Candida Isolation During COVID-19: Microbiological Findings of a Prospective Study in a Regional Hospital.

BACKGROUND: Prolonged hospitalization due to the COVID-19 pandemic gathered risk factors for developing invasive candidiasis. AIM: To describe Candida spp. isolated from patients with clinical suspicion of COVID treated in a public hospital specialized in COVID-19 during the pandemic, considering the susceptibility profiles and the risk factors related to the species detected in a positive yeast culture. METHODS: From different samples of 33 patients with comorbidities, 42 clinical isolates were identified by VITEKⓇ MS Plus. Antifungal susceptibility testing was performed using VITEKⓇ 2 Compact with the AST-YS08 card. RESULTS: The most frequently identified species were C. albicans and C. glabrata, which were also the most common co-infections, Saprochaete capitata, an uncommon yeast was isolated in one patient. 85% of the co-infections were COVID positive and 100% of patients with a co-infection required mechanical ventilation (MV) which has been described as one of the major predisposing factors to candidiasis. Candida species vary in their response to treatment. In this study, 44% of isolates identified as C. glabrata were fluconazole-resistant, which were also immediately susceptible to caspofungin; this profile limits therapeutic options and emphasizes the importance of evaluating the susceptibility profile. CONCLUSIONS: This work highlights the increase in isolation of different Candida species during COVID-19 and the importance of establishing criteria to declare Candida colonization or infection and the correct etiological identification to establish an agent-based antifungal treatment, to reduce the spreading risk of Candida spp. in the hospital environment, mortality, time, and cost of hospitalization.

Timed restricted feeding cycles drive daily rhythms in female rats maintained in constant light but only partially restore the estrous cycle.

Light at night is an emergent problem for modern society. Rodents exposed to light at night develop a loss of circadian rhythms, which leads to increased adiposity, altered immune response, and increased growth of tumors. In female rats, constant light (LL) eliminates the estrous cycle leading to a state of persistent estrus. The suprachiasmatic nucleus (SCN) drives circadian rhythms, and it interacts with the neuroendocrine network necessary for reproductive function. Timed restricted feeding (RF) exerts a powerful entraining influence on the circadian system, and it can influence the SCN activity and can restore rhythmicity or accelerate re-entrainment in experimental conditions of shift work or jet lag. The present study explored RF in female rats exposed to LL, with the hypothesis that this cyclic condition can rescue or prevent the loss of daily rhythms and benefit the expression of the estrous cycle. Two different feeding schedules were explored: 1. A 12-h food/12-h fasting schedule applied to arrhythmic rats after 3 weeks in LL, visualized as a rescue strategy (LL + RFR, 3 weeks), or applied simultaneously with the first day of LL as a preventive strategy (LL + RFP, 6 weeks). 2. A 12-h window of food intake with food given in four distributed pulses (every 3 h), applied after 3 weeks in LL, as a rescue strategy (LL + PR, 3 weeks) or applied simultaneously with the first day of LL as a preventive strategy (LL + PP, 6 weeks). Here, we present evidence that scheduled feeding can drive daily rhythms of activity and temperature in rats exposed to LL. However, the protocol of distributed feeding pulses was more efficient to restore the day-night activity and core temperature as well as the c-Fos day-night change in the SCN. Likewise, the distributed feeding partially restored the estrous cycle and the ovary morphology under LL condition. Data here provided indicate that the 12-h feeding/12-h fasting window determines the rest-activity cycle and can benefit directly the circadian and reproductive function. Moreover, this effect is stronger when food is distributed along the 12 h of subjective night.

Vacuolar proteases from Candida glabrata: acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression / Proteasas vacuolares de Candida glabrata: aspartil proteasa ácida PrA, proteasa neutra PrB y carboxipeptidasa CpY. La fuente de nitrógeno influye en sus niveles de expresión

Background. The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. Aims. The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Methods. Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Results. Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. Conclusions. The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen (AU)

Antecedentes. La vacuola de Saccharomyces cerevisiae está involucrada activamente en el mecanismo de autofagia, desarrollando una labor importante en la homeostasis, degradación, recambio proteico, desintoxicación y protección de la célula en condiciones de estrés. Por el contrario, las proteasas vacuolares de Candida glabrata aún no han sido estudiadas por completo. Objetivos. El presente trabajo describe por primera vez la actividad proteolítica vacuolar en C. glabrata. Métodos. Los estudios bioquímicos realizados en C. glabrata pusieron de manifiesto la presencia de diferentes actividades proteolíticas: aspartil proteinasa ácida, que actúa sobre sustratos como la albúmina y la hemoglobina ácida desnaturalizada; serín proteasa neutra, con actividad sobre el substrato de tipo colágeno hide powder azure, y serín carboxipeptidasa, que actúa sobre N-benzoil-tyr-pNa. Resultados. La obtención de una fracción subcelular mostró una elevada actividad enzimática específica de las tres proteasas, lo que permitió confirmar su localización vacuolar. Se realizaron análisis de la expresión de los genes CgPEP4 (CgAPR1), CgPRB1 y CgCPY1 (CgPRC1), codificantes de las actividades proteolíticas aspartil proteasa A, proteasa neutra B y carboxipeptidasa Y, respectivamente. Los resultados reflejan una regulación diferencial de la expresión de la proteasa, dependiendo de la fuente de nitrógeno. Conclusiones. Las proteasas codificadas por los genes CgPEP4, CgPRB1 y CgCPY1 podrían participar en el proceso de autofagia y supervivencia de este patógeno oportunista (AU)

Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression.

BACKGROUND: The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. AIMS: The present paper is the first report on proteolytic activity in the C. glabrata vacuole. METHODS: Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. RESULTS: Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. CONCLUSIONS: The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.

Identification and expression of nor efflux family genes in Staphylococcus epidermidis that act against gatifloxacin.

NorA, NorB, and NorC are efflux proteins in the Nor family that regulate the secretion of fluoroquinolones, and MgrA/NorR is a transcription factor of the Nor family. Overexpression of Nor family proteins provides fluoroquinolone resistance in Staphylococcus aureus. However, in coagulase-negative staphylococci (CNS), members of the Nor family had not been identified. In this work, the presence of Nor family proteins in Staphylococcus spp. and the expression of Nor family in gatifloxacin resistant S. epidermidis strains obtained from ocular infections (OI) were identified and analyzed. S. epidermidis strains from OIs (n = 44) and healthy skin (HS; n = 52) were isolated. The nor family genes were identified in CNS using PCR, sequencing and phylogenetic approaches. Nor family expression was determined by RT-PCR. NorA efflux activity was determined using the automated ethidium bromide method. In-silico analysis showed that norA, mgrA/norR, and "norB-like" and "norC-like" (norB/norC) genes are present in CNS. The nor family genes were distributed and constitutively expressed in all S. epidermidis strains studied. In one gatifloxacin resistant strain isolated from the endophthalmitis, treatment with gatifloxacin induced overexpression of the norA gene and resulted in high activity of NorA efflux. These results indicate that the Nor family of proteins is present in CNS, and the NorA efflux mechanism for gatifloxacin response occurs in at least one strain of S. epidermidis, contributing to gatifloxacin resistance.

Differential expression of Candida dubliniensis-secreted aspartyl proteinase genes (CdSAP1-4) under different physiological conditions and during infection of a keratinocyte culture.

The in vitro and keratinocyte (HaCAT cells) culture expression of four putative genes coding for secreted aspartyl proteases of Candida dubliniensis-CdSAP1, CdSAP2, CdSAP3, and CdSAP4 (CdSAP1-4) - is reported for the first time. In addition, CdSAP7, 8, 9, and 10, orthologous genes of Candida albicans, were recognized in C. dubliniensis genome. There are no orthologs of C. albicans SAP5 and 6 in C. dubliniensis. The expression of CdSAP1 and 2 was independent of the morphological stage of C. dubliniensis; they are expressed at both pH 4 and pH 7, and were induced with albumin as nitrogen source. CdSAP3 expression was regulated by the pH, and was related to the infection process of keratinocytes. Expression of CdSAP4 predominated during the mycelial phase and the initial stage of keratinocyte infection. During infection of the HaCaT cell line, only genes CdSAP3-4 were expressed, and keratinocytes were affected in their number and shape by the infection with C. dubliniensis; however, this effect decreased in the presence of pepstatin A (aspartyl protease inhibitor). Pepstatin A was not able to inhibit keratinocyte damage. Based on the aforementioned, we suggest that the Saps from C. dubliniensis could be considered a virulence factor just as those from C. albicans, and participants in the nitrogen metabolism of the yeast for nutrient acquisition.