Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis. The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds.

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.

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.

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

The alcohol content in wine has increased due to external factors in recent decades. In recent reports, some non-Saccharomyces yeast species have been confirmed to reduce ethanol during the alcoholic fermentation process. Thus, an efficient screening of non-Saccharomyces yeasts with low ethanol yield is required due to the broad diversity of these yeasts. In this study, we proposed a rapid method for selecting strains with a low ethanol yield from forty-five non-Saccharomyces yeasts belonging to eighteen species. Single fermentations were carried out for this rapid selection. Then, sequential fermentations in synthetic and natural must were conducted with the selected strains to confirm their capacity to reduce ethanol compared with that of Saccharomyces cerevisiae. The results showed that ten non-Saccharomyces strains were able to reduce the ethanol content, namely, Hanseniaspora uvarum (2), Issatchenkia terricola (1), Metschnikowia pulcherrima (2), Lachancea thermotolerans (1), Saccharomycodes ludwigii (1), Torulaspora delbrueckii (2), and Zygosaccharomyces bailii (1). Compared with S. cerevisiae, the ethanol reduction of the selected strains ranged from 0.29 to 1.39% (v/v). Sequential inoculations of M. pulcherrima (Mp51 and Mp FA) and S. cerevisiae reduced the highest concentration of ethanol by 1.17 to 1.39% (v/v) in synthetic or natural must. Second, sequential fermentations with Z. bailii (Zb43) and T. delbrueckii (Td Pt) performed in natural must yielded ethanol reductions of 1.02 and 0.84% (v/v), respectively.