Characterization of Candida species isolated from the hands of the healthcare workers in the neonatal intensive care unit.

Colonization of health professional hands by potentially pathogenic microorganisms capable to expressing virulence factors, such as Candida spp., is worrisome because of the high contact between patients and professionals. The study aims to evaluate, in vitro, the following virulence factors: hemolytic activity, DNAse expression, biofilm formation, and susceptibility for antifungal agents of Candida species isolated from health professionals hands of a neonatal intensive care unit (NICU). The study includes 50 isolates of Candida spp.: 19 C. parapsilosis sensu stricto, three C. metapsilosis, one C. orthopsilosis, seven C. albicans, six C. famata, five C. lusitaniae, three C. krusei, two C. kefyr, two C. tropicalis, one C. glabrata, and one C. guilliermondii. The hemolytic activity and DNAse were investigated using blood agar and DNAse agar, respectively. Biofilm production was evaluated through XTT sodium salt reduction ability, and the susceptibility of the isolates to antifungals through the microdilution methodology. Forty-nine isolates presented at least one of the three virulence factors investigated. C. albicans showed more intense hemolytic activity. DNAse production was statistically significant between the C. parapsilosis complex and C. albicans, as well as between the C. parapsilosis complex and C. krusei, even as between C. famata and C. albicans, and between C. famata and C. krusei. Forty-three isolates produced biofilm. Seventy-eight percent of the isolates were sensitive to the three antifungals tested. This study demonstrated that Candida isolated from healthcare professionals' hands has virulence.

Bothrops snake venom L-amino acid oxidases impair biofilm formation of clinically relevant bacteria.

The present work addressed the abilities of two L-amino acid oxidases isolated from Bothrops moojeni (BmooLAAO-I) and Bothrops jararacussu (BjussuLAAO-II) snake venoms to control the growth and prevent the biofilm formation of clinically relevant bacterial pathogens. Upon S. aureus (ATCC BAA44) and S. aureus (clinical isolates), BmooLAAO-I (MIC = 0.12 and 0.24 µg/mL, respectively) and BjussuLAAO-II (MIC = 0.15 µg/mL) showed a potent bacteriostatic effect. Against E. coli (ATCC BAA198) and E. coli (clinical isolates), BmooLAAO-I (MIC = 15.6 and 62.5 µg/mL, respectively) and BjussuLAAO-II (MIC = 4.88 and 9.76 µg/mL, respectively) presented a lower extent effect. Also, BmooLAAO-I (MICB50 = 0.195 µg/mL) and BjussuLAAO-II (MICB50 = 0.39 µg/mL) inhibited the biofilm formation of S. aureus (clinical isolates) in 88% and 89%, respectively, and in 89% and 53% of E. coli (clinical isolates). Moreover, scanning electron microscopy confirmed that the toxins affected bacterial morphology by increasing the roughness of the cell surface and inhibited the biofilm formation. Furthermore, analysis of the tridimensional structures of the toxins showed that the surface-charge distribution presents a remarkable positive region close to the glycosylation motif, which is more pronounced in BmooLAAO-I than BjussuLAAO-II. This region may assist the interaction with bacterial and biofilm surfaces. Collectively, our findings propose that venom-derived antibiofilm agents are promising biotechnological tools which could provide novel strategies for biofilm-associated infections.

Back to Nature: Medicinal Plants as Promising Sources for Antibacterial Drugs in the Post-Antibiotic Era.

Undoubtedly, the advent of antibiotics in the 19th century had a substantial impact, increasing human life expectancy. However, a multitude of scientific investigations now indicate that we are currently experiencing a phase known as the post-antibiotic era. There is a genuine concern that we might regress to a time before antibiotics and confront widespread outbreaks of severe epidemic diseases, particularly those caused by bacterial infections. These investigations have demonstrated that epidemics thrive under environmental stressors such as climate change, the depletion of natural resources, and detrimental human activities such as wars, conflicts, antibiotic overuse, and pollution. Moreover, bacteria possess a remarkable ability to adapt and mutate. Unfortunately, the current development of antibiotics is insufficient, and the future appears grim unless we abandon our current approach of generating synthetic antibiotics that rapidly lose their effectiveness against multidrug-resistant bacteria. Despite their vital role in modern medicine, medicinal plants have served as the primary source of curative drugs since ancient times. Numerous scientific reports published over the past three decades suggest that medicinal plants could serve as a promising alternative to ineffective antibiotics in combating infectious diseases. Over the past few years, phenolic compounds, alkaloids, saponins, and terpenoids have exhibited noteworthy antibacterial potential, primarily through membrane-disruption mechanisms, protein binding, interference with intermediary metabolism, anti-quorum sensing, and anti-biofilm activity. However, to optimize their utilization as effective antibacterial drugs, further advancements in omics technologies and network pharmacology will be required in order to identify optimal combinations among these compounds or in conjunction with antibiotics.

Cryptococcus liquefaciens isolated from the hand of a healthcare professional in a neonatal intensive care unit.

Fungal infections are responsible for high morbidity and mortality in neonatal patients, especially in premature newborns. Infections in neonates caused by Cryptococcus spp. are rare, but it has occurred in an immunocompromised population. This study aims to describe the isolation of Cryptococcus liquefaciens from the hands of a health professional in a neonatal intensive care unit, and to evaluate the production of biofilm and virulence factors and susceptibility to antifungals. Antifungal susceptibility tests were performed according to Clinical and Laboratory Standard Institute document M27-A3. Thermotolerance virulence factors and DNase, phospholipase, proteinase, and hemolytic activities were verified through phenotypic tests; biofilm was evaluated by determining the metabolic activity and biomass. The isolate did not produce any of the tested enzymes and was susceptible to all antifungals (amphotericin B, fluconazole, and micafungin). The growth at 37 °C was very weak; however, the isolate showed a strong biomass production and low metabolic activity. This is the first report of C. liquefaciens isolated from the hands of a health professional. The isolate did not express any of the studied virulence factors in vitro, except for the low growth at 37 °C in the first 48 h, and the strong production of biofilm biomass. Cryptococcus liquefaciens can remain in the environment for a long time and is a human pathogen because it tolerates temperature variations. This report draws attention to the circulation of rare species in critical locations, information that may help in a fast and correct diagnosis and, consequently, implementation of an appropriate treatment.

Quantification of biofilm produced by clinical, environment and hands' isolates Klebsiella species using colorimetric and classical methods.

Some species of Klebsiella, such as Klebsiella pneumoniae and Klebsiella oxytoca, are important nosocomial pathogens frequently involved in outbreaks in Neonatal Intensive Care Units (NICU) and have the ability to form a biofilm. This study aims to evaluate the biofilm production of K. pneumoniae and K. oxytoca isolates collected from the hands of health professionals, neonates' blood and the environment of a Brazilian NICU, using three colorimetric methods and a classical method of counting the colony-forming units and compare the analysis among these techniques. The biofilm formation was carried out by the microplate technique, using three colorimetric assays: crystal violet, safranin and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5 [(phenylamino) arbonyl] - 2H-tetrazolium hydroxide (XTT). Also, colony-forming units were determined. Twenty-eight isolates of K. pneumoniae were collected from the blood, hands and environment and five of K. oxytoca from the hands and environment. All of them were strong biofilm producers, but K. pneumoniae isolates produced more biofilm than K. oxytoca when compared to the American Type Culture Collection (ATCC) strains used as positive controls. The number of viable cells in the biofilm produced by K. pneumoniae isolated from blood was significantly higher than in the control sample. Regarding the three colorimetric tests used in the study, the violet crystal obtained a higher absorbance average. The use of crystal-violet and XTT in the evaluation of biofilm in vitro make possible a complete analysis, since that it can quantify the total biomass (including the extracellular matrix) and evaluate the metabolic activity. In conclusion, this study identified isolates of K. pneumoniae and K. oxytoca that produce biofilms in the NICU and the bloodstream of neonates. This fact deserves attention since these patients are immunocompromised. The best methods will be chosen to answer research questions by always adopting more than one method so that more than one parameter or component of the biofilm is analyzed.

High mortality by nosocomial infections caused by carbapenem-resistant P. aeruginosa in a referral hospital in Brazil: facing the perfect storm.

Introduction. Carbapenem-resistant Pseudomonas aeruginosa is responsible for increased patient mortality.Gap Statement. Five and 30 day in-hospital all-cause mortality in patients with P. aeruginosa infections were assessed, followed by evaluations concerning potential correlations between the type III secretion system (TTSS) genotype and the production of metallo-ß-lactamase (MBL).Methodology. This assessment comprised a retrospective cohort study including consecutive patients with carbapenem-resistant infections hospitalized in Brazil from January 2009 to June 2019. PCR analyses were performed to determine the presence of TTSS-encoding genes and MBL genes.Results. The 30-day and 5-day mortality rates for 262 patients were 36.6 and 17.9 %, respectively. The unadjusted survival probabilities for up to 5 days were 70.55 % for patients presenting exoU-positive isolates and 86 % for those presenting exo-negative isolates. The use of urinary catheters, as well as the presence of comorbidity conditions, secondary bacteremia related to the respiratory tract, were independently associated with death at 5 and 30 days. The exoS gene was detected in 64.8 % of the isolates, the presence of the exoT and exoY genes varied and exoU genes occurred in 19.3 % of the isolates. The exoU genotype was significantly more frequent among multiresistant strains. MBL genes were not detected in 92 % of the isolates.Conclusions. Inappropriate therapy is a crucial factor regarding the worse prognosis among patients with infections caused by multiresistant P. aeruginosa, especially those who died within 5 days of diagnosis, regardless of the genotype associated with TTSS virulence.

Candidemia by Candida parapsilosis in a neonatal intensive care unit: human and environmental reservoirs, virulence factors, and antifungal susceptibility.

The Candida parapsilosis complex has emerged as one of the main causes of candidemia worldwide. This study aims to evaluate possible C. parapsilosis sensu stricto reservoirs in a NICU, the expression of virulence factors, and antifungal susceptibility, and to analyze their genetic and phenotypic similarity. The study included 17 isolates of C. parapsilosis: seven environmental, one from a newborn's mother, and nine samples from six newborns. We used molecular and phenotypic tests to characterize the isolates and to trace possible routes of infection. The genetic similarity was determined by random amplified polymorphic DNA. The hemolytic and DNAse activity was determined using sheep's blood and DNAse agar, biofilm production by XTT method, and the susceptibility to antifungals through microdilution methodology. Two environmental strains isolated in the same month had high similarity. The 17 isolates expressed at least one of the three virulence factors studied, and one environmental isolate was resistant to fluconazole. This study shows that environmental contamination can be an important reservoir of potentially pathogenic microorganisms, since isolates of C. parapsilosis sensu stricto collected from the hospital environment were able to express virulence factors. Therefore, we emphasized the importance of determining the transmission routes in NICU in order to detect pathogen sources and reservoirs, as well as to establish prevention measures, such as adequate disinfection of the environment.

Evaluation of virulence factors of Candida albicans isolated from HIV-positive individuals using HAART.

The colonization by Candida species is one of the most important factors related to the development of oral candidiasis in HIV-infected individuals. The aim of the study was to evaluate and discuss the phospholipase, proteinase, DNAse and haemolytic activities of Candida albicans isolated from the oral cavity of HIV individuals with high efficiency antiretroviral therapy. Seventy-five isolates of C. albicans obtained from saliva samples of patients with HIV and 41 isolates from HIV-negative individuals were studied. Haemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture medium containing DNA base-agar, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. All isolates from the HIV patients group had haemolytic activity, 98% showed phospholipase activity, 92% were positive for proteinase and 32% DNAse activity. Regarding the group of indivídios HIV negative, all 41 isolates presented hemolytic activity, 90.2% showed phospholipase and proteinase activity and 12.2% were positive for DNAse. The phospholipase activity was more intense for the group of HIV positive individuals. DNase production was more frequently observed in the group of HIV-positive individuals. The percentage of isolates having DNAse activity was also significantly different between the groups of patients not using any antiretroviral therapy, those using transcriptase inhibitors and those using transcriptase inhibitor and protease inhibitor in combination.