It has been accepted that the infection by Mycobacterium tuberculosis (M. tuberculosis) can be more heterogeneous than considered. The emergence of clonal variants caused by microevolution events leading to population heterogeneity is a phenomenon largely unexplored. Until now, we could only superficially analyze this phenomenon by standard fingerprinting (RFLP and VNTR). In this study we applied whole genome sequencing for a more in-depth analysis of the scale of microevolution both at the intrapatient and interpatient scenarios. We found that the amount of variation accumulated within a patient can be as high as that observed between patients along a chain of transmission. Intrapatient diversity was found both at the extrapulmonary and respiratory sites, meaning that this variability can be transmitted and impact on the inference of transmission events. One of the events studied allowed us to track for a single strain the complete process of (i) interpatient microevolution, (ii) intrapatient respiratory variation, and (iii) isolation of different variants at different infected sites of this patient. 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.

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.

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)–variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS 6110 -based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS 6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Background: We present our experience in patients with hematologic malignancy and Pseudomonas aeruginosa infection treated with ceftolozane-tazobactam. Materials/methods: We performed a single-center case-control study including all patients with hematologic malignancy and P. aeruginosa infection treated with ceftolozane-tazobactam (study group) and compared them with similar patients not treated with ceftolozane-tazobactam (control group) to assess safety and efficacy. Results: Nineteen cases and 38 controls were analyzed. Cases were younger (45.6 y vs 57.6 y, p=0.012) and less frequently had bacteremia (52.6% vs 86.8%, p=0.008). They also had a worse MASCC score (10.2 vs 16.1, p=0.0001), more hospital-acquired infections (78.9% vs 47.4%, p=0.013), and more XDR P. aeruginosa (47.4% vs 21.1%, p=0.015). Cases received a median of 14 days (7-18) of ceftolozane-tazobactam (monotherapy in 11 cases [57.9.6%]). Ceftolozane-tazobactam was mostly used as targeted therapy (16; 84.2%) because of resistance (9; 47.4%), failure (4; 21.1%), and toxicity (3; 15.8%). Ten cases had bacteremia (52.6%). The sources were pneumonia (26.3%), catheter-related bacteremia (21.1%), primary bacteremia (21.1%), perianal/genital (15.7%), urinary (10.5%), and skin/soft tissue infection (5.3%). No toxicity was attributed to ceftolozane/tazobactam. More than 60% had neutropenia, and 15.8% fulfilled the criteria for sepsis. There were no significant differences in clinical cure at day 14 (89.5% vs 71.1%, p=0.183) or recurrence (15.8% vs 10.5%, p=0.675). Thirty-day mortality was lower among cases (5.3% vs 28.9%, p=0.045). Conclusions: Ceftolozane-tazobactam was well tolerated and at least as effective as other alternatives for P. aeruginosa infection in patients with hematologic malignancy, including neutropenic patients with sepsis caused by XDR strains.

Background: The analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective. Aim: To propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importations Methods: We selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster. Results: In five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country. Conclusion: We provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.