om as , F e r na n d o G on zález Candelas, SeqCOVID-SPAIN consortium, Tanja Stadler & Richard A. NeherThis is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS–MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS–MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.

Our study adds new data to the understanding of variability in M. tuberculosis in a wide clinical scenario and alerts about the difficulties of establishing thresholds to differentiate relatedness in M. tuberculosis with epidemiological purposes.

We evaluated 117 isolates of Mycobacterium tuberculosis for susceptibility to linezolid by the proportion and E-test methods. Linezolid showed high in vitro activity, with all the strains inhibited by <1 g of the drug per ml. E-test MICs were at least 4 dilutions lower than their equivalents by the standard proportion method.Linezolid is an oxazolidinone whose mechanism of action involves inhibition of protein synthesis at a very early stage (7). This new drug has shown good activity against some grampositive bacteria, including resistant staphylococci, enterococci, and pneumococci (2, 5, 9). However, information in the literature regarding the activity of linezolid against Mycobacterium tuberculosis strains is scarce.We evaluated the in vitro activities of linezolid using the standard and E-test methods against 117 clinical isolates of M. tuberculosis with different levels of susceptibility to first-line antituberculous drugs.(This study was presented in part at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 16 to 19 December 2001.) The active substance as reference powder and the linezolid E-test strips were provided by Pharmacia Upjohn (Barcelona, Spain). Stock solution was prepared at 10,000 g/ml in distilled water. Aliquots of the oxazolidinone were frozen at Ϫ70°C until use. Staphylococcus aureus strain ATCC 29213 was used as a quality control to assure the potency of the drug and the E-test activity.A total of 117 isolates of M. tuberculosis from different patients were selected from our laboratory collection (isolates collected in 1988 to 2000). Susceptibility testing on first-line antituberculous drugs (streptomycin, isoniazid, rifampin, and ethambutol [SIRE]) was performed by the agar proportion method at a reference laboratory. Of all the strains tested, 44 strains (38%) were resistant to at least one SIRE (R-SIRE), 12 strains were resistant to both isoniazid and rifampin, while the remainder were susceptible to the four SIRE drugs (S-SIRE).To prevent bias in the performance of the study, there was a single initial inoculum for each isolate for both the proportion and E-test methods. This inoculum was prepared in Middlebrook 7H9 and was adjusted to be equivalent to that of a McFarland no. 3 standard. Furthermore, both methods were performed in a double-blind manner.The agar proportion method was performed as recommended by the National Committee for Clinical Laboratory Standards (6). Briefly, linezolid was added to 7H10 agar medium (Difco) supplemented with OADC (oleic acid, albumin, dextrose, and catalase) (Becton Dickinson) at 50 to 56°C by doubling the dilutions to yield a final concentration of 0.125 to 4 g/ml. Five milliliters of each concentration of antimycobacterial agent-containing medium was dispensed into plastic quadrant petri dishes. As a growth control, one quadrant in each plate was filled with 7H10 agar medium with no drug. The inoculum of each isolate was prepared by diluting the initial inoculum in Middlebrook 7H9 until the absorbance was ...

Three patients with progressive multifocal leukoencephalopathy (PML) treated with highly active antiretroviral therapy (HAART) worsened clinically and radiologically. At the time of deterioration all three had reduced HIV viraemia and increased CD4 cell counts. Brain biopsy in all three disclosed PML and marked perivascular lymphoplasmacytic infiltration. We reviewed the slides of 28 brain biopsies diagnostic of PML. Inflammatory changes were observed in four out of nine patients on HAART and in one out of 19 patients not on HAART.