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

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

International audienceMDR 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. 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. 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. 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

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

International audiencePreviously, 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

Epidemiological, clinical, and bacteriological findings among tunisian patients with tuberculous cervical lymphadenitis

International audienceThe incidence of tuberculous cervical lymphadenitis (TCL) is likely to be on the rise in Tunisia over the last two decades. However, this pathological condition remains poorly characterized, in regard to involved mycobacterial species. Purpose: To study the etiology and treatment outcome of TCL among Tunisian patients; to indicate the myc-obecteria responsible for the majority of TCL cases. This prospective study has involved 114 patients, clinically diagnosed as TCL, presenting to a National referral hospital in Tunis, from November 2011 to January 2014. Results: 69 patients displayed typical cytological signs of TCL, whose mycobacterial etiology was confirmed in 23 cases. 4 cases may be a possible disseminated TB. Mycobacterial species assignment could be established for 15 culture-positive specimens, 11 of which were found to be Mycobacterium bovis, while the remaining were identified as tuberculosis. 6 of M. bovis isolates belonged to the BOVIS1 spoligopattern, and 3 of the M. tuberculosisisolates to the Haarlem3, one of the most prevalent genotype associated with pulmonary tuberculosis in Tunisia. Although all subjects lived in an urban area, the majority declared having consumed raw milk and derived products. The cure rate was low, as among patients that completed an anti-tubercular chemotherapy of at least 8 months, only 55.5% were cured. Conclusion: Our results are consistent with literature since positive cases demonstrated by AFB smear test don't exceed 37.4% and varied by culture between 19 and 71%. This is the first indication that M. bovis is a significant cause of TCL in Tunisia. Consumption of unpasteurized dairy products is the most likely source of transmission. The low cure rates among TCL cases should call health authorities for improved management and therapeutic schemes

Automated IS<i>6110</i>-based fingerprinting of <i>Mycobacterium tuberculosis</i>: Reaching unprecedented discriminatory power and versatility

<div><p>Background</p><p>Several technical hurdles and limitations have restricted the use of IS<i>6110</i> restriction fragment length polymorphism (IS<i>6110</i> RFLP), the most effective typing method for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS<i>6110</i>-5’3’FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5’ and 3’ IS<i>6110</i> polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS<i>6110</i>-5’3’FP to be used as an alternative to IS<i>6110</i> RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility.</p><p>Objectives</p><p>Here we introduced critical amendments to the original IS<i>6110</i>-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.</p><p>Methods</p><p>IS<i>6110</i>-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 <i>E</i>. <i>coli</i> 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.</p><p>Results</p><p>In doing so, the overall turnaround time of IS<i>6110</i>-5’3’FP was reduced to 4 hours. The new protocol allowed detecting almost all 5’ and 3’ IS<i>6110</i> polymorphic fragments of any given strain, including IS<i>6110</i> high-copy number Beijing strains. IS<i>6110</i>-5’3’FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the <i>M</i>. <i>tuberculosis</i> lineage 4.</p><p>Conclusions</p><p>The IS<i>6110</i>-5’3’FP protocol described herein reached the optimal discriminatory potential of IS<i>6110</i> 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.</p></div

Comparative cost estimates (USD).

<p>Comparative cost estimates (USD).</p

Comparison of the discriminatory power of IS<i>6110</i>-5’3’FP and 24-loci MIRU-VNTR.

<p>Comparison of the discriminatory power of IS<i>6110</i>-5’3’FP and 24-loci MIRU-VNTR.</p

Number of IS<i>6110</i>-5’3’FP-generated peaks relative to IS<i>6110</i> copies as determined from IS<i>6110</i> RFLP profiles.

<p>Number of IS<i>6110</i>-5’3’FP-generated peaks relative to IS<i>6110</i> copies as determined from IS<i>6110</i> RFLP profiles.</p

Box plot showing the sizes of IS<i>6110</i> polymorphic amplicons generated by IS<i>6110</i>-5’3’FP using the 11-banded laboratory reference strain genomic DNA digested either by <i>BstU</i>I or <i>Hinc</i>II.

<p>The IS<i>6110</i>-5’3’FP products were fractionated without being diluted on an ABI PRISM 3100 capillary DNA sequencer (Applied Biosystems Inc., CA, USA). The boxes show the 25% to 75% interquartile range.</p