Molecular Identification, Genotyping, Phenotyping, and Antifungal Susceptibilities of Medically Important Trichosporon, Apiotrichum, and Cutaneotrichosporon Species.

Recently, Trichosporon taxonomy has been reevaluated and new genera of the Trichosporonaceae family have been described. Here, 26 clinical isolates were submitted for identification via sequencing of the intergenic space 1 (IGS1) region, genotyping, and investigation of virulence factors. Antifungal susceptibility was determined using the CLSI broth microdilution method for fluconazole (FLC), itraconazole (ITC), and amphotericin B (AMB). Of these, 24 isolates were identified, including 12 T. asahii, 4 T. inkin, 3 T. faecale, 1 T. coremiiforme, 1 T. japonicum, 2 Cutaneotrichosporon dermatis (formerly T. dermatis), and 1 Apiotrichum mycotoxinivorans (formerly T. mycotoxinivorans). Species-level identification of 2 isolates was not successful; they were described as Trichosporon sp. We observed optimal colonial development at 35-40 °C. Lipase was the major extracellular enzyme produced (100%); caseinase was not produced (0%). Biofilms were produced by all isolates (classified as low). High AMB minimum inhibitory concentration (MIC) was observed, with all strains resistant. Fluconazole was the most active drug among the antifungals tested. However, high MICs for FLC were observed in C. dermatis and A. mycotoxinivorans species, which also showed resistance to ITC and AMB. This study, conducted in the Northern region of Brazil, identified 5 Trichosporon species along with C. dermatis and A. mycotoxinivorans and demonstrated their pathogenic potential through their ability to produce important virulence factors. This may contribute to our understanding of the epidemiology and factors related to the pathogeneses of species in the Trichosporonaceae family.

Molecular Epidemiology of Sporadic and Outbreak-Related Salmonella Typhi Isolates in the Brazilian North Region: A Retrospective Analysis from 1995 to 2013.

Typhoidal salmonellosis is a global public health problem occurring in developing endemic regions. In Brazil, cases are mostly registered in the North and Northeast regions. Molecular characterization of the strains is important to understand the epidemiology of disease infections and to design control strategies. The present study retrospectively evaluates the genotyping features of sporadic and outbreak-related Salmonella Typhi isolates from the Brazilian North region. Bacterial isolates were recovered from blood and a rectal swab of patients in the states of Acre and Pará, Brazilian North region, in the period of 1995 to 2013, and were submitted to genotyping by applying Multilocus sequence typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE) reference methods. MLST genotyping revealed the presence of epidemic clones ST1 and ST2, and 20 pulsotypes were identified by PFGE, including four distinct clusters (A-D), and six subclusters (A1-D1) with indistinguishable strains in different periods and locations. To conclude, the obtained data demonstrates the temporal stability, adaptation, and transmission of outbreak-related and sporadic S. Typhi strains over time, contributing to the transmission chain in the region.

Molecular epidemiology of mycobacteria among herds in Marajó Island, Brazil, reveals strains genetically related and potential zoonotic risk of clinical relevance.

Mycobacterium bovis is the main causative agent of bovine tuberculosis (bTB) being among the animal-adapted Mycobacterium tuberculosis complex. Herds can also be infected with non-tuberculous mycobacteria (NTM) causing a negative effect on the economy and on animal and human health through zoonotic infections. Molecular tools are required for mycobacteria identification; thus, it is laborious to determine the epidemiological information of mycobacteria among herds. We aimed to describe the mycobacterial pathogens associated with cases of suspected bTB lesions in cattle/buffaloes slaughtered for consumption and to investigate bTB transmission. We evaluated 74 lesion samples from 48 animals (27 bovine/21 buffaloes) from 16 mapped farms. Positives samples from nested-PCR were cultured in Lowenstein-Jensen (LJ), 2% pyruvate (LJ + P), and 2% glycerol (LJ + G) media, followed by Ziehl-Neelsen (ZN) staining technique and partial gene sequencing (hsp65, rpoB, and 16S-rRNA). Spoligotyping and 24-MIRU-VNTR were performed. The LJ + P increased the chance of obtaining bacilli. The respiratory tract and the oral cavity were the most important infection route. In addition, the calcified part of the lesions suggested chronic bTB. Spoligotypes of M. bovis (SIT986/SB0885) differed from others found in South America, and the MIRU-VNTR 24 loci suggested that bTB was associated to highly related strains. The NTM species found are of clinical importance in humans.

A case of pulmonary infection due to Mycobacterium paraffinicum from the Amazon Region.

M. paraffinicum, a slow-growing scotochromogenic mycobacterium that uses paraffinic hydrocarbons other than methane, i.e. inorganic carbon sources, was originally isolated from soil samples, but only in 2010 definitely achieved the species status. We have described here the first report of pulmonary disease due to M. paraffinicum in Amazon Region.

A fatal case of pulmonary infection by Mycobacterium colombiense in Para State, Amazon Region, Brazil.

Mycobacterium avium complex (MAC) is a heterogeneous group of species found in several environmental sources and that exhibit variable degrees of pathogenicity. Among the MAC members, Mycobacterium colombiense has been related to pulmonary disease and disseminated infection in HIV-infected patients in Colombia. Lymphadenopathy cases have also been reported. We have described a fatal case of M. colombiense pulmonary disease in a Brazilian patient without evidence of HIV infection or other known causes of immunosuppression.

Molecular identification of nontuberculous mycobacteria isolates in a Brazilian mycobacteria reference laboratory.

This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.

Molecular identification of rapidly growing mycobacteria isolates from pulmonary specimens of patients in the State of Pará, Amazon region, Brazil.

We isolated 44 strains of rapidly growing mycobacteria (RGM) from 19 patients with pulmonary infections assisted at the Instituto Evandro Chagas (Pará, Brazil) from 2004 to 2007. Identification at the species level was performed by PCR restriction fragment length polymorphism analysis (PRA) of a 441 bp hsp65 fragment and partial 16S rRNA, hsp65, and rpoB gene sequencing. Genotyping by PRA yielded 3 digestion patterns: one identical to Mycobacterium abscessus type I (group I); another to M. abscessus type II, Mycobacterium bolletii, and Mycobacterium massiliense (group II); and a third typical for Mycobacterium fortuitum type I (group III). When comparing analysis of the 3 genes, more discrimination was obtained by rpoB gene sequence, which allowed good distinction between group I, II, and III strains and subclassification of group II strains in SG IIa (M. bolletii) and SG IIb (M. massiliense). In this study, we show that the description of new RGM species requires the establishment of standardized procedures for RGM identification and the alert of the clinician about their involvement in pulmonary disease and the necessity of treatment for control and cure.