MALDI-TOF as a powerful tool for identifying and differentiating closely related microorganisms: the strange case of three reference strains of Paenibacillus polymyxa.

Accurate identification and typing of microbes are crucial steps in gaining an awareness of the biological heterogeneity and reliability of microbial material within any proprietary or public collection. Paenibacillus polymyxa is a bacterial species of great agricultural and industrial importance due to its plant growth-promoting activities and production of several relevant secondary metabolites. In recent years, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used as an alternative rapid tool for identifying, typing, and differentiating closely related strains. In this study, we investigated the diversity of three P. polymyxa strains. The mass spectra of ATCC 842T, DSM 292, and DSM 365 were obtained, analysed, and compared to select discriminant peaks using ClinProTools software and generate classification models. MALDI-TOF MS analysis showed inconsistent results in identifying DSM 292 and DSM 365 as belonging to P. polimixa species, and comparative analysis of mass spectra revealed the presence of highly discriminatory biomarkers among the three strains. 16S rRNA sequencing and Average Nucleotide Identity (ANI) confirmed the discrepancies found in the proteomic analysis. The case study presented here suggests the enormous potential of the proteomic-based approach, combined with statistical tools, to predict and explore differences between closely related strains in large microbial datasets.

Characterization of the Colletotrichum Species Causing Anthracnose in Andean Blackberry in Colombia.

Since 1992, anthracnose of Andean blackberry (Rubus glaucus) has generated losses as high as 40% for farmers in Colombia. In this study, our goal was to characterize 240 Colletotrichum isolates from Andean blackberry in eight areas of Colombia. These isolates were evaluated according to morphological characteristics, sensitivity to benomyl, pathogenicity, and genetic variability. Identification of the genus Colletotrichum was achieved by using species complex-specific polymerase chain reaction primers. A multilocus phylogeny approach was used to identify isolates to the species level with sequences from the ribosomal internal transcribed spacer region and partial sequences of the actin, ß-tubulin 2, calmodulin, chitin synthase 1, glutamine synthetase, and glyceraldehyde-3-phosphate dehydrogenase genes. Most of the isolates were identified as Colletotrichum gloeosporioides sensu lato, were associated with the Castilla ecotype, showed high sensitivity to benomyl, and were highly aggressive. Isolates identified as C. acutatum sensu lato were found mainly on the Thornless ecotype, were highly resistant to benomyl, and showed intermediate aggressiveness. Only three isolates were identified as C. boninense sensu lato. The species identified included C. fructicola, C. kahawae subsp. ciggaro, C. godetiae, C. karstii, C. brassicicola, and undetermined Colletotrichum spp. This study is the first report of these species associated with anthracnose in Andean blackberry.

Identification and Functional Characterization of GAA Mutations in Colombian Patients Affected by Pompe Disease.

Pompe disease (PD) is a recessive metabolic disorder characterized by acid α-glucosidase (GAA) deficiency, which results in lysosomal accumulation of glycogen in all tissues, especially in skeletal muscles. PD clinical course is mainly determined by the nature of the GAA mutations. Although ~400 distinct GAA sequence variations have been described, the genotype-phenotype correlation is not always evident.In this study, we describe the first clinical and genetic analysis of Colombian PD patients performed in 11 affected individuals. GAA open reading frame sequencing revealed eight distinct mutations related to PD etiology including two novel missense mutations, c.1106 T > C (p.Leu369Pro) and c.2236 T > C (p.Trp746Arg). In vitro functional studies showed that the structural changes conferred by both mutations did not inhibit the synthesis of the 110 kD GAA precursor form but affected the processing and intracellular transport of GAA. In addition, analysis of previously described variants located at this position (p.Trp746Gly, p.Trp746Cys, p.Trp746Ser, p.Trp746X) revealed new insights in the molecular basis of PD. Notably, we found that p.Trp746Cys mutation, which was previously described as a polymorphism as well as a causal mutation, displayed a mild deleterious effect. Interestingly and by chance, our study argues in favor of a remarkable Afro-American and European ancestry of the Colombian population. Taken together, our report provides valuable information on the PD genotype-phenotype correlation, which is expected to facilitate and improve genetic counseling of affected individuals and their families.

Diastasis congénita de la sínfisis púbica: reporte de un caso y análisis de la literatura médica / Congenital Diastasis of pubic sínfisis it: report of a case análisis y de la literature

Se presentó una niña que entró a los 8 meses de edad con extrofia cloacal y vesical e imposibilidad para el gateo y la marcha, que ingresó sentada con un arrastre continuo sobre un colchón visceral. Los estudios radiográficos iniciales de malformaciones óseas asociadas mostraron un doble sacro y displasia de las caderas en desarrollo, por lo cual se practicó una técnica quirúrgica de osteotomía bilateral pélvica tipo “pivote” parasacroilica. Se describe la historia clínica de la paciente que fue intervenida con la ayuda de cirugía pediátrica en la Clínica del Niño con un tiempo de seguimiento postoperatorio de dieciséis (16) meses, se analiza una forma de osteotomía pélvica como una opción más para estos pacientes. Igualmente se revisa la literatura de estas lesiones congénitas y su manejo.

Molecular and Pathogenicity Characterization of Sphaceloma manihoticola Isolates from South-Central Brazil.

Isolates of Sphaceloma manihoticola, the asexual stage of Elsinoe brasiliensis, were collected from several regions of south-central Brazil. The isolates were obtained from samples of leaves, stems, and petioles of cassava (Manihot esculenta) and the weedy Euphorbia heterophylla ("amendoim bravo") by directly plating infected tissue onto acidified potato dextrose agar. For pathogenicity studies, 19 isolates were inoculated onto each of two cassava cultivars, MBRA 703 as a susceptible cultivar and MBRA 12 as a resistant cultivar to S. manihoticola. MBRA 703, with the greatest pathogenicity to 58% (11) of the isolates, showed an intermediate pathogenic reaction to 16% (3) of the isolates, and was less pathogenic to 26% (5) of the isolates. MBRA 12, with a less pathogenic reaction to 63% (12) of the isolates, showed an intermediate pathogenic reaction to 16% (3) of the isolates, and was highly pathogenic to 21% (4) of the isolates. The isolates were verified as belonging to the genus Sphaceloma based on their morphological characteristics, including conidia and hyphae of monoconidial isolate. Conidia of isolates were small, thin-walled, ellipsoid to (rarely) globose, commonly with one or two gut-tules. Conidiophores were phialides, hyaline to slightly pigmented 0-to-1 septate; conidiophores from the weedy specie were phialides, hyaline to brown 0-to-2 septate producing hyaline conidia. The isolates also were verified as belonging to the genus Sphaceloma by using a poly-merase chain reaction (PCR) assay, which detected a 645-bp band in all isolates except two (1 and 6) for which the PCR product had 600 bp. Digestion of the amplified product with the enzymes MspI and CfoI allowed differences to be detected in restriction patterns among isolates. A homogeneous banding pattern was obtained for 17 of the isolates but a different restriction pattern was obtained for isolates 1 and 6 of E. heterophylla. This suggests the possibility of another species within this group of isolates. The results indicate the presence of pathogenic variation among isolates of the fungus and an isolate-host interaction, because statistically significant differences were observed between the two cassava cultivars in response to inoculation with the isolates of S. manihoticola.