Phenotypic and genomic hallmarks of a novel, potentially pathogenic rapidly growing Mycobacterium species related to the Mycobacterium fortuitum complex.

Previously, we have identified a putative novel rapidly growing Mycobacterium species, referred to as TNTM28, recovered from the sputum of an apparently immunocompetent young man with an underlying pulmonary disease. Here we provide a thorough characterization of TNTM28 genome sequence, which consists of one chromosome of 5,526,191 bp with a 67.3% G + C content, and a total of 5193 predicted coding sequences. Phylogenomic analyses revealed a deep-rooting relationship to the Mycobacterium fortuitum complex, thus suggesting a new taxonomic entity. TNTM28 was predicted to be a human pathogen with a probability of 0.804, reflecting the identification of several virulence factors, including export systems (Sec, Tat, and ESX), a nearly complete set of Mce proteins, toxin-antitoxins systems, and an extended range of other genes involved in intramacrophage replication and persistence (hspX, ahpC, sodA, sodC, katG, mgtC, ClpR, virS, etc.), some of which had likely been acquired through horizontal gene transfer. Such an arsenal of potential virulence factors, along with an almost intact ESX-1 locus, might have significantly contributed to TNTM28 pathogenicity, as witnessed by its ability to replicate efficiently in macrophages. Overall, the identification of this new species as a potential human pathogen will help to broaden our understanding of mycobacterial pathogenesis.

Successful expansion of Mycobacterium tuberculosis Latin American and Mediterranean sublineage (L4.3/LAM) in Tunisia mainly driven by a single, long-established clonal complex.

OBJECTIVES: To explore the evolutionary history of Mycobacterium tuberculosis Latin American and Mediterranean (L4.3/LAM) sublineage in Tunisia, where it predominates. METHODS: High-resolution genotyping of 252 L4.3/LAM clinical strains was undertaken, and whole-genome sequencing was performed on 31 representative isolates. RESULTS: Genotyping data coupled with Bayesian analyses split the Tunisian L4.3/LAM strain collection into two divergent entities (65.07% vs 34.92%): a major subpopulation, dominated by a single clonal complex (CC), TUN4.3_CC1 (94.51%); and a minor subpopulation, dominated by TUN4.3_CC2 (42.04%). TUN4.3_CC1 is clearly thriving in Tunisia, accounting for 61.5% of the L4.3/LAM sublineage. TUN4.3_CC1 displayed higher mean allelic richness compared with TUN4.3_CC2 and predominated throughout the entire region, indicating a long-established history. The very low proportion of drug resistance among TUN4.3_CC1 isolates is indicative of their intrinsic ability to spread successfully in the host population. Genomic analyses further confirmed the clear genetic separation between the two main CCs (pairwise fixation index 0.56), and suggested the relatively ancient origin of TUN4.3_CC1. Consistent with its successful expansion, TUN4.3_CC1 showed reduced mean pairwise genetic distance between genomes. CONCLUSIONS: These findings link the successful expansion of L4.3/LAM in Tunisia to a single long-established clone.

Nontuberculous mycobacteria isolated from specimens of pulmonary tuberculosis suspects, Northern Tunisia: 2002-2016.

BACKGROUND: Reports on the worldwide ascending trend of pulmonary nontuberculous mycobacteria (NTM) isolation rates and their effective role in respiratory tract infections are compelling. However, as yet, there are no such data relating to Tunisia. METHODS: Here we carried out a retrospective review of mycobacterial cultures originating from Northern Tunisia, which have been processed in the laboratory of mycobacteria of the Institut Pasteur de Tunis, during the time period 2002-2016. All pulmonary NTM (PNTM) isolates available for culture were characterized phenotypically and their taxonomic status was further established based on polymorphisms in rpoB, 16S rRNA, hsp65, and sodA DNA gene sequences. RESULTS: Of the 10,466 specimens collected from HIV-negative Tunisian patients with presumptive clinical pulmonary TB, 60 (0.6%) yielded PNTM isolates. An overall annual PNTM isolation prevalence of 0.2/100,000 was estimated. As far as could be ascertained, this isolation rate accounts amongst the lowest reported hitherto throughout the world. Among the 30 NTM isolates that were available for culture, 27 (90.0%) have been identified to the species level. The most commonly encountered species was Mycobacterium kansasii (23.3%) subtype 1. Strikingly, all M. kansasii cases were male patients originating from Bizerte, an industrialized region particularly known for iron industry. The remaining NTM species were M. fortuitum (16.6%), M. novocastrense (16.6%), M. chelonae (10.0%), M. gordonae (6.6%), M. gadium (6.6%), M. peregrinum (3.3%), M. porcinum (3.3%), and M. flavescens (3.3%). There were no bacteria of the M. avium complex, the most frequently isolated NTM globally, and the main driver of the rise of NTM-lung diseases. CONCLUSIONS: This study uncovered an exceptional low prevalence of PNTM isolation among HIV-negative TB suspects in Northern Tunisia, suggesting a very low burden of NTM pulmonary disease. However, the frequent isolation of M. kansasii subtype 1, the most pathogenic subtype, particularly from the industrialized region of Bizerte, strongly suggests its effective involvement in a typical pulmonary disease.

Automated IS6110-based fingerprinting of Mycobacterium tuberculosis: Reaching unprecedented discriminatory power and versatility.

BACKGROUND: Several technical hurdles and limitations have restricted the use of IS6110 restriction fragment length polymorphism (IS6110 RFLP), the most effective typing method for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS6110-5'3'FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5' and 3' IS6110 polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS6110-5'3'FP to be used as an alternative to IS6110 RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility. OBJECTIVES: Here we introduced critical amendments to the original IS6110-5'3'FP protocol and compared its performance to that of 24-loci multiple interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR), the current standard method for TB transmission analyses. METHODS: IS6110-5'3'FP protocol modifications involved: (i) the generation of smaller-sized polymorphic fragments for efficient cloning and PCR amplification, (ii) omission of the plasmid amplification step in E. coli for shorter turnaround times, (iii) the use of more stable fluorophores for increased sensitivity, (iv) automated subtraction of background fluorescent signals, and (v) the automated conversion of fluorescent peaks into binary data. RESULTS: In doing so, the overall turnaround time of IS6110-5'3'FP was reduced to 4 hours. The new protocol allowed detecting almost all 5' and 3' IS6110 polymorphic fragments of any given strain, including IS6110 high-copy number Beijing strains. IS6110-5'3'FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the M. tuberculosis lineage 4. CONCLUSIONS: The IS6110-5'3'FP protocol described herein reached the optimal discriminatory potential of IS6110 fingerprinting and proved more accurate than 24-loci MIRU-VNTR in estimating recent TB transmission. The method, which is highly cost-effective, was rendered versatile enough to prompt its evaluation as an automatized solution for a TB integrated molecular surveillance.

MDR-TB Outbreak among HIV-Negative Tunisian Patients followed during 11 Years.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) outbreaks that evolve, from the outset, in a context strictly negative for HIV infection deserve special consideration since they reflect the true intrinsic epidemic potential of the causative strain. To our knowledge, the long-term evolution of such exceptional outbreaks and the treatment outcomes for the involved patients has never been reported hitherto. Here we provide a thorough description, over an 11-year period, of an MDR-TB outbreak that emerged and expanded in an HIV-negative context, Northern Tunisia. METHODOLOGY/PRINCIPAL FINDINGS: From October 2001 to June 2011, the MDR-TB outbreak involved 48 HIV-negative individuals that are mainly young (mean age 31.09 yrs; 89.6% male) and noninstitutionalized. Drug susceptibility testing coupled to mutational analysis revealed that initial transmission involved an isolate that was simultaneously resistant to isoniazid, rifampicin, ethambutol, and streptomycin. The causative Haarlem3-ST50 outbreak strain expanded mainly as an 11-banded IS6110 RFLP profile (77.1%), from which a 12-banded subclone evolved. After undergoing a 2-year treatment with second-line drugs, 22 (45.8%) patients were cured and 3 (6.2%) completed treatment, thus yielding an overall treatment success rate of 52.1%. Among the patients that experienced unfavorable treatment outcomes, 10 (20.8%) failed treatment, 3 (6.2%) were lost to follow-up, 5 (10.4%) died, and 5 (10.4%) could not be evaluated. Poor adherence to treatment was found to be the main independent predictor of unfavorable outcomes (HR: 9.15; 95% CI 1.72-48.73; P = 0.014). Intriguingly, the evolved 12-banded subclone proved significantly associated with unfavorable outcomes (HR: 4.90; 95% CI 1.04-23.04, P = 0.044). High rate of fatality and relapse was further demonstrated at the long-term, since 70% of those whose treatment failed have died, and 24% among those deemed successfully treated have relapsed. CONCLUSIONS/SIGNIFICANCE: Taken together, the data obtained in this study indicate that MDR-TB clinical isolates could become fit enough to cause large and severe outbreaks in an HIV-negative context. Such MDR-TB outbreaks are characterized by low treatment success rates and could evolve towards increased severity, thus calling for early detection of cases and the necessity to raise the bar of surveillance throughout and beyond the treatment period.

Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain.

BACKGROUND: MDR Mycobacterium tuberculosis clinical strains that cause large outbreaks, particularly among HIV-negative patients, are likely to have undergone the most successful compensatory evolution. Hence, mutations secondary to the acquisition of drug resistance are worthy of consideration in these highly transmissible strains. Here, we assessed the role of a mutation within rpoB, rpoB V615M, secondary to the rifampicin resistance-conferring mutation rpoB S531L, which is associated with a major MDR tuberculosis outbreak strain that evolved in an HIV-negative context in northern Tunisia. METHODS: Using BCG as a model organism, we engineered strains harbouring either the rpoB S531L mutation alone or the double mutation rpoB S531L, V615M. Individual and competitive in vitro growth assays were performed in order to assess the relative fitness of each BCG mutant. RESULTS: The rpoB V615M mutation was found to be invariably associated with rpoB S531L. Structural analysis mapped rpoB V615M to the same bridge helix region as rpoB compensatory mutations previously described in Salmonella. Compared with the rpoB single-mutant BCG, the double mutant displayed improved growth characteristics and fitness rates equivalent to WT BCG. Strikingly, the rpoB double mutation conferred high-level resistance to rifampicin. CONCLUSIONS: Here, we demonstrated the fitness compensatory role of a mutation within rpoB, secondary to the rifampicin resistance mutation rpoB S531L, which is characteristic of an MDR M. tuberculosis major outbreak strain. The finding that this secondary mutation concomitantly increased the resistance level to rifampicin argues for its significant contribution to the successful transmission of the MDR-TB strain.

Genetic profiling of Mycobacterium tuberculosis in Tunisia: predominance and evidence for the establishment of a few genotypes.

Typing analyses of 378 Mycobacterium tuberculosis isolates collected between the years 2001 and 2005 from three northern representative regions of Tunisia revealed a highly homogeneous population. Indeed, 84.9 % of all tuberculosis (TB) cases were attributed to the Haarlem, LAM or T families. Strikingly, within each family, more than 60 % of TB cases were due to a single genotype. ST50 (Haarlem3) and ST42 (LAM9) genotypes were exceptionally predominant, representing 46.3 % of all typed isolates. ST50 showed an increased tendency for clustering and was more predominant in the extreme north of the country. By contrast, the more widespread ST42, which was apparently prevalent 17 years ago, displayed weak cluster individualization and a low transmission rate, consistent with its stable association with the Tunisian population. It is believed that both mass BCG vaccination, strictly applied for four decades, and the high endogamy rate that characterizes the Tunisian population could have profoundly shaped the population structure of M. tuberculosis by concurrently favouring the selection and accommodation of particular genotypes.

Tuberculosis due to resistant Haarlem strain, Tunisia.

Multidrug-resistant tuberculosis was diagnosed in 21 HIV-negative, nonhospitalized male patients residing in northern Tunisia. A detailed investigation showed accelerated transmission of a Mycobacterium tuberculosis clone of the Haarlem type in 90% of all patients. This finding highlights the epidemic potential of this prevalent genotype.