The ESX-4 substrates, EsxU and EsxT, modulate Mycobacterium abscessus fitness.

ESX type VII secretion systems are complex secretion machineries spanning across the mycobacterial membrane and play an important role in pathogenicity, nutrient uptake and conjugation. We previously reported the role of ESX-4 in modulating Mycobacterium abscessus intracellular survival. The loss of EccB4 was associated with limited secretion of two effector proteins belonging to the WXG-100 family, EsxU and EsxT, and encoded by the esx-4 locus. This prompted us to investigate the function of M. abscessus EsxU and EsxT in vitro and in vivo. Herein, we show that EsxU and EsxT are substrates of ESX-4 and form a stable 1:1 heterodimer that permeabilizes artificial membranes. While expression of esxU and esxT was up-regulated in M. abscessus-infected macrophages, their absence in an esxUT deletion mutant prevented phagosomal membrane disruption while maintaining M. abscessus in an unacidified phagosome. Unexpectedly, the esxUT deletion was associated with a hyper-virulent phenotype, characterised by increased bacterial loads and mortality in mouse and zebrafish infection models. Collectively, these results demonstrate that the presence of EsxU and EsxT dampens survival and persistence of M. abscessus during infection.

Treatment of MDR, pre-XDR, XDR and rifampicin-resistant tuberculosis or in case of intolerance to at least rifampicin in Austria, Germany and Switzerland - Amendment to the 2022 consensus-based guideline: Tuberculosis in adulthood by the German Central Committee against Tuberculosis (DZK) on behalf of the German Respiratory Society (DGP) dated 19.09.2023.

Based on the assessment of new evidence, the World Health Organization (WHO) updated its guidelines for the treatment of drug-resistant tuberculosis (TB) in December 2022. The new recommendations and the latest study data made it necessary to update the existing guideline on the treatment of at least rifampicin- (RR-TB) for the German-speaking countries, replacing the respective chapters of the treatment guidelines published 2022. A shortened treatment of proven RR-TB and multidrug-resistant (MDR)-TB for at least 6 months using the fixed and non-modifiable drug combination of bedaquiline, pretomanid, linezolid, and moxifloxacin (BPaLM) is now also recommended for Austria, Germany, and Switzerland under certain conditions considering the existing barriers for the implementation of the new treatment regimen. For the treatment of pre-extensively drug-resistant (pre-XDR)-TB, an individualized treatment for 18 months continues to be the primary recommendation. The non-modifiable drug combination of bedaquiline, pretomanid, and linezolid (BPaL) may be used alternatively in selected pre-XDR-TB cases, provided that all prerequisites are met. The necessary requirements for using BPaLM and BPaL are presented in detail in this amendment to the consensus-based TB treatment guideline for adult patients.

[Treatment of MDR, pre-XDR, XDR and rifampicin resistant tuberculosis or in case of intolerance to at least rifampicin in Austria, Germany and Switzerland - Amendment dated 19.09.2023 to the Sk2-Guideline: Tuberculosis in adulthood of the German Central Committee against Tuberculosis (DZK) on behalf of the German Respiratory Society (DGP)]. / Therapie bei MDR-, prä-XDR-, XDR-Tuberkulose und Rifampicin-Resistenz oder bei Medikamentenunverträglichkeit gegenüber mindestens Rifampicin.

In December 2022, based on the assessment of new evidence, the World Health Organization (WHO) updated its guidelines for the treatment of drug-resistant tuberculosis (TB). The evaluation of both, these recommendations, and the latest study data, makes it necessary to update the existing guidelines on the treatment of at least rifampicin-resistant tuberculosis for the German-speaking region, hereby replacing the respective chapters. A shortened MDR-TB treatment of at least 6 month using the fixed and non-modifiable drug combination of bedaquiline, pretomanid, linezolid, and moxifloxacin (BPaLM) is now also recommended for Germany, Austria, and Switzerland under certain conditions. This recommendation applies to TB cases with proven rifampicin resistance, including rifampicin monoresistance. For treatment of pre-extensively drug resistant TB (pre-XDR-TB), an individualized treatment for 18 months adjusted to resistance data continues to be the primary recommendation. The non-modifiable drug combination of bedaquiline, pretomanid, and linezolid (BPaL) may be used alternatively in pre-XDR TB if all prerequisites are met. The necessary prerequisites for the use of BPaLM and BPaL are presented in this amendment to the S2k guideline for 'Tuberculosis in adulthood'.

Novel Synergies and Isolate Specificities in the Drug Interaction Landscape of Mycobacterium abscessus.

Mycobacterium abscessus infections are difficult to treat and are often considered untreatable without tissue resection. Due to the intrinsic drug-resistant nature of the bacteria, combination therapy of three or more antibiotics is recommended. A major challenge in treating M. abscessus infections is the absence of a universal combination therapy with satisfying clinical success rates, leaving clinicians to treat infections using antibiotics lacking efficacy data. We systematically measured drug combinations in M. abscessus to establish a resource of drug interaction data and identify patterns of synergy to help design optimized combination therapies. We measured 191 pairwise drug combination effects among 22 antibacterials and identified 71 synergistic pairs, 54 antagonistic pairs, and 66 potentiator-antibiotic pairs. We found that commonly used drug combinations in the clinic, such as azithromycin and amikacin, are antagonistic in the lab reference strain ATCC 19977, whereas novel combinations, such as azithromycin and rifampicin, are synergistic. Another challenge in developing universally effective multidrug therapies for M. abscessus is the significant variation in drug response between isolates. We measured drug interactions in a focused set of 36 drug pairs across a small panel of clinical isolates with rough and smooth morphotypes. We observed strain-dependent drug interactions that cannot be predicted from single-drug susceptibility profiles or known drug mechanisms of action. Our study demonstrates the immense potential to identify synergistic drug combinations in the vast drug combination space and emphasizes the importance of strain-specific combination measurements for designing improved therapeutic interventions.

Emergence of bedaquiline resistance in a high tuberculosis burden country.

RATIONALE: Bedaquiline has been classified as a group A drug for the treatment of multidrug-resistant tuberculosis (MDR-TB) by the World Health Organization; however, globally emerging resistance threatens the effectivity of novel MDR-TB treatment regimens. OBJECTIVES: We analysed pre-existing and emerging bedaquiline resistance in bedaquiline-based MDR-TB therapies, and risk factors associated with treatment failure and death. METHODS: In a cross-sectional cohort study, we employed patient data, whole-genome sequencing (WGS) and phenotyping of Mycobacterium tuberculosis complex (MTBC) isolates. We could retrieve baseline isolates from 30.5% (62 out of 203) of all MDR-TB patients who received bedaquiline between 2016 and 2018 in the Republic of Moldova. This includes 26 patients for whom we could also retrieve a follow-up isolate. MEASUREMENTS AND MAIN RESULTS: At baseline, all MTBC isolates were susceptible to bedaquiline. Among 26 patients with available baseline and follow-up isolates, four (15.3%) patients harboured strains which acquired bedaquiline resistance under therapy, while one (3.8%) patient was re-infected with a second bedaquiline-resistant strain. Treatment failure and death were associated with cavitary disease (p=0.011), and any additional drug prescribed in the bedaquiline-containing regimen with WGS-predicted resistance at baseline (OR 1.92 per unit increase, 95% CI 1.15-3.21; p=0.012). CONCLUSIONS: MDR-TB treatments based on bedaquiline require a functional background regimen to achieve high cure rates and to prevent the evolution of bedaquiline resistance. Novel MDR-TB therapies with bedaquiline require timely and comprehensive drug resistance monitoring.

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid.

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.

Insertion and deletion evolution reflects antibiotics selection pressure in a Mycobacterium tuberculosis outbreak.

In genome evolution, genetic variants are the source of diversity, which natural selection acts upon. Treatment of human tuberculosis (TB) induces a strong selection pressure for the emergence of antibiotic resistance-conferring variants in the infecting Mycobacterium tuberculosis (MTB) strains. MTB evolution in response to treatment has been intensively studied and mainly attributed to point substitutions. However, the frequency and contribution of insertions and deletions (indels) to MTB genome evolution remains poorly understood. Here, we analyzed a multi-drug resistant MTB outbreak for the presence of high-quality indels and substitutions. We find that indels are significantly enriched in genes conferring antibiotic resistance. Furthermore, we show that indels are inherited during the outbreak and follow a molecular clock with an evolutionary rate of 5.37e-9 indels/site/year, which is 23 times lower than the substitution rate. Inherited indels may co-occur with substitutions in genes along related biological pathways; examples are iron storage and resistance to second-line antibiotics. This suggests that epistatic interactions between indels and substitutions affect antibiotic resistance and compensatory evolution in MTB.

Integrated use of laboratory services for multiple infectious diseases in the WHO European Region during the COVID-19 pandemic and beyond.

Technical advances in diagnostic techniques have permitted the possibility of multi-disease-based approaches for diagnosis and treatment monitoring of several infectious diseases, including tuberculosis (TB), human immunodeficiency virus (HIV), viral hepatitis and sexually transmitted infections (STI). However, in many countries, diagnosis and monitoring, as well as disease response programs, still operate as vertical systems, potentially causing delay in diagnosis and burden to patients and preventing the optimal use of available resources. With countries facing both human and financial resource constraints, during the COVID-19 pandemic even more than before, it is important that available resources are used as efficiently as possible, potential synergies are leveraged to maximise benefit for patients, continued provision of essential health services is ensured. For the infectious diseases, TB, HIV, hepatitis C (HCV) and STI, sharing devices and integrated services starting with rapid, quality-assured, and complete diagnostic services is beneficial for the continued development of adequate, efficient and effective treatment strategies. Here we explore the current and future potential (as well as some concerns), importance, implications and necessary implementation steps for the use of platforms for multi-disease testing for TB, HIV, HCV, STI and potentially other infectious diseases, including emerging pathogens, using the example of the COVID-19 pandemic.

[Tuberculosis in adulthood - The Sk2-Guideline of the German Central Committee against Tuberculosis (DZK) and the German Respiratory Society (DGP) for the diagnosis and treatment of adult tuberculosis patients]. / Tuberkulose im Erwachsenenalter.

In Germany tuberculosis is a rare disease and usually well treatable. Worldwide it is one of the most common infectious diseases with approximately 10 million new cases every year. Even with low incidences in Germany, tuberculosis is an important differential diagnosis especially due to international developments and migration movements. With a decreasing experience there's a continuous demand on accurate and up-to-date information. This guideline covers all aspects of microbiological diagnostics, basic principles of standard therapy, treatment of extrapulmonary tuberculosis, management of side effects, special features of diagnosis and treatment of resistant tuberculosis, and treatment in TB-HIV coinfection. Also, it explains when treatment in specialized centers is required, aspects of care and legal regulations and the diagnosis and preventive therapy of latent tuberculosis infection. The update of the S2k guideline "Tuberculosis in Adults" is intended to serve as a guideline for prevention, diagnosis, and treatment of tuberculosis for all those involved in tuberculosis care and to help meet the current challenges in dealing with tuberculosis in Germany.

Design of Multidrug-Resistant Tuberculosis Treatment Regimens Based on DNA Sequencing.

BACKGROUND: Comprehensive and reliable drug susceptibility testing (DST) is urgently needed to provide adequate treatment regimens for patients with multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). We determined whether next-generation sequencing (NGS) analysis of Mycobacterium tuberculosis complex isolates and genes implicated in drug resistance can guide the design of effective MDR/RR-TB treatment regimens. METHODS: NGS-based genomic DST predictions of M. tuberculosis complex isolates from MDR/RR-TB patients admitted to a TB reference center in Germany between 1 January 2015 and 30 April 2019 were compared with phenotypic DST results of mycobacteria growth indicator tubes (MGIT). Standardized treatment algorithms were applied to design individualized therapies based on either genomic or phenotypic DST results, and discrepancies were further evaluated by determination of minimal inhibitory drug concentrations (MICs) using Sensititre MYCOTBI and UKMYC microtiter plates. RESULTS: In 70 patients with MDR/RR-TB, agreement among 1048 pairwise comparisons of genomic and phenotypic DST was 86.3%; 76 (7.2%) results were discordant, and 68 (6.5%) could not be evaluated due to the presence of polymorphisms with yet unknown implications for drug resistance. Importantly, 549 of 561 (97.9%) predictions of drug susceptibility were phenotypically confirmed in MGIT, and 27 of 64 (42.2%) false-positive results were linked to previously described mutations mediating a low or moderate MIC increase. Virtually all drugs (99.0%) used in combination therapies that were inferred from genomic DST were confirmed to be susceptible by phenotypic DST. CONCLUSIONS: NGS-based genomic DST can reliably guide the design of effective MDR/RR-TB treatment regimens.

Secretome characterization of clinical isolates from the Mycobacterium abscessus complex provides insight into antigenic differences.

BACKGROUND: Mycobacterium abscessus (MAB) is a widely disseminated pathogenic non-tuberculous mycobacterium (NTM). Like with the M. tuberculosis complex (MTBC), excreted / secreted (ES) proteins play an essential role for its virulence and survival inside the host. Here, we used a robust bioinformatics pipeline to predict the secretome of the M. abscessus ATCC 19977 reference strain and 15 clinical isolates belonging to all three MAB subspecies, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. RESULTS: We found that ~ 18% of the proteins encoded in the MAB genomes were predicted as secreted and that the three MAB subspecies shared > 85% of the predicted secretomes. MAB isolates with a rough (R) colony morphotype showed larger predicted secretomes than isolates with a smooth (S) morphotype. Additionally, proteins exclusive to the secretomes of MAB R variants had higher antigenic densities than those exclusive to S variants, independent of the subspecies. For all investigated isolates, ES proteins had a significantly higher antigenic density than non-ES proteins. We identified 337 MAB ES proteins with homologues in previously investigated M. tuberculosis secretomes. Among these, 222 have previous experimental support of secretion, and some proteins showed homology with protein drug targets reported in the DrugBank database. The predicted MAB secretomes showed a higher abundance of proteins related to quorum-sensing and Mce domains as compared to MTBC indicating the importance of these pathways for MAB pathogenicity and virulence. Comparison of the predicted secretome of M. abscessus ATCC 19977 with the list of essential genes revealed that 99 secreted proteins corresponded to essential proteins required for in vitro growth. CONCLUSIONS: This study represents the first systematic prediction and in silico characterization of the MAB secretome. Our study demonstrates that bioinformatics strategies can help to broadly explore mycobacterial secretomes including those of clinical isolates and to tailor subsequent, complex and time-consuming experimental approaches accordingly. This approach can support systematic investigation exploring candidate proteins for new vaccines and diagnostic markers to distinguish between colonization and infection. All predicted secretomes were deposited in the Secret-AAR web-server ( http://microbiomics.ibt.unam.mx/tools/aar/index.php ).

Microevolution of Mycobacterium tuberculosis Subpopulations and Heteroresistance in a Patient Receiving 27 Years of Tuberculosis Treatment in Germany.

Preexisting and newly emerging resistant pathogen subpopulations (heteroresistance) are potential risk factors for treatment failure of multi/extensively drug resistant (MDR/XDR) tuberculosis (TB). Intrapatient evolutionary dynamics of Mycobacterium tuberculosis complex (Mtbc) strains and their implications on treatment outcomes are still not completely understood. To elucidate how Mtbc strains escape therapy, we analyzed 13 serial isolates from a German patient by whole-genome sequencing. Sequencing data were compared with phenotypic drug susceptibility profiles and the patient's collective 27-year treatment history to further elucidate factors fostering intrapatient resistance evolution. The patient endured five distinct TB episodes, ending in resistance to 16 drugs and a nearly untreatable XDR-TB infection. The first isolate obtained, during the patient's 5th TB episode, presented fixed resistance mutations to 7 anti-TB drugs, including isoniazid, rifampin, streptomycin, pyrazinamide, prothionamide, para-aminosalicylic acid, and cycloserine-terizidone. Over the next 13 years, a dynamic evolution with coexisting, heterogeneous subpopulations was observed in 6 out of 13 sequential bacterial isolates. The emergence of drug-resistant subpopulations coincided with frequent changes in treatment regimens, which often included two or fewer active compounds. This evolutionary arms race between competing subpopulations ultimately resulted in the fixation of a single XDR variant. Our data demonstrate the complex intrapatient microevolution of Mtbc subpopulations during failing MDR/XDR-TB treatment. Designing effective treatment regimens based on rapid detection of (hetero) resistance is key to avoid resistance development and treatment failure.

Conserved and specialized functions of Type VII secretion systems in non-tuberculous mycobacteria.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in Mycobacterium smegmatis, to ESX-5 structures in Mycobacterium xenopi and Mycobacterium tuberculosis. In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of Mycobacterium abscessus; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.

Use of a whole genome sequencing-based approach for Mycobacterium tuberculosis surveillance in Europe in 2017-2019: an ECDC pilot study.

Whole genome sequencing (WGS) can be used for molecular typing and characterisation of Mycobacterium tuberculosis complex (MTBC) strains. We evaluated the systematic use of a WGS-based approach for MTBC surveillance involving all European Union/European Economic Area (EU/EEA) countries and highlight the challenges and lessons learnt to be considered for the future development of a WGS-based surveillance system.WGS and epidemiological data of patients with rifampicin-resistant (RR) and multidrug-resistant (MDR) tuberculosis (TB) were collected from EU/EEA countries between January 2017 and December 2019. WGS-based genetic relatedness analysis was performed using a standardised approach including both core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP)-based calculation of distances on all WGS data that fulfilled minimum quality criteria to ensure data comparability.A total of 2218 RR/MDR-MTBC isolates were collected from 25 countries. Among these, 56 cross-border clusters with increased likelihood of recent transmission (≤5 SNPs distance) comprising 316 RR/MDR-MTBC isolates were identified. The cross-border clusters included between two and 30 resistant isolates from two to six countries, demonstrating different RR/MDR-TB transmission patterns in Western and Eastern EU countries.This pilot study shows that a WGS-based surveillance system is not only feasible but can efficiently elucidate the dynamics of in-country and cross-border RR/MDR-TB transmission across EU/EEA countries. Lessons learnt from this study highlight that the establishment of an EU/EEA centralised WGS-based surveillance system for TB will require strengthening of national integrated systems performing prospective WGS surveillance and the development of clear procedures to facilitate international collaboration for the investigation of cross-border clusters.

Prediction of anti-tuberculosis treatment duration based on a 22-gene transcriptomic model.

BACKGROUND: The World Health Organization recommends standardised treatment durations for patients with tuberculosis (TB). We identified and validated a host-RNA signature as a biomarker for individualised therapy durations for patients with drug-susceptible (DS)- and multidrug-resistant (MDR)-TB. METHODS: Adult patients with pulmonary TB were prospectively enrolled into five independent cohorts in Germany and Romania. Clinical and microbiological data and whole blood for RNA transcriptomic analysis were collected at pre-defined time points throughout therapy. Treatment outcomes were ascertained by TBnet criteria (6-month culture status/1-year follow-up). A whole-blood RNA therapy-end model was developed in a multistep process involving a machine-learning algorithm to identify hypothetical individual end-of-treatment time points. RESULTS: 50 patients with DS-TB and 30 patients with MDR-TB were recruited in the German identification cohorts (DS-GIC and MDR-GIC, respectively); 28 patients with DS-TB and 32 patients with MDR-TB in the German validation cohorts (DS-GVC and MDR-GVC, respectively); and 52 patients with MDR-TB in the Romanian validation cohort (MDR-RVC). A 22-gene RNA model (TB22) that defined cure-associated end-of-therapy time points was derived from the DS- and MDR-GIC data. The TB22 model was superior to other published signatures to accurately predict clinical outcomes for patients in the DS-GVC (area under the curve 0.94, 95% CI 0.9-0.98) and suggests that cure may be achieved with shorter treatment durations for TB patients in the MDR-GIC (mean reduction 218.0 days, 34.2%; p<0.001), the MDR-GVC (mean reduction 211.0 days, 32.9%; p<0.001) and the MDR-RVC (mean reduction of 161.0 days, 23.4%; p=0.001). CONCLUSION: Biomarker-guided management may substantially shorten the duration of therapy for many patients with MDR-TB.

Evaluation of the Roche cobas MTB and MTB-RIF/INH Assays in Samples from Germany and Sierra Leone.

The Roche cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens. A total of 325 frozen, leftover respiratory samples collected from treatment-naive patients with presumptive TB in Germany (n = 280) and presumptive RIF-resistant TB in Sierra Leone (n = 45) were used in this study. cobas MTB results for detection of MTBC DNA from N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH)-treated samples were compared to culture results. Predictions of RIF and INH resistance by the cobas MTB-RIF/INH assay were compared to a composite reference standard (phenotypic drug susceptibility testing and line probe assay). Whole-genome sequencing was used to resolve discordances. The overall sensitivity of cobas MTB for detection of MTBC DNA in culture-positive samples (n = 102) was 89.2% (95% confidence interval [CI], 81.7 to 93.9%). The specificity of cobas MTB was 98.6% (95% CI, 96.1 to 99.5%). Sensitivity and specificity for detection of RIF and INH resistance were 88.4% (95% CI, 75.5 to 94.9%) and 97.6% (95% CI, 87.4 to 99.6%) and 76.6% (95% CI, 62.8 to 86.4%) and 100.0% (95% CI, 90.8 to 100.0%), respectively. Discordant results for RIF and INH resistance were mainly due to uncommon mutations in samples from Sierra Leone that were not covered by the cobas MTB-RIF/INH assay. In conclusion, cobas MTB and MTB-RIF/INH assays provide accurate detection of MTBC DNA and resistance-associated mutations in respiratory samples. The influence of regional variations in the prevalence of resistance-conferring mutations requires further investigation.

Emergence of linezolid-resistance in vancomycin-resistant Enterococcus faecium ST117 associated with increased linezolid-consumption.

OBJECTIVE: We aim to describe the epidemiological, clinical and microbiological characteristics of the linezolid- and vancomycin- resistant Enterococcus faecium (LVRE) in a tertiary care hospital in Germany. METHODS: We conducted a retrospective analysis of 196 LVRE cases observed from 1st January 2012 to 31th December 2018. Patients' medical charts were reviewed and available LVRE (n = 102) were subjected to whole-genome-sequencing. Antibiotic consumption was measured in defined daily dose (DDD)/100 bed-days (BD). RESULTS: The prevalence of LVRE isolates among VRE was 6.3 % in 2018. Most patients had an onco-hematological disease (134/196, 68.4 %). From 2012-2018 an increase of +356.7 % of linezolid defined daily dose/100 bed-days was observed. In 71.4 % (90/126, 70 missing values) of the patients, linezolid was prescribed in the previous 6 months. The median exposure to linezolid was 15 days (Interquartile, IQR 9-23). 42/196 (21.4 %) patients had an LVRE-related infection with an overall 30-day mortality rate of 33 %. In 121/196 (61.7 %) patients, linezolid-susceptible VREfm were isolated before LVRE, suggesting secondary acquisition of linezolid resistance. Genetic analysis revealed that most isolates belonged to ST117 (64/102 available isolates, 62.7 %). The G2576T 23S rDNA mutation was identified as the most common resistance mechanism (96/102, 94.1 %). poxtA was identified in two isolates, while cfr, and optrA were not detected. CONCLUSIONS: Incidence of LVRE related to 23S rDNA mutations is rising and probably associated with antibiotic consumption. Restrictions in the use of linezolid may be needed in order to retain therapeutic options in VRE.

Impact of the COVID-19 pandemic on tuberculosis national reference laboratory services in the WHO European Region, March to November 2020.

We assessed the impact of COVID-19 on diagnostic services for tuberculosis (TB) by national reference laboratories in the WHO European Region. Of 35 laboratories, 30 reported declines in TB sample numbers, amounting up to > 50% of the pre-COVID-19 volumes. Sixteen reported reagent or consumable shortages. Nineteen reallocated ressources to SARS-CoV-2 testing, resulting in an overall increase in workload, largely without a concomitant increase in personnel (n = 14). This poses a risk to meeting the 2025 milestones of the End TB Strategy.

Improving tuberculosis surveillance by detecting international transmission using publicly available whole genome sequencing data.

IntroductionImproving the surveillance of tuberculosis (TB) is especially important for multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. The large amount of publicly available whole genome sequencing (WGS) data for TB gives us the chance to re-use data and to perform additional analyses at a large scale.AimWe assessed the usefulness of raw WGS data of global MDR/XDR Mycobacterium tuberculosis isolates available from public repositories to improve TB surveillance.MethodsWe extracted raw WGS data and the related metadata of M. tuberculosis isolates available from the Sequence Read Archive. We compared this public dataset with WGS data and metadata of 131 MDR- and XDR M. tuberculosis isolates from Germany in 2012 and 2013.ResultsWe aggregated a dataset that included 1,081 MDR and 250 XDR isolates among which we identified 133 molecular clusters. In 16 clusters, the isolates were from at least two different countries. For example, Cluster 2 included 56 MDR/XDR isolates from Moldova, Georgia and Germany. When comparing the WGS data from Germany with the public dataset, we found that 11 clusters contained at least one isolate from Germany and at least one isolate from another country. We could, therefore, connect TB cases despite missing epidemiological information.ConclusionWe demonstrated the added value of using WGS raw data from public repositories to contribute to TB surveillance. Comparing the German with the public dataset, we identified potential international transmission events. Thus, using this approach might support the interpretation of national surveillance results in an international context.

TREATMENT OF MYCOBACTERIOSIS CAUSED BY MYCOBACTERIUM AVIUM SSP. HOMINISSUIS IN A GROUP OF CAPTIVE LOWLAND TAPIRS (TAPIRUS TERRESTRIS).

Tapirs are a taxonomic group with a high susceptibility to mycobacterial diseases. However, successful therapy has only been documented sporadically. Here treatment of mycobacteriosis diagnosed in three, one male and two female, lowland tapirs (Tapirus terrestris) in a zoo in Germany is reported. Two of the animals showed chronic mild respiratory signs, and conventional therapy did not improve the condition. Culture of broncho-alveolar lavage (BAL) samples was positive for Mycobacterium avium ssp. hominissuis. Upon airway endoscopy, bronchial edema and increased mucus production were visible. Initially, all three infected tapirs received oral antimycobacterial therapy consisting of 5 mg/kg body weight isoniazid, 10 mg/kg rifampicin, and 10 mg/kg clarithromycin q24h. Based on therapeutic drug level monitoring, the doses of rifampicin were adjusted to 12 and 15 mg/kg in the females and the male, respectively. The treatment with all three drugs was continued for 11 mon. Six months into treatment, the clinical condition resolved, and repeated BAL samples of all three tapirs tested negative for mycobacteria by culture. Here the approach for a treatment protocol with minimal side effects suitable to control infections with nontuberculous mycobacteria in lowland tapirs is reported.