed for individualised therapy, and eventually could allow differential drug dosing. However, variability of MIC data for some M tuberculosis complex isolates carrying identical mutations also reveals limitations of our understanding of the genotype and phenotype relationships (eg, including epistasis and strain genetic background).