Outbreak of Intermediate Species Leptospira venezuelensis Spread by Rodents to Cows and Humans in L. interrogans-Endemic Region, Venezuela.

Leptospirosis is a common but underdiagnosed zoonosis. We conducted a 1-year prospective study in La Guaira State, Venezuela, analyzing 71 hospitalized patients who had possible leptospirosis and sampling local rodents and dairy cows. Leptospira rrs gene PCR test results were positive in blood or urine samples from 37/71 patients. Leptospira spp. were isolated from cultured blood or urine samples of 36/71 patients; 29 had L. interrogans, 3 L. noguchii, and 4 L. venezuelensis. Conjunctival suffusion was the most distinguishing clinical sign, many patients had liver involvement, and 8/30 patients with L. interrogans infections died. The Leptospira spp. found in humans were also isolated from local rodents; L. interrogans and L. venezuelensis were isolated from cows on a nearby, rodent-infested farm. Phylogenetic clustering of L. venezuelensis isolates suggested a recently expanded outbreak strain spread by rodents. Increased awareness of leptospirosis prevalence and rapid diagnostic tests are needed to improve patient outcomes.

Local adaptation of Mycobacterium tuberculosis on the Tibetan Plateau.

During its global dispersal, Mycobacterium tuberculosis (Mtb) has encountered varied geographic environments and host populations. Although local adaptation seems to be a plausible model for describing long-term host-pathogen interactions, genetic evidence for this model is lacking. Here, we analyzed 576 whole-genome sequences of Mtb strains sampled from different regions of high-altitude Tibet. Our results show that, after sequential introduction of a few ancestral strains, the Tibetan Mtb population diversified locally while maintaining strict separation from the Mtb populations on the lower altitude plain regions of China. The current population structure and estimated past population dynamics suggest that the modern Beijing sublineage strains, which expanded over most of China and other global regions, did not show an expansion advantage in Tibet. The mutations in the Tibetan strains showed a higher proportion of A > G/T > C transitions than strains from the plain regions, and genes encoding DNA repair enzymes showed evidence of positive selection. Moreover, the long-term Tibetan exclusive selection for truncating mutations in the thiol-oxidoreductase encoding sseA gene suggests that Mtb was subjected to local selective pressures associated with oxidative stress. Collectively, the population genomics of Mtb strains in the relatively isolated population of Tibet provides genetic evidence that Mtb has adapted to local environments.

Transcriptomic Analysis of Mycobacterial Infected Macrophages Reveals a High MOI Specific Type I IFN Signaling.

Macrophage (MΦ) infection models are important tools for studying host-mycobacterial interactions. Although the multiplicity of infection (MOI) is an important experimental variable, the selection of MOI in mycobacterial infection experiments is largely empirical, without reference to solid experimental data. To provide relevant data, we used RNA-seq to analyze the gene expression profiles of MΦs 4 or 24 h after infection with Mycobacterium marinum (M. m) at MOIs ranging from 0.1 to 50. Analysis of differentially expressed genes (DEGs) showed that different MOIs are linked to distinct transcriptomic changes and only 10% of DEGs were shared by MΦ infected at all MOIs. KEGG pathway enrichment analysis revealed that type I interferon (IFN)-related pathways were inoculant dose-dependent and enriched only at high MOIs, whereas TNF pathways were inoculant dose-independent and enriched at all MOIs. Protein-protein interaction (PPI) network alignment showed that different MOIs had distinct key node genes. By fluorescence-activated cell sorting and follow-up RT-PCR analysis, we could separate infected MΦs from uninfected MΦs and found phagocytosis of mycobacteria to be the determinant factor for type I IFN production. The distinct transcriptional regulation of RAW264.7 MΦ genes at different MOIs was also seen with Mycobacterium tuberculosis (M.tb) infections and primary MΦ infection models. In summary, transcriptional profiling of mycobacterial infected MΦs revealed that different MOIs activate distinct immune pathways and the type I IFN pathway is activated only at high MOIs. This study should provide guidance for selecting the MOI most appropriate for different research questions.

Lesion Heterogeneity and Long-Term Heteroresistance in Multidrug-Resistant Tuberculosis.

Tuberculosis heteroresistance, in which only a fraction of the bacteria in a patient with tuberculosis contains drug-resistant mutations, has been a rising concern. However, its origins and prevalence remain elusive. Here, whole-genome sequencing was performed on 83 serial isolates from 31 patients with multidrug-resistant tuberculosis, and heteroresistance was detected in isolates from 21 patients (67.74%). Heteroresistance persisted in the host for long periods, spanning months to years, and was associated with having multiple tubercular lesions. Our findings indicate that heteroresistance is common and persistent in patients with multidrug-resistant tuberculosis and may affect the success of their treatment regimens.

Citywide Transmission of Multidrug-resistant Tuberculosis Under China's Rapid Urbanization: A Retrospective Population-based Genomic Spatial Epidemiological Study.

BACKGROUND: Population movement could extend multidrug-resistant tuberculosis (MDR-TB) transmission and complicate its global prevalence. We sought to identify the high-risk populations and geographic sites of MDR-TB transmission in Shenzhen, the most common destination for internal migrants in China. METHODS: We performed a population-based, retrospective study in patients diagnosed with MDR-TB in Shenzhen during 2013-2017. By defining genomic clusters with a threshold of 12-single-nucleotide polymorphism distance based on whole-genome sequencing of their clinical strains, the clustering rate was calculated to evaluate the level of recent transmission. Risk factors were identified by multivariable logistic regression. To further delineate the epidemiological links, we invited the genomic-clustered patients to an in-depth social network investigation. RESULTS: In total, 105 (25.2%) of the 417 enrolled patients with MDR-TB were grouped into 40 genome clusters, suggesting recent transmission of MDR strains. The adjusted risk for student to have a clustered strain was 4.05 (95% confidence interval, 1.06-17.0) times greater than other patients. The majority (70%, 28/40) of the genomic clusters involved patients who lived in different districts, with residences separated by an average of 8.76 kilometers. Other than household members, confirmed epidemiological links were also identified among classmates and workplace colleagues. CONCLUSIONS: These findings demonstrate that local transmission of MDR-TB is a serious problem in Shenzhen. While most transmission occurred between people who lived distant from each other, there was clear evidence that transmission occurred in schools and workplaces, which should be included as targeted sites for active case finding.The average residential distance between genomic-clustered cases was more than 8 kilometers, while schools and workplaces, identified as sites of transmission in this study, deserve increased vigilance for targeted case finding of multidrug-resistant tuberculosis.

Fitness Cost and Compensatory Evolution in Levofloxacin-Resistant Mycobacterium aurum.

We isolated spontaneous levofloxacin-resistant strains of Mycobacterium aurum to study the fitness cost and compensatory evolution of fluoroquinolone resistance in mycobacteria. Five of six mutant strains with substantial growth defects showed restored fitness after being serially passaged for 18 growth cycles, along with increased cellular ATP level. Whole-genome sequencing identified putative compensatory mutations in the glgC gene that restored the fitness of the resistant strains, presumably by altering the bacterial energy metabolism.

Use of GeneXpert Remnants for Drug Resistance Profiling and Molecular Epidemiology of Tuberculosis in Libreville, Gabon.

Multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis pose major problems for global health. The GeneXpert MTB/RIF (Xpert) assay rapidly detects resistance to rifampin (RIFr), but for detection of the additional resistance that defines MDR-TB (MDR tuberculosis) and XDR-TB, and for molecular epidemiology, specimen cultures and a biosafe infrastructure are generally required. We sought to determine whether the remnants of sputa prepared for the Xpert assay could be used directly to find mutations associated with drug resistance and to study molecular epidemiology, thus providing precise characterization of MDR-TB cases in countries lacking biosafety level 3 (BSL3) facilities for M. tuberculosis cultures. After sputa were processed and run on the Xpert instrument, the leftovers of the samples prepared for the Xpert assay were used for PCR amplification and sequencing or for a line probe assay to detect mutations associated with resistance to additional drugs, as well as for molecular epidemiology with spoligotyping and selective mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Of 130 sputum samples from Gabon tested with the Xpert assay, 124 yielded interpretable results; 21 (17%) of these were determined to be RIFr Amplification and sequencing or a line probe assay of the Xpert remnants confirmed 18/21 samples as MDR, corresponding to 12/116 (9.5%) new and 6/8 (75%) previously treated TB patients. Spoligotyping and MIRU typing with hypervariable loci identified an MDR Beijing strain present in five samples. We conclude that the remnants of samples processed for the Xpert assay can be used in PCRs to find mutations associated with the resistance to the additional drugs that defines MDR and XDR-TB and to study molecular epidemiology without the need for culturing or a biosafe infrastructure.

Temporal and cellular analysis of granuloma development in mycobacterial infected adult zebrafish.

Because granulomas are a hallmark of tuberculosis pathogenesis, the study of the dynamic changes in their cellular composition and morphological character can facilitate our understanding of tuberculosis pathogenicity. Adult zebrafish infected with Mycobacterium marinum form granulomas that are similar to the granulomas in human patients with tuberculosis and therefore have been used to study host-mycobacterium interactions. Most studies of zebrafish granulomas, however, have focused on necrotic granulomas, while a systematic description of the different stages of granuloma formation in the zebrafish model is lacking. Here, we characterized the stages of granulomas in M. marinum-infected zebrafish, including early immune cell infiltration, nonnecrotizing granulomas, and necrotizing granulomas, using corresponding samples from patients with pulmonary tuberculosis as references. We combined hematoxylin and eosin staining and in situ hybridization to identify the different immune cell types and follow their spatial distribution in the different stages of granuloma development. The macrophages in zebrafish granulomas were shown to belong to distinct subtypes: epithelioid macrophages, foamy macrophages, and multinucleated giant cells. By defining the developmental stages of zebrafish granulomas and the spatial distribution of the different immune cells they contain, this work provides a reference for future studies of mycobacterial granulomas and their immune microenvironments.

Genomic investigation of bone tuberculosis highlighted the role of subclinical pulmonary tuberculosis in transmission.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) without symptomatic pulmonary involvement has been thought to be non-transmissible, but EPTB with asymptomatic pulmonary tuberculosis (PTB) could transmit tuberculosis (TB). Genomic investigation of Mycobacterium tuberculosis (Mtb) isolates from EPTB may provide insight into its epidemiological role in TB transmission. METHODS: Between January 2017 and May 2020, 107 Mtb isolates were obtained from surgical drainage of bone TB patients at the Beijing Chest Hospital, and 218 Mtb strains were isolated from PTB cases. These 325 Mtb isolates were whole-genome sequenced to reconstruct a phylogenetic tree, identify transmission clusters, and infer transmission links using a Bayesian approach. Possible subclinical PTB in the bone TB patients was investigated with chest imaging by two independent experts. RESULTS: Among 107 bone TB patients, 10 were in genomic clusters (≤12 SNPs). Phylogenetic analysis suggested that three bone TB patients transmitted the infection to secondary cases, supported by epidemiological investigations. Pulmonary imaging of 44 bone TB patients revealed that 79.5 % (35/44) had radiological abnormalities suggestive of subclinical PTB. CONCLUSIONS: This study provides genomic evidence that bone TB patients without clinically diagnosed PTB can be sources of TB transmission, underscoring the importance of screening for subclinical, transmissible PTB among EPTB cases.

The mutational signatures of poor treatment outcomes on the drug-susceptible Mycobacterium tuberculosis genome.

Drug resistance is a known risk factor for poor tuberculosis (TB) treatment outcomes, but the contribution of other bacterial factors to poor outcomes in drug-susceptible TB is less well understood. Here, we generate a population-based dataset of drug-susceptible Mycobacterium tuberculosis (MTB) isolates from China to identify factors associated with poor treatment outcomes. We analyzed whole-genome sequencing (WGS) data of MTB strains from 3196 patients, including 3105 patients with good and 91 patients with poor treatment outcomes, and linked genomes to patient epidemiological data. A genome-wide association study (GWAS) was performed to identify bacterial genomic variants associated with poor outcomes. Risk factors identified by logistic regression analysis were used in clinical models to predict treatment outcomes. GWAS identified fourteen MTB fixed mutations associated with poor treatment outcomes, but only 24.2% (22/91) of strains from patients with poor outcomes carried at least one of these mutations. Isolates from patients with poor outcomes showed a higher ratio of reactive oxygen species (ROS)-associated mutations compared to isolates from patients with good outcomes (26.3% vs 22.9%, t-test, p=0.027). Patient age, sex, and duration of diagnostic delay were also independently associated with poor outcomes. Bacterial factors alone had poor power to predict poor outcomes with an AUC of 0.58. The AUC with host factors alone was 0.70, but increased significantly to 0.74 (DeLong's test, p=0.01) when bacterial factors were also included. In conclusion, although we identified MTB genomic mutations that are significantly associated with poor treatment outcomes in drug-susceptible TB cases, their effects appear to be limited.

Whole-Genome Sequencing Exhibits Better Diagnostic Performance than Variable-Number Tandem Repeats for Identifying Mixed Infections of Mycobacterium tuberculosis.

Mixed infections of Mycobacterium tuberculosis, defined as the coexistence of multiple genetically distinct strains within a single host, have been associated with unfavorable treatment outcomes. Different methods have been used to detect mixed infections, but their performances have not been carefully evaluated. To compare the sensitivity of whole-genome sequencing (WGS) and variable-number tandem repeats (VNTR) typing to detect mixed infections, we prepared 10 artificial samples composed of DNA mixtures from two strains in different proportions and retrospectively collected 1,084 clinical isolates. The limit of detection (LOD) for the presence of a minor strain was 5% for both WGS and VNTR typing. The overall clinical detection rate of mixed infections was 3.7% (40/1,084) for the two methods combined, WGS identified 37/1,084 (3.4%), and VNTR typing identified 14/1,084 (1.3%), including 11 also identified by WGS. Multivariate analysis demonstrated that retreatment patients had a 2.7 times (95% confidence interval [CI], 1.2 to 6.0) higher risk of mixed infections than new cases. Collectively, WGS is a more reliable tool to identify mixed infections than VNTR typing, and mixed infections are more common in retreated patients. IMPORTANCE Mixed infections of M. tuberculosis have the potential to render treatment regimens ineffective and affect the transmission dynamics of the disease. VNTR typing, currently the most widely used method for the detection of mixed infections, detects mixed infections only by interrogating a small fraction of the M. tuberculosis genome, which necessarily limits sensitivity. With the introduction of WGS, it became possible to study the entire genome, but no quantitative comparison has yet been undertaken. Our systematic comparison of the ability of WGS and VNTR typing to detect mixed infections, using both artificial samples and clinical isolates, revealed the superior performance of WGS at a high sequencing depth (~100×) and found that mixed infections are more common in patients being retreated for tuberculosis (TB) in the populations studied. This provides valuable information for the application of WGS in the detection of mixed infections and the implications of mixed infections for tuberculosis control.

The Global Success of Mycobacterium tuberculosis Modern Beijing Family Is Driven by a Few Recently Emerged Strains.

Strains of the Mycobacterium tuberculosis complex (MTBC) Beijing family aroused concern because they were often found in clusters and appeared to be exceptionally transmissible. However, it was later found that strains of the Beijing family were heterogeneous, and the transmission advantage was restricted to sublineage L2.3 or modern Beijing. In this study, we analyzed the previously published genome sequences of 7,896 L2.3 strains from 51 different countries. Using BEAST software to approximate the temporal emergence of L2.3, our calculations suggest that L2.3 initially emerged in northern East Asia during the early 15th century and subsequently diverged into six phylogenetic clades, identified as L2.3.1 through L2.3.6. Using terminal branch length and genomic clustering as proxies for transmissibility, we found that the six clades displayed distinct population dynamics, with the three recently emerged clades (L2.3.4 to L2.3.6) exhibiting significantly higher transmissibility than the older three clades (L2.3.1 to L2.3.3). Of the Beijing family strains isolated outside East Asia, 83.1% belonged to the clades L2.3.4 to L2.3.6, which were also associated with more cross-border transmission. This work reveals the heterogeneity in sublineage L2.3 and demonstrates that the global success of Beijing family strains is driven by the three recently emerged L2.3 clades. IMPORTANCE The recent population dynamics of the global tuberculosis epidemic are heavily shaped by Mycobacterium tuberculosis complex (MTBC) strains with enhanced transmissibility. The infamous Beijing family strain stands out because it has rapidly spread throughout the world. Identifying the strains responsible for the global expansion and tracing their evolution should help to understand the nature of high transmissibility and develop effective strategies to control transmission. In this study, we found that the L2.3 sublineage diversified into six phylogenetic clades (L2.3.1 to L2.3.6) with various transmission characteristics. Clades L2.3.4 to L2.3.6 exhibited significantly higher transmissibility than clades L2.3.1 to L2.3.3, which helps explain why more than 80% of Beijing family strains collected outside East Asia belong to these three clades. We conclude that the global success of L2.3 was not caused by the entire L2.3 sublineage but rather was due to the rapid expansion of L2.3.4 to L2.3.6. Tracking the transmission of L2.3.4 to L2.3.6 strains can help to formulate targeted TB prevention and control.

The Evolution and Transmission Dynamics of Multidrug-Resistant Tuberculosis in an Isolated High-Plateau Population of Tibet, China.

On the Tibetan Plateau, most tuberculosis is caused by indigenous Mycobacterium tuberculosis strains with a monophyletic structure and high-level drug resistance. This study investigated the emergence, evolution, and transmission dynamics of multidrug-resistant tuberculosis (MDR-TB) in Tibet. The whole-genome sequences of 576 clinical strains from Tibet were analyzed with the TB-profiler tool to identify drug-resistance mutations. The evolution of the drug resistance was then inferred based on maximum-likelihood phylogeny and dated trees that traced the serial acquisition of mutations conferring resistance to different drugs. Among the 576 clinical M. tuberculosis strains, 346 (60.1%) carried at least 1 resistance-conferring mutation and 231 (40.1%) were MDR-TB. Using a pairwise distance of 50 single nucleotide polymorphisms (SNPs), most strains (89.9%, 518/576) were phylogenetically separated into 50 long-term transmission clusters. Eleven large drug-resistant clusters contained 76.1% (176/231) of the local multidrug-resistant strains. A total of 85.2% of the isoniazid-resistant strains were highly transmitted with an average of 6.6 cases per cluster, of which most shared the mutation KatG Ser315Thr. A lower proportion (71.6%) of multidrug-resistant strains were transmitted, with an average cluster size of 2.9 cases. The isoniazid-resistant clusters appear to have undergone substantial bacterial population growth in the 1970s to 1990s and then subsequently accumulated multiple rifampicin-resistance mutations and caused the current local MDR-TB burden. These findings highlight the importance of detecting and curing isoniazid-resistant strains to prevent the emergence of endemic MDR-TB. IMPORTANCE Emerging isoniazid resistance in the 1970s allowed M. tuberculosis strains to spread and form into large multidrug-resistant tuberculosis clusters in the isolated plateau of Tibet, China. The epidemic was driven by the high risk of transmission as well as the potential of acquiring further drug resistance from isoniazid-resistant strains. Eleven large drug-resistant clusters consisted of the majority of local multidrug-resistant cases. Among the clusters, isoniazid resistance overwhelmingly evolved before all the other resistance types. A large bacterial population growth of isoniazid-resistant clusters occurred between 1970s and 1990s, which subsequently accumulated rifampicin-resistance-conferring mutations in parallel and accounted for the local multidrug-resistant tuberculosis burden. The results of our study indicate that it may be possible to restrict MDR-TB evolution and dissemination by prioritizing screening for isoniazid (INH)-resistant TB strains before they become MDR-TB and by adopting measures that can limit their transmission.

Comprehensive genomic analysis of Mycobacterium tuberculosis reveals limited impact of high-fitness genotypes on MDR-TB transmission.

OBJECTIVES: Environmental and host-related factors that contribute to the transmission of multidrug-resistant tuberculosis (MDR-TB) have become an increasing concern, but the impact of bacterial genetic factors associated with bacterial fitness on MDR-TB transmission is poorly understood. Here, we present a global view of the correlation between common fitness-related genotypes and MDR-TB transmission by analyzing a representative number of MDR-TB isolates. METHODS: We assembled a global whole genome sequencing (WGS) dataset of MDR-TB strains collected through retrospective cohorts or population-based approaches using public databases and literature curation. WGS-based clusters were defined as groups of strains with genomic difference of ≤ 5 SNPs. RESULTS: We curated high-quality WGS data of 4696 MDR-TB isolates from 17 countries with a mean clustering rate of 48% (range 0-100%). Correlational analysis showed that increased risk of MDR-TB strain clustering was not associated with compensatory mutations (OR 1.07, 95% CI 0.72-1.59), low-fitness cost drug-resistant mutations (katG S315T: OR 1.42, 95% CI 0.82-2.47; rpoB S450L: OR 1.26, 95% CI 0.87-1.83) or Lineage 2 (OR 1.50, 95% CI 0.95-2.39). CONCLUSIONS: The factors most commonly thought to increase bacterial fitness were not significantly associated with increased MDR-TB transmission, and thus do not appear to be major contributors to the current epidemic of MDR-TB.

Modern Beijing sublineage of Mycobacterium tuberculosis shift macrophage into a hyperinflammatory status.

The high prevalence of the modern Beijing sublineage of Mycobacterium tuberculosis may be related to increased virulence, although the responsible mechanisms remain poorly understood. We previously described enhanced triacylglycerol accumulation in modern Beijing strains. Here we show that modern Beijing strains grow faster in vitro and trigger a vigorous immune response and pronounced macrophage infiltration. Transcriptomic analysis of bone marrow derived macrophages infected with modern Beijing lineage strains revealed a significant enrichment of infection, cholesterol homeostasis and amino acid metabolic pathways. The upregulation of proinflammatory / bactericidal cytokines was confirmed by RT-PCR analysis, which is also in consistent with the reduced bacterial burden in modern strains infected macrophages. These results suggest that modern Beijing strains elicit a hyperinflammatory response which might indicate a stronger virulence and contribute to their extensive global prevalence.

Negative Regulator Nlrc3-like Maintain the Balanced Innate Immune Response During Mycobacterial Infection in Zebrafish.

The NOD-like receptors (NLRs) have been shown to be involved in infection and autoinflammatory disease. Previously, we identified a zebrafish NLR, nlrc3-like, required for macrophage homeostasis in the brain under physiological conditions. Here, we found that a deficiency of nlrc3-like leads to decreased bacterial burden at a very early stage of Mycobacterium marinum infection, along with increased production of pro-inflammatory cytokines, such as il-1ß and tnf-α. Interestingly, myeloid-lineage specific overexpression of nlrc3-like achieved the opposite effects, suggesting that the impact of nlrc3-like on the host anti-mycobacterial response is mainly due to its expression in the innate immune system. Fluorescence-activated cell sorting (FACS) and subsequent gene expression analysis demonstrated that inflammasome activation-related genes were upregulated in the infected macrophages of nlrc3-like deficient embryos. By disrupting asc, encoding apoptosis-associated speck-like protein containing a CARD, a key component for inflammasome activation, the bacterial burden increased in asc and nlrc3-like double deficient embryos compared with nlrc3-like single deficient embryos, implying the involvement of inflammasome activation in infection control. We also found extensive neutrophil infiltration in the nlrc3-like deficient larvae during infection, which was associated with comparable bacterial burden but increased tissue damage and death at a later stage that could be alleviated by administration of dexamethasone. Our findings uncovered an important role of nlrc3-like in the negative regulation of macrophage inflammasome activation and neutrophil infiltration during mycobacterial infection. This highlights the importance of a balanced innate immune response during mycobacterial infection and provides a potential molecular basis to explain how anti-inflammatory drugs can improve treatment outcomes in TB patients whose infection is accompanied by a hyperinflammatory response.

Evaluation of fluoromycobacteriophages for detecting drug resistance in Mycobacterium tuberculosis.

We tested a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). After promising results in preliminary studies, the EGFP-phage was used to detect isoniazid (INH), rifampin (RIF), and streptomycin (STR) resistance in 155 strains of M. tuberculosis, and the results were compared to the resazurin microplate technique, with the proportion method serving as the reference standard. The resazurin technique yielded sensitivities of 94% for INH and RIF and 98% for STR and specificities of 97% for INH, 95% for RIF, and 98% for STR. The sensitivity of EGFP-phage was 94% for all three antibiotics, with specificities of 90% for INH, 93% for RIF, and 95% for STR. The EGFP-phage results were available in 2 days for RIF and STR and in 3 days for INH, with an estimated cost of ∼2$ to test the three antibiotics. Using a more stringent criterion for resistance improved the specificity of the EGFP-phage for INH and RIF without affecting the sensitivity. In preliminary studies, the EGFP-phage could also effectively detect resistance to the fluoroquinolones. The EGFP-phage method has the potential to be a valuable rapid and economic screen for detecting drug-resistant tuberculosis if the procedure can be simplified, if it can be adapted to clinical material, and if its sensitivity can be improved.

Highly transmitted M. tuberculosis strains are more likely to evolve MDR/XDR and cause outbreaks, but what makes them highly transmitted?

Multi-Drug-Resistant strains of Mycobacterium tuberculosis (MDR-TB) are a serious obstacle to global TB eradication. While most MDR-TB strains are infrequently transmitted, a few cause large transmission clusters that contribute substantially to local MDR-TB burdens. Here we examine whether the known mutations in these strains can explain their success. Drug resistance mutations differ in fitness costs and strains can also acquire compensatory mutations (CM) to restore fitness, but some highly transmitted MDR strains have no CM. The acquisition of resistance mutations that maintain high transmissibility seems to occur by chance and are more likely in strains that are intrinsically highly transmitted and cause many cases. Modern Beijing lineage strains have caused several large outbreaks, but MDR outbreaks are also caused by ancient Beijing and lineage 4 strains, suggesting the lineage is less important than the characteristics of the individual strain. The development of fluoroquinolone resistance appears to represent another level of selection, in which strains must surmount unknown fitness costs of gyrA mutations. The genetic determinants of high transmission are poorly defined but may involve genes encoding proteins involved in molybdenum acquisition and the Esx systems. In addition, strains eliciting lower cytokine responses and producing more caseating granulomas may have advantages for transmission. Successful MDR/XDR strains generally evolve from highly transmitted drug sensitive parent strains due to selection pressures from deficiencies in local TB control programs. Until TB incidence is considerably reduced, there will likely be highly transmitted strains that develop resistance to any new antibiotic.

Drug resistance, fitness and compensatory mutations in Mycobacterium tuberculosis.

For tuberculosis to be eradicated, the transmission of Multi-Drug-Resistant and eXtensively Drug Resistant strains of Mycobacterium tuberculosis (MDR and XDR-TB) must be considerably reduced. Drug resistant strains were initially thought to have reduced fitness, and the majority of resistant strains may actually have compromised fitness because they are found in only one or a few patients. In contrast, some MDR/XDR-TB strains are highly transmitted and cause large outbreaks. Most antibiotics target essential bacterial functions and the mutations that confer resistance to anti-TB drugs can incur fitness costs manifested as slower growth and reduced viability. The fitness costs vary with different resistance mutations and the bacilli can also accumulate secondary mutations that compensate for the compromised functions and partially or fully restore lost fitness. The compensatory mutations (CM) are different for each antibiotic, as they mitigate the deleterious effects of the specific functions compromised by the resistance mutations. CM are generally more common in strains with resistance mutations incurring the greatest fitness costs, but for RIF resistance, CM are most frequent in strains with the mutation carrying the least fitness cost, Ser450Leu. Here, we review what is known about fitness costs, CM and mechanisms of resistance to the drugs that define a strain as MDR or XDR-TB. The relative fitness costs of the resistance mutations and the mitigating effects of CM largely explain why certain mutations are frequently found in highly transmitted clusters while others are less frequently, rarely or never found in clinical isolates. The CM illustrate how drug resistance affects bacteria and how bacteria evolve to overcome the effects of the antibiotics, and thus a paradigm for how mycobacteria can evolve in response to stress.

Deep whole-genome sequencing reveals no evidence for heteroresistance influencing treatment outcomes among drug-susceptible tuberculosis patients.

The purpose of this study was to investigate the minimum level of heteroresistance that predicts poor tuberculosis treatment outcomes. This retrospective study enrolled 45 new tuberculosis patients with varied treatment outcomes and 16 drug-susceptible retreatment cases. Pretreatment isolates from these 61 patients were whole genome sequenced to detect heteroresistance. Heteroresistance was not found in isolates from any of the new patients, but was detected in isolates from retreatment patients who were nevertheless cured. The results of our small series of patients suggest that heteroresistance <1%, the threshold used to define resistance with the phenotypic proportion method, is not associated with poor treatment outcomes.