Unraveling the role of high-intensity resistance training on left ventricle proteome: Is there a shift towards maladaptation?

High-intensity resistance training (RT) induces adaptations that improve physiological function. However, high intensity, volume and/or frequency may lead to injury and other health issues such as adverse cardiac effects. The aim of this study was to evaluate the effect of RT on left ventricle proteome, and to identify the pathways involved on the harmful adaptations induced by this protocol. Male Wistar rats were randomized into 2 groups: Trained (T) and Sedentary (S). Animals from T group were trained for 6weeks, and then all the animals were sacrificed and left ventricle was isolated for analysis. We identified 955 proteins, and 93 proteins were considered; 36 were expressed exclusively in T group, and 4 in S group. Based on quantitative analysis, 42 proteins were found overexpressed and 11 underexpressed in T group compared with S group. Using the Gene Ontology to relate the biological processes in which these proteins are involved, we conclude that RT protocol promotes changes similar to those found in the initial phase of heart failure, but we also observed a concomitant increased expression of protective proteins, suggesting the activation of pathways to avoid major damages on left ventricle and delay the onset of pathological hypertrophy. STATEMENT OF SIGNIFICANCE OF THE STUDY: Our study shows that high-intensity RT protocol changes left ventricle proteome, modifying metabolic profile of heart tissue and inducing the expression of proteins that acts against cardiac injury. We hypothesize that these adaptations occur to prevent the onset of cardiac dysfunction. Despite highly significant, it remains to be determined whether these adaptations are sufficient to further keep left ventricle function and exert cardioprotection, and whether this panel will be shifted towards maladaptation, and heart failure.

Multiple species of Trichosporon produce biofilms highly resistant to triazoles and amphotericin B.

Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.

Breakthrough candidemia due to multidrug-resistant Candida glabrata during prophylaxis with a low dose of micafungin.

We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS2 gene was found in all five isolates. This mutation yielded a 1,3-ß-D-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

Molecular identification, antifungal susceptibility profile, and biofilm formation of clinical and environmental Rhodotorula species isolates.

Rhodotorula species are emergent fungal pathogens capable of causing invasive infections, primarily fungemia. They are particularly problematic in immunosuppressed patients when using a central venous catheter. In this study, we evaluated the species distribution of 51 clinical and 8 environmental Rhodotorula species isolates using the ID32C system and internal transcribed spacer (ITS) sequencing. Antifungal susceptibility testing and biofilm formation capability using a crystal violet staining assay were performed. Using ITS sequencing as the gold standard, the clinical isolates were identified as follows: 44 R. mucilaginosa isolates, 2 R. glutinis isolates, 2 R. minuta isolates, 2 R. dairenensis isolates, and 1 Rhodosporidium fluviale isolate. The environmental isolates included 7 R. mucilaginosa isolates and 1 R. slooffiae isolate. Using the ID32C system, along with a nitrate assimilation test, only 90.3% of the isolates tested were correctly identified. In the biofilm formation assay, R. mucilaginosa and R. minuta exhibited greater biofilm formation ability compared to the other Rhodotorula species; the clinical isolates of R. mucilaginosa showed greater biofilm formation compared to the environmental isolates (P = 0.04). Amphotericin B showed good in vitro activity (MIC ≤ 1 µg/ml) against planktonic cells, whereas voriconazole and posaconazole showed poor activity (MIC(50)/MIC(90), 2/4 µg/ml). Caspofungin and fluconazole MICs were consistently high for all isolates tested (≥64 µg/ml and ≥ 4 µg/ml, respectively). In this study, we emphasized the importance of molecular methods to correctly identify Rhodotorula species isolates and non-R. mucilaginosa species in particular. The antifungal susceptibility profile reinforces amphotericin B as the antifungal drug of choice for the treatment of Rhodotorula infections. To our knowledge, this is the first study evaluating putative differences in the ability of biofilm formation among different Rhodotorula species.

In vitro susceptibility of a large collection of Candida Strains against fluconazole and voriconazole by using the CLSI disk diffusion assay.

We evaluated all Candida spp. isolates obtained from patients admitted to two tertiary care hospitals between 1999 and 2003 in the city of São Paulo, Brazil. The in vitro activities of fluconazole (FCZ) and voriconazole were determined by the agar disk diffusion test using the Clinical and Laboratory Standards Institute M44-A guidelines. The inhibition zone diameters were read and interpreted automatically by the BIOMIC(®) image-analysis plate reader system. We tested a total of 4,625 strains, including 2,393 strains of C. albicans (51.7%), 658 of C. tropicalis (14.2%), 503 of C. glabrata (10.9%), 495 of C. parapsilosis (10.7%), 292 of C. rugosa (6.3%), 195 of C. guilliermondii (4.2%) and 89 of other Candida species (2.0%). Only 2.0% of the strains tested were classified as dose-dependent susceptible (DDS), and 5.8% of them were resistant to FCZ. The resistance or DDS to fluconazole was verified mainly among C. glabrata (7.8%), C. krusei (67.9%) and C. rugosa (65.1%). Voriconazole exhibited better activity in vitro than fluconazole, even in isolates fluconazole resistant. The resistance of fluconazole and voriconazole did not increase in the isolates of Candida spp. during the evaluated period.

In vitro evaluation of putative virulence attributes of oral isolates of Candida spp. obtained from elderly healthy individuals.

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.

Characteristics of biofilm formation by Candida tropicalis and antifungal resistance.

Candida tropicalis is a common species related to nosocomial candidemia and candiduria. Most Candida spp. infections are associated with biofilm formation on implanted medical devices or on host epithelial cell surfaces. Sessile cells display phenotypic traits dramatically different from those of their free-living, planktonic counterparts, such as increased resistance to antimicrobial agents and to host defenses. The characteristics of C. tropicalis biofilm formation in vitro are described. By an XTT-reduction assay, an increase in metabolic activity was observed up to 24 h of biofilm formation, and this activity showed a linear relationship with sessile cell density. Scanning electron microscopy was used to further characterize C. tropicalis biofilms. The initial adherence of yeast cells was followed by germination, microcolony formation, filamentation and maturation at 24-48 h. Mature biofilms consisted of a dense network of yeast cells and filamentous forms of C. tropicalis. Increased resistance of sessile cells against fluconazole and amphotericin B was also demonstrated. Real-time reverse transcription-PCR quantification showed that sessile cells overexpressed ERG11 (coding for lanosterol 14 alpha-demethylase) and MDR1 (coding for an efflux protein belonging to the major facilitator superfamily). These mechanisms may contribute to the fluconazole resistance of the C. tropicalis biofilm.

Utilização de um meio cromogênico e da técnica de semi-nested PCR para identificação de espécies de Candida / Use of chromogenic medium and semi-nested PCR-based assay to identify Candida species

Nesse trabalho, o meio cromogênico CHROMagar Candida e a técnica de semi-nested PCR (sn-PCR) foram comparados quanto a sua capacidade de identificar a espécie de 52 isolados clínicos de Candida sp. Com o emprego do meio cromogênico, 39 (75%) isolados foram identificados presuntivamente como C. albicans (n = 22), C. glabrata (n = 9), C. tropicalis (n = 5) e C. krusei (n = 3). Treze isolados (25%) não puderam ser identificados. Por meio da técnica de sn-PCR, que se baseia na amplificação de uma região do cluster gênico do RNA ribossomal (5.8S – 28S), 43 (83%) isolados foram identificados como C.albicans (n = 24), C. glabrata (n = 11), C. tropicalis (n = 5), C. parapsilosis (n = 3) e nove isolados não puderam ser identificados em nível de espécie. Entre os 52 isolados analisados, 34 (65,4%) apresentaram resultados concordantes pelos dois métodos, 12 (23,1%) apresentaram resultados discrepantes, e 6 (11,5%) não puderam ser identificadas por nenhuma das duas metodologias. Os resultados indicam que ambas as técnicas apresentam limitações, mas o método de sn-PCR é mais adequado que o meio cromogênico para a identificação de espécies do gênero Candida, devido ao menor número de isolados que não puderam ser identificados.