Candida parapsilosis Virulence and Antifungal Resistance Mechanisms: A Comprehensive Review of Key Determinants.

Candida parapsilosis is the second most common Candida species isolated in Asia, Southern Europe, and Latin America and is often involved in invasive infections that seriously impact human health. This pathogen is part of the psilosis complex, which also includes Candida orthopsilosis and Candida metapsilosis. C. parapsilosis infections are particularly prevalent among neonates with low birth weights, individuals who are immunocompromised, and patients who require prolonged use of a central venous catheter or other indwelling devices, whose surfaces C. parapsilosis exhibits an enhanced capacity to adhere to and form biofilms. Despite this well-acknowledged prevalence, the biology of C. parapsilosis has not been as extensively explored as that of Candida albicans. In this paper, we describe the molecular mechanistic pathways of virulence in C. parapsilosis and show how they differ from those of C. albicans. We also describe the mode of action of antifungal drugs used for the treatment of Candida infections, namely, polyenes, echinocandins, and azoles, as well as the resistance mechanisms developed by C. parapsilosis to overcome them. Finally, we stress the importance of the ongoing search for species-specific features that may aid the development of effective control strategies and thus reduce the burden on patients and healthcare costs.

Genomic evolution towards azole resistance in Candida glabrata clinical isolates unveils the importance of CgHxt4/6/7 in azole accumulation.

The increasing prevalence of candidosis caused by Candida glabrata is related to its ability to acquire azole resistance. Although azole resistance mechanisms are well known, the mechanisms for azole import into fungal cells have remained obscure. In this work, we have characterized two hexose transporters in C. glabrata and further investigate their role as potential azole importers. Three azole susceptible C. glabrata clinical isolates were evolved towards azole resistance and the acquired resistance phenotype was found to be independent of CgPDR1 or CgERG11 mutations. Through whole-genome sequencing, CgHXT4/6/7 was found to be mutated in the three evolved strains, when compared to their susceptible parents. CgHxt4/6/7 and the 96% identical CgHxt6/7 were found to confer azole susceptibility and increase azole accumulation in C. glabrata cells, strikingly rescuing the susceptibility phenotype imposed by CgPDR1 deletion, while the identified loss-of-function mutation in CgHXT4/6/7, leads to increased azole resistance. In silico docking analysis shows that azoles display a strong predicted affinity for the glucose binding site of CgHxt4/6/7. Altogether, we hypothesize that hexose transporters, such as CgHxt4/6/7 and CgHxt6/7, may constitute a family of azole importers, involved in clinical drug resistance in fungal pathogens, and constituting promising targets for improved antifungal therapy.

Clinical azole cross-resistance in Candida parapsilosis is related to a novel MRR1 gain-of-function mutation.

OBJECTIVES: Hereby, we describe the molecular mechanisms underlying the acquisition of azole resistance by a Candida parapsilosis isolate following fluconazole treatment due to candiduria. METHODS: A set of three consecutive C. parapsilosis isolates were recovered from the urine samples of a patient with candiduria. Whole-genome sequencing and antifungal susceptibility assays were performed. The expression of MRR1, MDR1, ERG11 and CDR1B (CPAR2_304370) was quantified by RT-qPCR. RESULTS: The initial isolate CPS-A was susceptible to all three azoles tested (fluconazole, voriconazole and posaconazole); isolate CPS-B, collected after the second cycle of treatment, exhibited a susceptible-dose-dependent phenotype to fluconazole and isolate CPS-C, recovered after the third cycle, exhibited a cross-resistance profile to fluconazole and voriconazole. Whole-genome sequencing revealed a putative resistance mechanism in isolate CPS-C, associated with a G1810A nucleotide substitution, leading to a G604R change in the Mrr1p transcription factor. Introducing this mutation into the susceptible CPS-A isolate (MRR1RI) resulted in resistance to fluconazole and voriconazole, as well as up-regulation of MRR1 and MDR1. Interestingly, the susceptible-dose-dependent phenotype exhibited by isolate CPS-B was associated with an increased copy number of the CDR1B gene. The expression of CDR1B was increased in both isolates CPS-B and CPS-C and in the MRR1RI strain, harbouring the gain-of-function mutation. CONCLUSIONS: Our results describe clinical azole cross-resistance acquisition in C. parapsilosis due to a G1810A (G604R) gain-of-function mutation, resulting in MRR1 hyperactivation and consequently, MDR1 efflux pump overexpression. We also associated amplification of the CDR1B gene with decreased fluconazole susceptibility and showed that it is a putative target of the MRR1 gain-of-function mutation.

The Role of Phage Therapy in Burn Wound Infections Management: Advantages and Pitfalls.

Burn wound infections are often the source of bacteria responsible for systemic infections, including bloodstream infections and pneumonia that ultimately can result in multisystem organ failure and death. Any rapid change in the burn wound appearance or the clinical condition of the burn patient may herald burn wound infection or sepsis. The revival of phage therapy, either in single mode or in combination with conventional antibiotics may represent a valuable alternative, to treat specific bacterial infections such as burn wound infections, including those caused by multidrug-resistant organisms. This systematic review addresses the: 1) general characteristics of bacteriophages; 2) activity of bacteriophages vs conventional antibiotics; 3) activity of bacteriophages against biofilms; 4) bacteriophage administration; and 5) use of bacteriophages in burn wound infections. Although several scientific organizations/societies recognized that phage therapy could be of key value in modern wound care, specific aspects are critical for a burn surgeon and might represent pitfalls discouraging phage therapy adoption in burn wound management; in particular, the unavailability of consensual therapeutic guidelines/regulatory policies and the lack of laboratorial support that might be predictive of its efficacy. The availability of a product/formulation convenient to use, with adequate stability and shelf half-life is also a key condition.

Evaluation of FASTinov Ultrarapid Flow Cytometry Antimicrobial Susceptibility Testing Directly from Positive Blood Cultures.

The FASTinov flow cytometry kit, an ultrarapid antimicrobial susceptibility test, was directly evaluated on positive blood cultures (BC) at two sites: (i) FASTinov, S.A., in Porto, Portugal, using BC spiked with well-characterized bacteria, and (ii) Ramón y Cajal University Hospital in Madrid, Spain, using positive BC from patients. Two kits were evaluated, FASTgramneg (Enterobacterales, Pseudomonas, Acinetobacter) and FASTgrampos (Staphylococcus, Enterococcus). Dedicated software for cytometric data analysis and interpretative reporting, including both CLSI and EUCAST criteria, was used. The FASTgramneg kit also provides information about the presence of resistant mechanisms, including extended-spectrum beta-lactamases (ESBLs) and carbapenemases. After 1 h of incubation at 37°C, bacteria were analyzed using a CytoFLEX cytometer (Beckman, CA). Disk diffusion was performed as the reference susceptibility method. Overall, 447 positive BC were included, 100 from hospitalized patients. Categorical agreement values for the FASTgramneg panel were 96.8% based on EUCAST criteria and 96.4% based on CLSI criteria. For the FASTgrampos panel, categorical agreement was 98.6% when using both criteria. When EUCAST criteria were used, the percentages of errors for the FASTgramneg panel were 2.1% minor errors (mE), 1.3% major errors (ME), and 0.6% very major errors (VME). When CLSI criteria were used, 2.9% mE, 0.9% ME, and 0.4% VME were found. VME were mainly observed with amoxicillin-clavulanate, cefotaxime, ceftazidime, and gentamicin. The FASTgrampos panel showed 0.3% mE, 1.4% ME, and 0.4% VME when EUCAST criteria were used (VME with respect to gentamicin and Staphylococcus) and 0.4% mE, 1.4% ME, and no VME when CLSI criteria were used. The FASTinov flow cytometry kits represent a rapid alternative for direct antimicrobial susceptibility testing from positive BC, showing time to results of <2 h, and can be used to personalize antibiotic and stewardship practices.

The transcription factor Ndt80 is a repressor of Candida parapsilosis virulence attributes.

Candida parapsilosis is an emergent opportunistic yeast among hospital settings that affects mainly neonates and immunocompromised patients. Its most remarkable virulence traits are the ability to adhere to prosthetic materials, as well as the formation of biofilm on abiotic surfaces. The Ndt80 transcription factor was identified as one of the regulators of biofilm formation by C. parapsilosis; however, its function in this process was not yet clarified. By knocking out NDT80 (CPAR2-213640) gene, or even just one single copy of the gene, we observed substantial alterations of virulence attributes, including morphogenetic changes, adhesion and biofilm growth profiles. Both ndt80Δ and ndt80ΔΔ mutants changed colony and cell morphologies from smooth, yeast-shaped to crepe and pseudohyphal elongated forms, exhibiting promoted adherence to polystyrene microspheres and notably, forming a higher amount of biofilm compared to wild-type strain. Interestingly, we identified transcription factors Ume6, Cph2, Cwh41, Ace2, Bcr1, protein kinase Mkc1 and adhesin Als7 to be under Ndt80 negative regulation, partially explaining the phenotypes displayed by the ndt80ΔΔ mutant. Furthermore, ndt80ΔΔ pseudohyphae adhered more rapidly and were more resistant to murine macrophage attack, becoming deleterious to such cells after phagocytosis. Unexpectedly, our findings provide the first evidence for a direct role of Ndt80 as a repressor of C. parapsilosis virulence attributes. This finding shows that C. parapsilosis Ndt80 functionally diverges from its homolog in the close related fungal pathogen C. albicans.

Experience in Management of Burn Injury During Pregnancy in a Burn Unit.

Burns injuries during pregnancy are rarely reported in developed countries, but an increasing in mortality and morbidity has been observed. The authors describe their experience in the treatment of pregnant women in a burn unit. A 12-year retrospective study of burns in pregnant women hospitalized was conducted. Since 2008, two pregnant women were admitted in their unit. Patient 1, a 32-year-old pregnant woman on second trimester (27s6d), suffered a second-degree burn injury, 16% total body surface area (TBSA), caused by fire. She was admitted in their burn unit and submitted to medical treatment, wound dressing, and surgical treatment. Cerium nitrate and silver sulfadiazine were used in burn lesions and the patient was submitted to debridement and skin graft surgery. No uneventful events occurred with the fetus. Patient 2 was a 32-year-old pregnant woman on second trimester (26s), HVC positive, admitted with a second-degree flash burn, 8% TBSA. She was submitted to endotracheal intubation before arriving to the hospital due to risk of airway burn. Dexamethasone was administered for fetus lung maturation. No uneventful events were observed. The incidence of thermal injury in pregnancy in Portugal is low. Active medical treatment together with conservative wound care should be the standard in each trimester of pregnancy. Although there is limited safety information on cerium nitrate or silver sulfadiazine during pregnancy, those were used with no adverse effects on one of their patients. Obstetrical management should be individualized.

Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii.

OBJECTIVES: Both EUCAST and CLSI recommend broth microdilution for antimicrobial susceptibility testing of colistin, but this method is cumbersome and takes 16-24 h to give results. Our objective was to evaluate a rapid quantitative colistin MIC susceptibility assay based on flow cytometry analysis (FASTcolistin MIC) in comparison with standard broth microdilution assay. METHODS: One hundred and sixteen Gram-negative bacilli (78 Enterobacterales, 28 Pseudomonas aeruginosa and 10 Acinetobacter baumannii) were studied in parallel using standard broth microdilution following EUCAST recommendations and FASTcolistin MIC kit. In the last one, a bacteria suspension (0.5 MacFarland) was prepared, diluted in Muller-Hinton broth, incubated in the susceptibility panel containing different colistin concentrations (range 0.125-64 mg/L) with a fluorescent probe and incubated 1 h at 35ºC. After that, a flow cytometry analysis using CytoFLEX (Beckmam) was performed. Using a dedicated software (BioFAST) an automated MIC result was obtained after 1.5 h. Performance evaluation was performed according to the ISO standard 20776-2. Reproducibility and repeatability, categorical (CA) and essential agreement (EA), and lot-to-lot variation and operator-to-operator variability, as well as time to results were determined. RESULTS: Overall, 100% CA (CI 97-100%) and 95.7% EA (CI 90-98%) was obtained with high repeatability (100%; CI 80-100%)and reproducibility (97%; (CI 83-99%)). Absence of lot-to-lot variations or differences in the operators' performance was observed. CONCLUSIONS: FASTcolistin MIC is an accurate, reliable and ultra-rapid method (1 h incubation versus 24 h) for susceptibility testing of colistin of common Gram-negative bacilli recovered in clinical laboratories.

A Rapid Flow Cytometric Antimicrobial Susceptibility Assay (FASTvet) for Veterinary Use - Preliminary Data.

A rapid flow cytometric antimicrobial susceptibility test for bacteria isolated from companion animals - the FASTvet assay, developed by FASTinov®, was evaluated. Bacterial strains isolated from different biological samples of companion animals with infectious diseases in progress were obtained from several veterinary clinical laboratories across the country. A total of 115 strains, comprising 65 Gram-negative and 50 Gram positive isolates, were incubated with 13 antimicrobial drugs (ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefpodoxime, imipenem, enrofloxacin, gentamicin, amikacin for Gram-negative; penicillin, cefoxitin, enrofloxacin, vancomycin and ampicillin for Gram-positive) at breakpoint concentrations following CLSI protocol (CLSI Vet 01, 2018) for 1 h and analyzed by flow cytometry. The overall categorical agreement was 95.6% in case of Gram-negative and of 96.7% in Gram-positive isolates when compared to microdilution. FASTvet kits contribute to reduce the turnaround time (2 vs. 24 h) with early determination of the antimicrobial susceptibility profile. The correct and rapid choice of the target antibiotic therapy, will have a positive impact on animal care, contributing for preventing antimicrobial resistance. In conclusion, FASTinov® vet kits showed an excellent performance, both for Gram-negative and Gram-positive isolates encouraging us to enlarge the sample size and planning multicentric studies.

Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from positive blood cultures.

The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.

Candida albicans Antifungal Resistance and Tolerance in Bloodstream Infections: The Triad Yeast-Host-Antifungal.

Candida albicans represents the most frequent isolated yeast from bloodstream infections. Despite the remarkable progress in diagnostic and therapeutic approaches, these infections continue to be a critical challenge in intensive care units worldwide. The economic cost of bloodstream fungal infections and its associated mortality, especially in debilitated patients, remains unacceptably high. Candida albicans is a highly adaptable microorganism, being able to develop resistance following prolonged exposure to antifungals. Formation of biofilms, which diminish the accessibility of the antifungal, selection of spontaneous mutations that increase expression or decreased susceptibility of the target, altered chromosome abnormalities, overexpression of multidrug efflux pumps and the ability to escape host immune defenses are some of the factors that can contribute to antifungal tolerance and resistance. The knowledge of the antifungal resistance mechanisms can allow the design of alternative therapeutically options in order to modulate or revert the resistance. We have focused this review on the main factors that are involved in antifungal resistance and tolerance in patients with C. albicans bloodstream infections.

Malassezia colonisation on a reconstructed human epidermis: Imaging studies.

BACKGROUND: Biofilm formation represents a major microbial virulence attribute especially at epithelial surfaces such as the skin. Malassezia biofilm formation at the skin surface has not yet been addressed. OBJECTIVE: The present study aimed to evaluate Malassezia colonisation pattern on a reconstructed human epidermis (RhE) by imaging techniques. METHODS: Malassezia clinical isolates were previously isolated from volunteers with pityriasis versicolor and seborrhoeic dermatitis. Yeast of two strains of M furfur and M sympodialis were inoculated onto the SkinEthic™ RHE. The tissues were processed for light microscopy, wide-field fluorescence microscopy and scanning electron microscopy. RESULTS: Colonisation of the RhE surface with aggregates of Malassezia yeast entrapped in a multilayer sheet with variable amount of extracellular matrix was unveiled by imaging techniques following 24, 48, 72 and 96 hours of incubation. Whenever yeast were suspended in RPMI medium supplemented with lipids, the biofilm substantially increased with a dense extracellular matrix in which the yeast cells were embedded. Slight differences were found in the biofilm architectural structure between the two tested species with an apparently higher entrapment and viscosity in M furfur biofilm. CONCLUSION: Skin isolates of M furfur and M sympodialis were capable of forming biofilm in vitro at the epidermal surface simulating in vivo conditions. Following 24 hours of incubation, without added lipids, rudimental matrix was barely visible, conversely to the reported at plastic surfaces. The amount of biofilm apparently increased progressively from 48 to 96 hours. A structural heterogeneity of biofilm between species was found.

Evaluation of Physiological Effects Induced by Manuka Honey Upon Staphylococcus aureus and Escherichia coli.

Several studies have explored the antimicrobial properties of manuka honey (MkH). However, the data available regarding antibacterial action mechanisms are scarcer. The aim of this study was to scrutinize and characterize primary effects of manuka honey (MkH) upon the physiological status of Staphylococcus aureus and Escherichia coli (as Gram-positive and Gram-negative bacteria models, respectively), using flow cytometry (FC) to reveal its antibacterial action mechanisms. Effects of MkH on membrane potential, membrane integrity and metabolic activity were assessed using different fluorochromes in a 180 min time course assay. Time-kill experiments were carried out under the same conditions. Additionally, MkH effect on efflux pumps was also studied in an E. coli strain with an over-expression of several efflux pumps. Exposure of bacteria to MkH resulted in physiological changes related to membrane potential and membrane integrity; these effects displayed slight differences among bacteria. MkH induced a remarkable metabolic disruption as primary physiological effect upon S. aureus and was able to block efflux pump activity in a dose-dependent fashion in the E. coli strain.

Draft Genome Sequences of Three Clinical Isolates of the Pathogenic Yeast Candida glabrata.

Here, we report the draft genome sequences of three Candida glabrata clinical isolates, 040, 044, and OL152. The isolates were recovered from patients admitted to Centro Hospitalar de S. João (CHSJ) in Porto, Portugal. Isolates 040 and 044 were taken from blood samples, while isolate OL152 was collected from urine.

Malassezia interaction with a reconstructed human epidermis: Keratinocyte immune response.

The immediate immune response developed by the keratinocytes against Malassezia yeasts has been addressed yielding conflicting results. This study aims the assessment of cytokines and antimicrobial peptides gene expression elicited by M. sympodialis and M. furfur once in contact with a reconstructed human epidermis. A yeast suspension was prepared in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with Tween 60 and oleic acid to obtain approximately 1 × 106 cells in a volume of 100 µL. Clinical isolates of M. sympodialis (from pityriasis versicolor) and M. furfur (from seborrhoeic dermatitis) were inoculated, separately, onto a reconstructed human epidermis. A distinct expression pattern was found between the two tested species, with a tendency for overexpression of pro-inflammatory cytokines very soon after infection, whereas no significant expression or gene downregulation was often noticed following 24 and 48 h of incubation. A possible Malassezia species-dependent immune response pattern is highlighted.

Assessing the impact of Medical Microbiology classes using active strategies on short- and long-term retention on medical students: an innovative study.

One of teachers' concerns, with students in general and medical students in particular, is to ensure as much as possible that information goes from students' short-term memories to their long-term memories. The present study focuses on knowledge retention in Medical Microbiology and assesses the effectiveness of some strategies implemented for short- and long-term retention. A pre- and post-test was used to assess student's learning. This study involved students of Porto University (test group). Test group participants were all attending the third year of the Medicine Degree Program. The results of post-test 1 were considered very positive and support the importance of these applied active activities and/or methodologies in Medical Microbiology for short-term retention. However, the results obtained in post-test 2 showed that knowledge retention after 9 months, despite substantial, decreases.

A Transcriptomics Approach To Unveiling the Mechanisms of In Vitro Evolution towards Fluconazole Resistance of a Candida glabrata Clinical Isolate.

Candida glabrata is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of a C. glabrata clinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in the C. glabrataPDR1 gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of a C. glabrata population toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.

Efficacy of UV-C Ray Sterilization of Calliphora vicina (Diptera: Calliphoridae) Eggs for Use in Maggot Debridement Therapy.

Maggot debridement therapy (MDT) is a simple wound debridement technique. It is a natural treatment licensed by the Food and Drug Administration (FDA) and is increasingly used in the United States and in Europe. This treatment is safe when the larvae originate from laboratory stocks of eggs that have been sterilized. In this study, a simple, inexpensive microbe decontamination technique is described. It yields eggs that are free of chemical residues and are easy to handle, meeting the growing demand for medicinal larvae in hospitals or medical centers. Three treatments (T1, T2, T3) involving 3, 6, and 12 min of exposure to ultraviolet (UV-C) rays, respectively, were compared. Egg sterility was evaluated by culture in thioglycollate broth, incubated at 32°C ± 2.5°C under aerobic conditions for up to 14 d. The UV-C radiation sterilization process obtained satisfactory results after 12 min exposure (treatment 3). Larval viability was 57%, pupal viability was 54%, and 54% of the adults emerged. The sex ratio was 50%, within the expected values. There were no morphological abnormalities associated to the UV-C treatment in the flies. In conclusion sterilization by UV-C rays is indicated to obtain sterile larvae destined for MDT.

Potential Impact of Flow Cytometry Antimicrobial Susceptibility Testing on the Clinical Management of Gram-Negative Bacteremia Using the FASTinov® Kit.

Laboratory assessment of antimicrobial susceptibility is a prerequisite for adequate management of infections. The aim of this research was to evaluate the performance of the novel FASTinov® kit for antimicrobial susceptibility testing (AST) of Gram negative bacilli directly on positive blood cultures. One hundred and two positive blood cultures from patients of a Portuguese University Hospital were included. AST were performed with routine method, Vitek2, with FASTinov® kit, and with the gold standard microdilution. Bacteria directly extracted from blood cultures were used to inoculate the FASTinov® kit. Time-to-result as well as the number of patients receiving initially inappropriate therapy (and those in whom de-escalation would have been done) and length of stay (LOS) was recorded. Seventy percent of patients were over 70 years old and 18.6% were admitted in intensive care units. Regarding the isolates, 88.2% were Enterobacteriaceae, 9.8% Pseudomonas spp. and 1% Acinetobacter spp. Extended spectrum ß-lactamases producing-Enterobacteriaceae were found in 7.8% of cases and 10.8% were multi-drug resistant. Fifty-one hours was the mean of time-to-result for routine test (Vitek2) vs. 2 h response regarding Fastinov® test. The overall agreement between FASTinov® and the reference microdilution method was 98%. According to the susceptibility phenotype, 16.7% of patients received initially inappropriate therapy and the mean hospital LOS of these patients was significantly higher. FASTinov® kit revealed an excellent correlation with the AST standard method and provided much earlier results than Vitek2.

Unveiling the Synergistic Interaction Between Liposomal Amphotericin B and Colistin.

Patients with multiple comorbidities are often administered simultaneously or sequentially antifungals and antibacterial agents, without full knowledge of the consequences of drug interactions. Considering the clinical relevance of liposomal amphotericin B (L-AMB), the association between L-AMB and six antibacterial agents was evaluated against four clinical isolates and one type strain of Candida spp. and two clinical isolates and one type strain of Aspergillus fumigatus. In order to evaluate such combined effects, the minimal inhibitory concentration (MIC) of L-AMB was determined in the presence of 0.5-, 1-, 2-, and 4-fold peak plasma concentrations of each of the antibacterial drugs. Since the L-AMB/colistin (CST) association was the most synergic, viability assays were performed and the physiological status induced by this association was characterized. In addition, computational molecular dynamics studies were also performed in order to clarify the molecular interaction. The maximum synergistic effect with all antibacterial agents, except CST, was reached at fourfold the usual peak plasma concentrations, resulting in 2-to 8-fold L-AMB MIC reduction for Candida and 2-to 16-fold for Aspergillus. For CST, the greatest synergism was registered at peak plasma concentration (3 mg/L), with 4-to 8-fold L-AMB MIC reduction for Candida and 16-to 32-fold for Aspergillus. L-AMB at subinhibitory concentration (0.125 mg/L) combined with CST 3 mg/L resulted in: a decrease of fungal cell viability; an increase of cell membrane permeability; an increase of cellular metabolic activity soon after 1 h of exposure, which decreased until 24 h; and an increase of ROS production up to 24 h. From the molecular dynamics studies, AMB and CST molecules shown a propensity to form a stable molecular complex in solution, conferring a recognition and binding added value for membrane intercalation. Our results demonstrate that CST interacts synergistically with L-AMB, forming a stable complex, which promotes the fungicidal activity of L-AMB at low concentration.