Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance.

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.

Tuberculosis Variant with Rifampin Resistance Undetectable by Xpert MTB/RIF, Botswana.

GeneXpert MTB/RIF, a tool widely used for diagnosing tuberculosis, has limitations for detecting rifampin resistance in certain variants. We report transmission of a pre-extensively drug-resistant variant in Botswana that went undetected by GeneXpert. The public health impact of misdiagnosis emphasizes the need for comprehensive molecular testing to identify resistance and guide treatment.

Limited Nosocomial Transmission of Drug-Resistant Tuberculosis, Moldova.

Applying whole-genome-sequencing, we aimed to detect transmission events of multidrug-resistant/rifampin-resistant strains of Mycobacterium tuberculosis complex at a tuberculosis hospital in Chisinau, Moldova. We recorded ward, room, and bed information for each patient and monitored in-hospital transfers over 1 year. Detailed molecular and patient surveillance revealed only 2 nosocomial transmission events.

Use of High-Resolution Geospatial and Genomic Data to Characterize Recent Tuberculosis Transmission, Botswana.

Combining genomic and geospatial data can be useful for understanding Mycobacterium tuberculosis transmission in high-burden tuberculosis (TB) settings. We performed whole-genome sequencing on M. tuberculosis DNA extracted from sputum cultures from a population-based TB study conducted in Gaborone, Botswana, during 2012-2016. We determined spatial distribution of cases on the basis of shared genotypes among isolates. We considered clusters of isolates with ≤5 single-nucleotide polymorphisms identified by whole-genome sequencing to indicate recent transmission and clusters of ≥10 persons to be outbreaks. We obtained both molecular and geospatial data for 946/1,449 (65%) participants with culture-confirmed TB; 62 persons belonged to 5 outbreaks of 10-19 persons each. We detected geospatial clustering in just 2 of those 5 outbreaks, suggesting heterogeneous spatial patterns. Our findings indicate that targeted interventions applied in smaller geographic areas of high-burden TB identified using integrated genomic and geospatial data might help interrupt TB transmission during outbreaks.

Using genetic data to identify transmission risk factors: Statistical assessment and application to tuberculosis transmission.

Identifying host factors that influence infectious disease transmission is an important step toward developing interventions to reduce disease incidence. Recent advances in methods for reconstructing infectious disease transmission events using pathogen genomic and epidemiological data open the door for investigation of host factors that affect onward transmission. While most transmission reconstruction methods are designed to work with densely sampled outbreaks, these methods are making their way into surveillance studies, where the fraction of sampled cases with sequenced pathogens could be relatively low. Surveillance studies that use transmission event reconstruction then use the reconstructed events as response variables (i.e., infection source status of each sampled case) and use host characteristics as predictors (e.g., presence of HIV infection) in regression models. We use simulations to study estimation of the effect of a host factor on probability of being an infection source via this multi-step inferential procedure. Using TransPhylo-a widely-used method for Bayesian estimation of infectious disease transmission events-and logistic regression, we find that low sensitivity of identifying infection sources leads to dilution of the signal, biasing logistic regression coefficients toward zero. We show that increasing the proportion of sampled cases improves sensitivity and some, but not all properties of the logistic regression inference. Application of these approaches to real world data from a population-based TB study in Botswana fails to detect an association between HIV infection and probability of being a TB infection source. We conclude that application of a pipeline, where one first uses TransPhylo and sparsely sampled surveillance data to infer transmission events and then estimates effects of host characteristics on probabilities of these events, should be accompanied by a realistic simulation study to better understand biases stemming from imprecise transmission event inference.

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.

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.

Role of Epistasis in Amikacin, Kanamycin, Bedaquiline, and Clofazimine Resistance in Mycobacterium tuberculosis Complex.

Antibiotic resistance among bacterial pathogens poses a major global health threat. Mycobacterium tuberculosis complex (MTBC) is estimated to have the highest resistance rates of any pathogen globally. Given the low growth rate and the need for a biosafety level 3 laboratory, the only realistic avenue to scale up drug susceptibility testing (DST) for this pathogen is to rely on genotypic techniques. This raises the fundamental question of whether a mutation is a reliable surrogate for phenotypic resistance or whether the presence of a second mutation can completely counteract its effect, resulting in major diagnostic errors (i.e., systematic false resistance results). To date, such epistatic interactions have only been reported for streptomycin that is now rarely used. By analyzing more than 31,000 MTBC genomes, we demonstrated that the eis C-14T promoter mutation, which is interrogated by several genotypic DST assays endorsed by the World Health Organization, cannot confer resistance to amikacin and kanamycin if it coincides with loss-of-function (LoF) mutations in the coding region of eis. To our knowledge, this represents the first definitive example of antibiotic reversion in MTBC. Moreover, we raise the possibility that mmpR (Rv0678) mutations are not valid markers of resistance to bedaquiline and clofazimine if these coincide with an LoF mutation in the efflux pump encoded by mmpS5 (Rv0677c) and mmpL5 (Rv0676c).

Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Beijing Clades, Ukraine, 2015.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) is an emerging threat to TB control in Ukraine, a country with the third highest XDR TB burden globally. We used whole-genome sequencing of a convenience sample to identify bacterial genetic and patient-related factors associated with MDR/XDR TB in this country. MDR/XDR TB was associated with 3 distinct Mycobacterium tuberculosis complex lineage 2 (Beijing) clades, Europe/Russia W148 outbreak, Central Asia outbreak, and Ukraine outbreak, which comprised 68.9% of all MDR/XDR TB strains from southern Ukraine. MDR/XDR TB was also associated with previous treatment for TB and urban residence. The circulation of Beijing outbreak strains harboring broad drug resistance, coupled with constraints in drug supply and limited availability of phenotypic drug susceptibility testing, needs to be considered when new TB management strategies are implemented in Ukraine.

Towards a whole-genome sequence for rye (Secale cereale L.).

We report on a whole-genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole-genome shotgun sequencing of the 7.9-Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low-copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high-density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high-density genetic map and establishing a synteny-based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome-wide map of selection signals differentiating the divergent gene pools. This rye whole-genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome-based breeding in rye.

Genomic and phenotypic differentiation of Arabidopsis thaliana along altitudinal gradients in the North Italian Alps.

Altitudinal gradients in mountain regions are short-range clines of different environmental parameters such as temperature or radiation. We investigated genomic and phenotypic signatures of adaptation to such gradients in five Arabidopsis thaliana populations from the North Italian Alps that originated from 580 to 2350 m altitude by resequencing pools of 19-29 individuals from each population. The sample includes two pairs of low- and high-altitude populations from two different valleys. High-altitude populations showed a lower nucleotide diversity and negative Tajima's D values and were more closely related to each other than to low-altitude populations from the same valley. Despite their close geographic proximity, demographic analysis revealed that low- and high-altitude populations split between 260 000 and 15 000 years before present. Single nucleotide polymorphisms whose allele frequencies were highly differentiated between low- and high-altitude populations identified genomic regions of up to 50 kb length where patterns of genetic diversity are consistent with signatures of local selective sweeps. These regions harbour multiple genes involved in stress response. Variation among populations in two putative adaptive phenotypic traits, frost tolerance and response to light/UV stress was not correlated with altitude. Taken together, the spatial distribution of genetic diversity reflects a potentially adaptive differentiation between low- and high-altitude populations, whereas the phenotypic differentiation in the two traits investigated does not. It may resemble an interaction between adaptation to the local microhabitat and demographic history influenced by historical glaciation cycles, recent seed dispersal and genetic drift in local populations.

Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing: From Sequence Data to Resistance Profiles.

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.

Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing.

Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.

Emergence of bedaquiline-resistant tuberculosis and of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains with rpoB Ile491Phe mutation not detected by Xpert MTB/RIF in Mozambique: a retrospective observational study.

BACKGROUND: In 2021, an estimated 4800 people developed rifampicin-resistant tuberculosis in Mozambique, 75% of which went undiagnosed. Detailed molecular data on rifampicin-resistant and multidrug-resistant (MDR) tuberculosis are not available. Here, we aimed at gaining precise data on the determinants of rifampicin-resistant and MDR tuberculosis in Mozambique. METHODS: In this retrospective observational study, we performed whole-genome sequencing of 704 rifampicin-resistant Mycobacterium tuberculosis complex (Mtbc) strains submitted to the National Tuberculosis Reference Laboratory (NTRL) in Maputo, Mozambique, between 2015 and 2021. Phylogenetic strain classification, genomic resistance prediction, and cluster analysis were performed. FINDINGS: Between Jan 1, 2015, and July 31, 2021, 2606 Mtbc isolates with an isoniazid or rifampicin resistance were identified in the NTRL biobank, of which, 1483 (56·9%) were from men, 1114 (42·7%) from women, and nine (0·4%) were unknown. Genome-based drug-resistant prediction classified 704 Mtbc strains as rifampicin resistant. 628 (89%) of the 704 Mtbc strains were classified MDR; of those, 146 (23%) were pre-extensively drug resistant (pre-XDR; additional fluoroquinolone resistance), and 24 (4%) extensively drug resistant (XDR; combined fluoroquinolone and bedaquiline resistance). Overall, 61 (9%) of 704 strains revealed resistance to bedaquiline: five (7%) of 76 rifampicin resistant plus bedaquiline resistant, 32 (7%) of 458 MDR plus bedaquiline resistant, and 24 (100%) of 24 XDR. Prevalence of bedaquiline resistance increased from 3% in 2016 to 14% in 2021. The cluster rate (12 single-nucleotide polymorphism threshold) was 42% for rifampicin-resistant strains, 78% for MDR strains, 94% for pre-XDR strains, and 96% for XDR Mtbc strains. 31 (4%) of 704 Mtbc strains, belonging to a diagnostic escape outbreak strain previously described in Eswatini (group_56), had an rpoB Ile491Phe mutation which is not detected by Xpert MTB/RIF (no other rpoB mutation). Of these, 23 (74%) showed additional resistance to bedaquiline, 13 (42%) had bedaquiline and fluoroquinolone resistance, and two (6%) were bedaquiline, fluoroquinolone, and delamanid resistant. INTERPRETATION: Pre-XDR resistance is highly prevalent among MDR Mtbc strains in Mozambique and so is bedaquiline resistance; and the frequency of bedaquiline resistance quadrupled over time and was found even in Mtbc strains without fluoroquinolone resistance. Importantly, strains with Ile491Phe mutation were frequent, accounting for 31% (n=10) of MDR plus bedaquiline-resistant strains and 54% (n=13) of XDR Mtbc strains. Given the current diagnostic algorithms and treatment regimens, both the emergence of rifampicin resistance due to Ile491Phe and bedaquiline resistance might jeopardise MDR tuberculosis prevention and care unless sequencing-based technology is rolled out. The potential cross border spread of diagnostic escape strains needs further investigation. FUNDING: The German Ministry of Health through the Seq_MDRTB-Net project, the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy Precision Medicine in Inflammation and the Research Training Group 2501 TransEvo, the Leibniz Science Campus Evolutionary Medicine of the Lung, and the German Ministry of Education and Research via the German Center for Infection Research.

Molecular determinants of multidrug-resistant tuberculosis in Sierra Leone.

Multidrug-resistant tuberculosis (MDR-TB) management has become a serious global health challenge. Understanding its epidemic determinants on the regional level is crucial for developing effective control measures. We used whole genome sequencing data of 238 of Mycobacterium tuberculosis complex (MTBC) strains to determine drug resistance profiles, phylogeny, and transmission dynamics of MDR/rifampicin-resistant (RR) MTBC strains from Sierra Leone. Forty-two strains were classified as RR, 196 as MDR, 5 were resistant to bedaquiline (BDQ) and clofazimine (CFZ), but none was found to be resistant to fluoroquinolones. Sixty-one (26%) strains were resistant to all first-line drugs, three of which had additional resistance to BDQ/CFZ. The strains were classified into six major MTBC lineages (L), with strains of L4 being the most prevalent, 62% (n = 147), followed by L6 (Mycobacterium africanum) strains, (21%, n = 50). The overall clustering rate (using ≤d12 single-nucleotide polymorphism threshold) was 44%, stratified into 31 clusters ranging from 2 to 16 strains. The largest cluster (n = 16) was formed by sublineage 2.2.1 Beijing Ancestral 3 strains, which developed MDR several times. Meanwhile, 10 of the L6 strains had a primary MDR transmission. We observed a high diversity of drug resistance mutations, including borderline resistance mutations to isoniazid and rifampicin, and mutations were not detected by commercial assays. In conclusion, one in five strains investigated was resistant to all first-line drugs, three of which had evidence of BDQ/CFZ resistance. Implementation of interventions such as rapid diagnostics that prevent further resistance development and stop MDR-TB transmission chains in the country is urgently needed. IMPORTANCE: A substantial proportion of MDR-TB strains in Sierra Leone were resistant against all first line drugs; however this makes the all-oral-six-month BPaLM regimen or other 6-9 months all oral regimens still viable, mainly because there was no FQ resistance.Resistance to BDQ was detected, as well as RR, due to mutations outside of the hotspot region. While the prevalence of those resistances was low, it is still cause for concern and needs to be closely monitored.

Transmission Dynamics of a Mycobacterium tuberculosis Complex Outbreak in an Indigenous Population in the Colombian Amazon Region.

Whole genome sequencing (WGS) has become the main tool for studying the transmission of Mycobacterium tuberculosis complex (MTBC) strains; however, the clonal expansion of one strain often limits its application in local MTBC outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis could potentially increase the resolution, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported MTBC outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March to October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct MTBC strain belonging to lineage 4.3.3. Employing a reference genome from an outbreak strain and highly confident single nucleotide polymorphisms (SNPs) in repetitive genomic regions, e.g., the proline-glutamic acid/proline-proline-glutamic-acid (PE/PPE) gene family, increased the phylogenetic resolution compared to a classical H37Rv reference mapping approach. Specifically, the number of differentiating SNPs increased from 890 to 1,094, which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 5 versus 9 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites, suggesting that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and employment of a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal MTBC populations and help elucidate within-host MTBC diversity. IMPORTANCE 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. Here, we employed a whole-genome sequencing-based outbreak investigation in order to improve the phylogenetic resolution and gain new insights into the transmission dynamics in this remote Colombian Amazon Region. The inclusion of well-supported single nucleotide polymorphisms in repetitive regions and a de novo-assembled local reference genome provided a more granular picture of the circulating outbreak strain and revealed new transmission chains. Multiple patients from different settlements were possibly infected with at least two different clones in this high-incidence setting. Thus, our results have the potential to improve molecular surveillance studies in other high-burden settings, especially regions with few clonal multidrug-resistant (MDR) MTBC lineages/clades.

In vitro activity of new combinations of ß-lactam and ß-lactamase inhibitors against the Mycobacterium tuberculosis complex.

As meropenem-clavulanic acid is recommended for the treatment of drug-resistant tuberculosis, the repurposing of new carbapenem combinations may provide new treatment options, including oral alternatives. Therefore, we studied the in vitro activities of meropenem-vaborbactam, meropenem-clavulanic acid, and tebipenem-clavulanic acid. One hundred nine Mycobacterium tuberculosis complex (MTBC) clinical isolates were tested, of which 69 were pan-susceptible and the remaining pyrazinamide- or multidrug-resistant. Broth microdilution MICs were determined using the EUCAST reference method. Meropenem and tebipenem were tested individually and in combination with vaborbactam 8 mg/L and clavulanic-acid 2 and 4 mg/L, respectively. Whole-genome sequencing was performed to explore resistance mechanisms. Clavulanic acid lowered the modal tebipenem MIC approximately 16-fold (from 16 to 1 mg/L). The modal meropenem MIC was reduced twofold by vaborbactam compared with an approximately eightfold decrease by clavulanic acid. The only previously described high-confidence carbapenem resistance mutation, crfA T62A, was shared by a subgroup of lineage 4.3.4.1 isolates and did not correlate with elevated MICs. The presence of a ß-lactamase inhibitor reduced the MTBC MICs of tebipenem and meropenem. The resulting MIC distribution was lowest for the orally available drugs tebipenem-clavulanic acid. Whether this in vitro activity translates to similar or greater clinical efficacy of tebipenem-clavulanic acid compared with the currently WHO-endorsed meropenem-clavulanic acid requires clinical studies. IMPORTANCE Repurposing of already approved antibiotics, such as ß-lactams in combination with ß-lactamase inhibitors, may provide new treatment alternatives for drug-resistant tuberculosis. Meropenem-clavulanic acid was more active in vitro compared to meropenem-vaborbactam. Notably, tebipenem-clavulanic acid showed even better activity, raising the potential of an all-oral treatment option. Clinical data are needed to investigate whether the better in vitro activity of tebipenem-clavulanic acid correlates with greater clinical efficacy compared with the currently WHO-endorsed meropenem-clavulanic acid.

High fluoroquinolone resistance proportions among multidrug-resistant tuberculosis driven by dominant L2 Mycobacterium tuberculosis clones in the Mumbai Metropolitan Region.

BACKGROUND: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB (further resistance against bedaquiline and/or linezolid) rates. Still, factors driving the MDR/pre-XDR epidemic in India are not well defined. METHODS: In a retrospective study, we analyzed 1852 consecutive MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai by whole genome sequencing (WGS). Univariate and multivariate statistics was used to investigate factors associated with pre-XDR. Core genome multi locus sequence typing, time scaled haplotypic density (THD) method and homoplasy analysis were used to analyze epidemiological success, and positive selection in different strain groups, respectively. RESULTS: In total, 1016 MTBC strains were MDR, out of which 703 (69.2%) were pre-XDR and 45 (4.4%) were XDR. Cluster rates were high among MDR (57.8%) and pre-XDR/XDR (79%) strains with three dominant L2 (Beijing) strain clusters (Cl 1-3) representing half of the pre-XDR and 40% of the XDR-TB cases. L2 strains were associated with pre-XDR/XDR-TB (P < 0.001) and, particularly Cl 1-3 strains, had high first-line and FQ resistance rates (81.6-90.6%). Epidemic success analysis using THD showed that L2 strains outperformed L1, L3, and L4 strains in short- and long-term time scales. More importantly, L2 MDR and MDR + strains had higher THD success indices than their not-MDR counterparts. Overall, compensatory mutation rates were highest in L2 strains and positive selection was detected in genes of L2 strains associated with drug tolerance (prpB and ppsA) and virulence (Rv2828c). Compensatory mutations in L2 strains were associated with a threefold increase of THD indices, suggesting improved transmissibility. CONCLUSIONS: Our data indicate a drastic increase of FQ resistance, as well as emerging bedaquiline resistance which endangers the success of newly endorsed MDR-TB treatment regimens. Rapid changes in treatment and control strategies are required to contain transmission of highly successful pre-XDR L2 strains in the Mumbai Metropolitan region but presumably also India-wide.