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1.
BMC Microbiol ; 24(1): 187, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38802760

ABSTRACT

BACKGROUND: Rapid antimicrobial susceptibility testing (AST) is urgently needed to provide safer treatment to counteract antimicrobial resistance. This is critical in septic patients, because resistance increases empiric therapy uncertainty and the risk of a poor outcome. We validate a novel 2h flow cytometry AST assay directly from positive blood cultures (PBC) by using a room temperature stable FASTgramneg and FASTgrampos kits (FASTinov® Porto, Portugal) in three sites: FASTinov (site-1), Hospital Ramon y Cajal, Madrid, Spain (site-2) and Centro Hospitalar S. João, Porto, Portugal (site-3). A total of 670 PBC were included: 333 spiked (site-1) and 337 clinical PBC (151 site-2 and 186 site-3): 367 gram-negative and 303 gram-positive. Manufacturer instructions were followed for sample preparation, panel inoculation, incubation (1h/37ºC) and flow cytometry analysis using CytoFlex (Site-1 and -2) or DxFlex (site-3) both instruments from Beckman-Coulter, USA. RESULTS: A proprietary software (bioFAST) was used to immediately generate a susceptibility report in less than 2 h. In parallel, samples were processed according to reference AST methods (disk diffusion and/or microdilution) and interpreted with EUCAST and CLSI criteria. Additionally, ten samples were spiked in all sites for inter-laboratory reproducibility. Sensitivity and specificity were >95% for all antimicrobials. Reproducibility was 96.8%/95.0% for FASTgramneg and 95.1%/95.1% for FASTgrampos regarding EUCAST/CLSI criteria, respectively. CONCLUSION: FASTinov® kits consistently provide ultra-rapid AST in 2h with high accuracy and reproducibility on both Gram-negative and Gram-positive bacteria. This technology creates a new paradigm in bacterial infection management and holds the potential to significantly impact septic patient outcomes and antimicrobial stewardship.


Subject(s)
Anti-Bacterial Agents , Blood Culture , Flow Cytometry , Microbial Sensitivity Tests , Humans , Flow Cytometry/methods , Anti-Bacterial Agents/pharmacology , Microbial Sensitivity Tests/methods , Microbial Sensitivity Tests/instrumentation , Blood Culture/methods , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/isolation & purification , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/isolation & purification , Time Factors , Portugal , Spain , Reproducibility of Results
2.
J Hosp Infect ; 127: 7-14, 2022 Sep.
Article in English | MEDLINE | ID: mdl-35594987

ABSTRACT

BACKGROUND: The prevention of healthcare-associated infections requires continuous effort. In order to achieve better practical results, the control of environmental microbial biofilms with effective disinfection strategies should be addressed. AIM: To test the efficacy of different time cycles of nebulized hydrogen peroxide (H2O2) against bacterial and yeast dry biofilms. METHODS: The efficacies of a standard cycle (SC) and a fast cycle (FC) of nebulized H2O2 were compared. Microbial biofilms were grown on different material coupons. The metabolic activity of biofilms was determined by XTT assay, and the total biomass of biofilms was determined by crystal violet assay. FINDINGS: Regarding the efficacy of nebulized H2O2 against biofilms, the mean reduction in metabolic activity for the SC was 55.2% [standard deviation (SD) 19.4%], compared with 50.4% (SD 17.7%) for the FC. The mean reduction in total biomass for the SC was 45.5% (SD 22.7%), compared with 46.7% (SD 21.7%) for the FC. No significant differences were found between the tested cycles and materials. CONCLUSION: H2O2 nebulization was found to exhibit good efficacy against healthcare-associated microbial dry biofilms. Moreover, similar efficacies were found between the SC and the FC.


Subject(s)
Cross Infection , Hydrogen Peroxide , Biofilms , Disinfection/methods , Humans , Hydrogen Peroxide/pharmacology , Saccharomyces cerevisiae
3.
J Eur Acad Dermatol Venereol ; 35(10): 2007-2021, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34146427

ABSTRACT

In the late 90s, a sharp increase of treatment failures of Trichomonas vaginalis (TV) infections with metronidazole (MTZ) was reported, representing a problem due to limited treatment options. We proposed to review the available evidence on the frequency of MTZ resistance by TV isolates and the relationship between treatment failure and in vitro resistance to MTZ. A systematic review based on the PRISMA guidelines was conducted by searching published studies in three different databases (PubMed, Scopus and Web of Science) up to December 2020. The extracted studies were uploaded to Covidence software; screening was guided based on inclusion and exclusion criteria. Additionally, different articles were included through other sources. For each article, study design, objectives, study population and key outcomes were summarized. We found 403 references from the databases and four extra studies. After duplicate removal and screening of title, abstract and full text, 27 studies were included. The selected studies were published between 1983 and 2019; all except one addressed only vaginal TV infection. We identified four major populations in vitro MTZ resistance: two studies evaluated female adolescents; other two assessed HIV-positive women. Fifteen studies considered MTZ resistance in newly diagnosed vaginal TV infection. Finally, eight articles studied in vitro susceptibility of isolates from women with clinical resistant trichomoniasis. High level of in vitro MTZ resistance was rare; low-moderate level was described in most of the cases. Although clinical resistance to MTZ of trichomoniasis was widely reported, there was a paucity of prospective controlled studies. Our review unveiled the need to standardize susceptibility testing, to define breakpoints for detection of MTZ-resistant isolates and to correlate with clinical outcome. It is important to establish criteria to define clinical resistance to MTZ. Such a consensus would foster the development of surveillance studies about clinical and microbiological response to MTZ treatment.


Subject(s)
Trichomonas Infections , Trichomonas Vaginitis , Trichomonas vaginalis , Adolescent , Drug Resistance , Female , Humans , Metronidazole/pharmacology , Prospective Studies , Trichomonas Infections/drug therapy , Trichomonas Vaginitis/drug therapy
4.
J Hosp Infect ; 113: 155-163, 2021 Jul.
Article in English | MEDLINE | ID: mdl-33989740

ABSTRACT

BACKGROUND: Hydrogen peroxide (H2O2)-based technology is currently used with the aim of controlling microbial contamination in hospital settings. However, the long cycles required result in prolonged room turnover time, thus precluding a wider implementation of the technology. AIM: To assess the efficacy of a shorter cycle of nebulized H2O2 against healthcare-associated micro-organisms, further comparing among multidrug-resistant and multidrug-susceptible strains. METHODS: The efficacy of a standard cycle (1 h) and of a faster cycle (15 min) of a 7% H2O2 nebulized solution was compared against bacteria and yeasts. MDR and MDS strains were inoculated on polyvinyl chloride, stainless steel, linoleum, napa leather, and formica coupons, and their growth ability was compared. FINDINGS: Globally, the mean efficacy of the standard cycle ranged between 82.5% (±17.0) and 95.9% (±8.3), while the efficacy of the fast cycle ranged between 84.4% (±17.0) and 95.7% (±10.5). No statistically significant differences were found for the majority of the tested cycles and materials. For all the tested strains, no differences were found regarding the efficacy of cycles. CONCLUSION: The very high disinfection efficacy of the fast cycle was found to be similar to that of the standard cycle. Moreover, a similar efficacy was also demonstrated when comparing between multidrug-resistant and multidrug-susceptible strains. This study supports a wider implementation of the technology, with the expected advantages of reducing room turnover time, costs, and indirect infection transmission. Further assessment of the efficacy of this faster cycle against other emergent microbial global threats would be highly recommended.


Subject(s)
Disinfectants , Hydrogen Peroxide , Delivery of Health Care , Disinfection , Humans , Stainless Steel
5.
Braz J Med Biol Res ; 54(5): e10543, 2021.
Article in English | MEDLINE | ID: mdl-33729391

ABSTRACT

We evaluated the effects of exercise training (ET) on the profile of mood states (POMS), heart rate variability, spontaneous baroreflex sensitivity (BRS), and sleep disturbance severity in patients with obstructive sleep apnea (OSA). Forty-four patients were randomized into 2 groups, 18 patients completed the untrained period and 16 patients completed the exercise training (ET). Beat-to-beat heart rate and blood pressure were simultaneously collected for 5 min at rest. Heart rate variability (RR interval) was assessed in time domain and frequency domain (FFT spectral analysis). BRS was analyzed with the sequence method, and POMS was analyzed across the 6 categories (tension, depression, hostility, vigor, fatigue, and confusion). ET consisted of 3 weekly sessions of aerobic exercise, local strengthening, and stretching exercises (72 sessions, achieved in 40±3.9 weeks). Baseline parameters were similar between groups. The comparisons between groups showed that the changes in apnea-hypopnea index, arousal index, and O2 desaturation in the exercise group were significantly greater than in the untrained group (P<0.05). The heart rate variability and BRS were significantly higher in the exercise group compared with the untrained group (P<0.05). ET increased peak oxygen uptake (P<0.05) and reduced POMS fatigue (P<0.05). A positive correlation (r=0.60, P<0.02) occurred between changes in the fatigue item and OSA severity. ET improved heart rate variability, BRS, fatigue, and sleep parameters in patients with OSA. These effects were associated with improved sleep parameters, fatigue, and cardiac autonomic modulation, with ET being a possible protective factor against the deleterious effects of hypoxia on these components in patients with OSA.


Subject(s)
Autonomic Nervous System , Sleep Apnea, Obstructive , Baroreflex , Exercise , Heart Rate , Humans , Sleep Apnea, Obstructive/therapy
6.
Med Vet Entomol ; 35(2): 225-229, 2021 06.
Article in English | MEDLINE | ID: mdl-33063897

ABSTRACT

Antibiotic-resistant bacteria pose a major threat to global health in the 21st century, requiring a quick, cheap and effective response from public health officials. This study evaluated the antimicrobial activity of native excretions/secretions (NES) produced by third instar (3 days old) larvae of Calliphora vicina using a protocol adapted from the Institute of Clinical and Laboratory Standards (CLSI). The microorganisms tested were: Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and the fungus Candida albicans. After the incubation period, the suspensions were diluted and spread on nutrient agar plates to count the colony-forming units. A turbidimetric test also was carried out to test the action of the NES of C. vicina against S. aureus, a very common bacterial species, with an enormous capacity for adaption and resistance, being one of the bacteria of medical importance that causes the most hospital and community infections in the world. According to our results, the NES of C. vicina exhibits antimicrobial activity at different dilutions, being most effective against the gram-negative bacteria E. coli and K. pneumoniae.


Subject(s)
Anti-Bacterial Agents , Antifungal Agents , Biological Products , Calliphoridae/metabolism , Gram-Negative Bacteria/drug effects , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Biological Products/metabolism , Biological Products/pharmacology , Candida albicans , Diptera , Escherichia coli/drug effects , Klebsiella pneumoniae/drug effects , Larva/metabolism , Microbial Sensitivity Tests/methods , Staphylococcus aureus/drug effects
7.
Braz. j. med. biol. res ; 54(5): e10543, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153549

ABSTRACT

We evaluated the effects of exercise training (ET) on the profile of mood states (POMS), heart rate variability, spontaneous baroreflex sensitivity (BRS), and sleep disturbance severity in patients with obstructive sleep apnea (OSA). Forty-four patients were randomized into 2 groups, 18 patients completed the untrained period and 16 patients completed the exercise training (ET). Beat-to-beat heart rate and blood pressure were simultaneously collected for 5 min at rest. Heart rate variability (RR interval) was assessed in time domain and frequency domain (FFT spectral analysis). BRS was analyzed with the sequence method, and POMS was analyzed across the 6 categories (tension, depression, hostility, vigor, fatigue, and confusion). ET consisted of 3 weekly sessions of aerobic exercise, local strengthening, and stretching exercises (72 sessions, achieved in 40±3.9 weeks). Baseline parameters were similar between groups. The comparisons between groups showed that the changes in apnea-hypopnea index, arousal index, and O2 desaturation in the exercise group were significantly greater than in the untrained group (P<0.05). The heart rate variability and BRS were significantly higher in the exercise group compared with the untrained group (P<0.05). ET increased peak oxygen uptake (P<0.05) and reduced POMS fatigue (P<0.05). A positive correlation (r=0.60, P<0.02) occurred between changes in the fatigue item and OSA severity. ET improved heart rate variability, BRS, fatigue, and sleep parameters in patients with OSA. These effects were associated with improved sleep parameters, fatigue, and cardiac autonomic modulation, with ET being a possible protective factor against the deleterious effects of hypoxia on these components in patients with OSA.


Subject(s)
Humans , Autonomic Nervous System , Sleep Apnea, Obstructive/therapy , Exercise , Baroreflex , Heart Rate
8.
Clin Hemorheol Microcirc ; 73(1): 195-201, 2019.
Article in English | MEDLINE | ID: mdl-31561347

ABSTRACT

BACKGROUND: In cardiovascular research small pigs breeds like Göttingen® minipigs (GM) are established animal models, but systematic data about the micromorphology of the GM vasculature at different ages are scarce. OBJECTIVE: The study was aimed at gaining knowledge about the micromorphology of the femoral artery (FA) from German Landrace pigs (DL) and GM during the period of growth over a body weight range of 10-40 kg. METHODS: FA samples from DL aged two or three months were compared to GM ones, aged 18 or 40 months using transmitted light microscopy. RESULTS: All FA samples showed typical characteristics of muscular arteries. Growth was associated with increased vessel wall thickness. In the GM this resulted in a slight decrease of the luminal diameter (LD), while in the DL pigs, an increase of the LD and smooth muscle cell content (10%) with decreased elastic fiber content (10%) has been detected. In contrast, within the 22 months lasting growth period of the GM, the tunica media content of smooth muscle cells and elastic fibers remained stable. CONCLUSIONS: FA maturation strongly depends on the pig breed and age. It can be different from what is described in humans.


Subject(s)
Femoral Artery/growth & development , Tunica Media/growth & development , Animals , Swine
9.
Int J Antimicrob Agents ; 54(6): 820-823, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31425793

ABSTRACT

Accurate assessment of colistin susceptibility is crucial with the increasing number of multidrug-resistant Gram-negative bacteria and simultaneously increasing colistin resistance. Both EUCAST and CLSI recommend broth microdilution (BMD) to determine colistin susceptibility, however it is cumbersome and growth-dependent. In this study, a rapid flow cytometry method (FASTinovⓇ) to determine colistin susceptibility directly from positive blood cultures (BCs) was evaluated. BCs were spiked with 204 Gram-negative bacilli (137 Enterobacterales, 35 Pseudomonas spp. and 32 Acinetobacter baumannii) at a concentration of 2 × 103 cells/bottle, inoculated with human donor blood and incubated until flagged positive. As quality control strains, two susceptible (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) and two resistant (colistin-resistant mcr-1-positive E. coli NCTC 13846 and Serratia marcescens ATCC 14756) were used. Bacteria were extracted according to assay instructions and were incubated for 1 h at 37 °C with 2 and 4 mg/L colistin and a fluorescent dye, previously optimised. Cells were analysed on CytoFLEX (Beckman Coulter) and AccuriTM C6 Plus (BD Biosciences) flow cytometers. Colistin susceptibility results were automatically provided by BioFAST software (FASTinovⓇ) and compared with those obtained with standard BMD. Overall categorical agreement between this new flow cytometry method and BMD was 99.0%. No very major errors were detected as well as no discrepancies between both flow cytometers. Here we describe a rapid and accurate assay for colistin susceptibility directly from positive BCs with a turnaround time of 2 h versus 48 h required for BMD. This method represents an accurate alternative to standard BMD.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Colistin/pharmacology , Drug Resistance, Bacterial , Gram-Negative Bacteria/drug effects , Blood Culture , Humans , Microbial Sensitivity Tests
11.
Eur J Clin Microbiol Infect Dis ; 36(11): 2053-2062, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28647859

ABSTRACT

Despite considerable efforts, healthcare-associated infections (HAIs) continue to be globally responsible for serious morbidity, increased costs and prolonged length of stay. Among potentially preventable sources of microbial pathogens causing HAIs, patient care items and environmental surfaces frequently touched play an important role in the chain of transmission. Microorganisms contaminating such high-touch surfaces include Gram-positive and Gram-negative bacteria, viruses, yeasts and parasites, with improved cleaning and disinfection effectively decreasing the rate of HAIs. Manual and automated surface cleaning strategies used in the control of infectious outbreaks are discussed and current trends concerning the prevention of contamination by the use of antimicrobial surfaces are taken into consideration in this manuscript.


Subject(s)
Bacteria/growth & development , Cross Infection/prevention & control , Disinfection/methods , Equipment and Supplies, Hospital/microbiology , Equipment and Supplies, Hospital/parasitology , Fungi/growth & development , Parasites/growth & development , Viruses/growth & development , Animals , Bacteria/drug effects , Cross Infection/microbiology , Cross Infection/mortality , Decontamination/methods , Disinfectants/pharmacology , Fungi/drug effects , Humans , Parasites/drug effects , Viruses/drug effects
12.
Clin Microbiol Infect ; 23(8): 575.e1-575.e8, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28196695

ABSTRACT

OBJECTIVES: Candida parapsilosis is a healthcare-related fungal pathogen particularly common among immunocompromised patients. Our understanding of antifungal resistance mechanisms in C. parapsilosis remains very limited. We previously described an azole-resistant strain of C. parapsilosis (BC014RPSC), obtained following exposure in vitro to posaconazole. Resistance was associated with overexpression of ergosterol biosynthetic genes (ERG genes), together with the transcription factors UPC2 (CPAR2-207280) and NDT80 (CPAR2-213640). The aim of this study was to identify the mechanisms underlying posaconazole resistance of the BC014RPSC strain. METHODS: To identify the causative mutation, we sequenced the genomes of the susceptible (BC014S) and resistant (BC014RPSC) isolates, using Illumina technology. Ergosterol content was assessed in both strains by mass spectrometry. UPC2 and NDT80 genes were deleted in BC014RPSC strain. Mutants were characterized regarding their azole susceptibility profile and ERG gene expression. RESULTS: One homozygous missense mutation (R135I) was found in ERG3 (CPAR2-105550) in the azole-resistant isolate. We show that Erg3 activity is completely impaired, resulting in a build up of sterol intermediates and a failure to generate ergosterol. Deleting UPC2 and NDT80 in BC014RPSC reduces the expression of ERG genes and restores susceptibility to azole drugs. CONCLUSIONS: A missense mutation in the ERG3 gene results in azole resistance and up-regulation of ERG genes expression. We propose that this mutation prevents the formation of toxic intermediates when cells are treated with azoles. Resistance can be reversed by deleting Upc2 and Ndt80 transcription factors. UPC2 plays a stronger role in C. parapsilosis azole resistance than does NDT80.


Subject(s)
Antifungal Agents/pharmacology , Azoles/pharmacology , Candida parapsilosis/drug effects , Drug Resistance, Fungal , Ergosterol/metabolism , Mutation, Missense , Transcription Factors/metabolism , Candida parapsilosis/chemistry , Candida parapsilosis/genetics , Ergosterol/analysis , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Deletion , Gene Expression Profiling , Mass Spectrometry , Microbial Sensitivity Tests , Oxidoreductases/genetics , Oxidoreductases/metabolism , Provitamins/metabolism , Transcription Factors/genetics , Whole Genome Sequencing
14.
Eur J Clin Microbiol Infect Dis ; 33(12): 2241-7, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25012821

ABSTRACT

This is the first Portuguese multicenter observational and descriptive study that provides insights on the species distribution and susceptibility profiles of yeast isolates from fungemia episodes. Ten district hospitals across Portugal contributed by collecting yeast isolates from blood cultures and answering questionnaires concerning patients' data during a 12-month period. Molecular identification of cryptic species of Candida parapsilosis and C. glabrata complex was performed. The susceptibility profile of each isolate, considering eight of the most often used antifungals, was determined. Both Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocols were applied. The incidence of 240 episodes of fungemia was 0.88/1,000 admissions. Fifteen different species were found, with C. albicans (40 %) being the most prevalent, followed by C. parapsilosis (23 %) and C. glabrata (13 %). Most isolates were recovered from patients admitted to surgical wards or intensive care units, with 57 % being males and 32 % aged between 41 and 60 years. For both the CLSI and EUCAST protocols, the overall susceptibility rates ranged from 74 to 97 % for echinocandins and from 84 to 98 % for azoles. Important resistance rate discrepancies between protocols were observed in C. albicans and C. glabrata for echinocandins and in C. parapsilosis and C. tropicalis for azoles. Death associated with fungemia occurred in 25 % of the cases, with more than half of C. glabrata infections being fatal. The great number of Candida non-albicans is noteworthy despite a relatively low antifungal resistance rate. Studies like this are essential in order to improve empirical treatment guidelines.


Subject(s)
Antifungal Agents/pharmacology , Candida/drug effects , Candida/isolation & purification , Candidemia/microbiology , Adolescent , Adult , Aged , Candidemia/epidemiology , Child , Child, Preschool , Drug Resistance, Fungal , Female , Humans , Incidence , Infant , Infant, Newborn , Male , Microbial Sensitivity Tests , Middle Aged , Portugal/epidemiology , Young Adult
15.
J Med Microbiol ; 63(Pt 9): 1167-1173, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24913563

ABSTRACT

Biofilms are commonly involved in medical device-related infections. The purpose of this study was to determine the antimicrobial and anti-biofilm activity of polyethyleneimine (PEI) and PEI-based nanoparticles (nanoPEI) against Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii and Candida albicans (clinical and ATCC strains), and to evaluate their effect upon biofilm formation on polyurethane (PUR)-like catheters. MICs and minimal lethal concentrations of PEI and nanoPEI were determined according to CLSI microdilution reference protocols. For PEI, the MIC value was 195.31 mg l(-1) for all the bacteria and 48.83 mg l(-1) for the yeast strains. For nanoPEI, the MIC value was 1250 mg l(-1) for all the strains except A. baumannii, for which it was 2500 mg l(-1). Biofilm formation was assessed with PUR-like catheter segments and biofilm metabolic activity was quantified by colorimetry with a tetrazolium reduction assay. Plasma membrane integrity and membrane potential were assessed by flow cytometry after staining microbial cells with a membrane-impermeable dye, propidium iodide, and a membrane-potential marker, DiBAC4(3). PEI inhibited growth of all microbial species; higher concentrations of nanoPEI were needed to inhibit growth of all species. Biofilm formation in the presence of anti-bacterial PEI activity was dose-dependent (except for S. epidermidis) and species-related. NanoPEI at 0.5×MIC and MIC significantly reduced the metabolic activity of biofilms of S. aureus, S. epidermidis and A. baumannii, whereas 2×MIC was required in order to inhibit biofilm metabolic activity.


Subject(s)
Acinetobacter baumannii/drug effects , Anti-Infective Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Nanoparticles , Polyethyleneimine/pharmacology , Staphylococcus/drug effects , Acinetobacter baumannii/growth & development , Acinetobacter baumannii/physiology , Biofilms/growth & development , Candida albicans/growth & development , Candida albicans/physiology , Catheters/microbiology , Cell Membrane/drug effects , Energy Metabolism/drug effects , Membrane Potentials/drug effects , Microbial Sensitivity Tests , Microbial Viability/drug effects , Polyurethanes , Staphylococcus/growth & development , Staphylococcus/physiology
16.
Med Microbiol Immunol ; 203(1): 25-33, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24013184

ABSTRACT

Candida invasive infections have increased in frequency during the last decades. Such infections are often associated to medical indwelling devices like central venous catheter. The recurrent nature and difficulties in the treatment of these infections are often related to biofilm formation. The objective of this study was to investigate the anti-biofilm activity of low-molecular weight chitosan hydrogel (LMWCH), a natural biopolymer obtained from the N-deacylation of crustacean chitin, upon clinical relevant Candida species. The in vitro ability of LMWCH to impair biofilm formation and to disorganize a preformed biofilm was tested in polystyrene microplates and quantified by the semi quantitative XTT assay and by the crystal violet assay. LMWCH in vivo efficacy as a coating for medical indwelling devices was evaluated for the first time for Candida parapsilosis, using a mouse subcutaneous foreign body model using polyurethane catheter segments. Scanning electron microscopy was used to access biofilm architecture after LMWCH treatment. We found that LMWCH efficiently impaired biofilm formation of all Candida species, also promoting biofilm disaggregation. Most importantly, LMWCH was able to significantly inhibit biofilm formation by C. parapsilosis in an in vivo catheter mouse model. SEM images showed biofilm collapsed cells compatible with membrane damage, suggesting that this could be one of the possible mechanisms underlying biofilm impairment. LMWCH revealed to be a promising compound for treatment of candidiasis or its prevention through medical device coating.


Subject(s)
Biofilms/drug effects , Candida/drug effects , Candida/physiology , Chitosan/pharmacology , Hydrogel, Polyethylene Glycol Dimethacrylate , Animals , Candida/ultrastructure , Candidiasis/drug therapy , Candidiasis/microbiology , Chitosan/administration & dosage , Chitosan/chemistry , Female , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Mice , Microbial Sensitivity Tests , Molecular Weight , Plankton/drug effects
17.
J Antimicrob Chemother ; 68(1): 126-30, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22991425

ABSTRACT

OBJECTIVES: Catheter-related bloodstream infections (CRBSIs) are common healthcare-associated infections associated with increased morbidity and medical costs. Antiseptic- and antibiotic-coated central venous catheters (CVCs) have been proposed to reduce the incidence of CRBSIs, with variable success. The aim of this study was to determine the in vivo antibiofilm activity of biocompatible and inexpensive compounds, such as cerium nitrate, chitosan and hamamelitannin, against usual agents of CRBSIs. METHODS: The antibiofilm effect of cerium nitrate, chitosan and hamamelitannin was tested against Staphylococcus epidermidis, Staphylococcus aureus, Acinetobacter baumannii and Candida albicans in a mouse foreign body infection model, using polyurethane catheter segments. Biofilm formation was assessed with a crystal violet assay to quantify the total biomass, with a tetrazolium reduction assay to quantify the metabolic activity and with scanning electron microscopy. RESULTS: At subinhibitory concentrations, cerium nitrate significantly reduced biofilm formation by C. albicans, chitosan significantly decreased biofilm formation by S. epidermidis and C. albicans, and hamamelitannin significantly inhibited all bacterial biofilms. DISCUSSION: The in vivo antibiofilm effect of cerium nitrate against C. albicans and of chitosan against C. albicans and S. epidermidis, at subinhibitory concentrations, makes them promising alternatives to coat CVCs. Moreover, the microbicidal effect on a wider range of CVC colonizers was previously reported in vitro for both compounds, at higher concentrations. For all bacterial strains, the highest in vivo antibiofilm efficacy was achieved with hamamelitannin. For A. baumannii, this is the first report of in vivo inhibition.


Subject(s)
Anti-Infective Agents/pharmacology , Biofilms/drug effects , Catheter-Related Infections/drug therapy , Cerium/pharmacology , Chitosan/pharmacology , Gallic Acid/analogs & derivatives , Hexoses/pharmacology , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/growth & development , Animals , Anti-Infective Agents/therapeutic use , Biofilms/growth & development , Candida albicans/drug effects , Candida albicans/growth & development , Candidiasis/drug therapy , Candidiasis/microbiology , Catheter-Related Infections/microbiology , Cerium/therapeutic use , Chitosan/therapeutic use , Female , Gallic Acid/pharmacology , Gallic Acid/therapeutic use , Hexoses/therapeutic use , Mice , Mice, Inbred BALB C , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcus epidermidis/drug effects , Staphylococcus epidermidis/growth & development
18.
Clin Microbiol Infect ; 19(1): E8-E15, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23145853

ABSTRACT

The rapid detection of extended-spectrum beta-lactamases (ESBLs) is a challenge for most clinical microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control. The aim of our study was to develop a rapid detection assay of ESBL producers based upon flow cytometric analysis. Antimicrobial susceptibility testing followed by molecular characterization of bla(TEM) , bla(SHV) or bla(CTX-M) genes was performed on clinical isolates (41 ESBL positive and 20 ESBL negative) and isolates expressing well-characterized beta-lactamases, including ESBLs (n = 13), plasmid AmpCs (n = 3), oxacillinases (n = 5) and carbapenemases (n = 3). Additionally, two ATCC strains recommended by CLSI for susceptibility testing were used as controls. The flow cytometry analysis protocol involved an incubation of bacterial cells with different concentrations of ceftazidime (1, 2 and 4 mg/L) and cefotaxime (4, 8 and 16 mg/L) for 1 and 2 hours, in the presence and absence of clavulanic acid; subsequently, cells were stained with the fluorescent dye Bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4) (3)], a lipophilic anion able to diffuse across depolarized membranes. Additionally, CFU counts were performed. Susceptible isolates displayed increased fluorescence after 1 hour of incubation; conversely, the increase of the depolarized population was only observed after incubation with clavulanic acid associated with ceftazidime or cefotaxime in ESBL producers. An excellent correlation was obtained between the number of non-depolarized bacteria quantified by flow cytometry and by conventional CFU assays. A novel, accurate and fast flow cytometric assay is available to detect the presence of ESBLs.


Subject(s)
Enterobacteriaceae/enzymology , Enterobacteriaceae/isolation & purification , Flow Cytometry/methods , Microbial Sensitivity Tests/methods , beta-Lactamases/analysis , Anti-Bacterial Agents/pharmacology , Cefotaxime/pharmacology , Ceftazidime/pharmacology , Colony Count, Microbial , Drug Resistance, Bacterial/genetics , Drug Resistance, Bacterial/physiology , Enterobacteriaceae/drug effects , Enterobacteriaceae/genetics , Enterobacteriaceae Infections/microbiology , Genes, Bacterial , Humans , Microbial Viability/drug effects , Phenotype , beta-Lactamases/genetics
19.
Gynecol Obstet Invest ; 74(2): 120-4, 2012.
Article in English | MEDLINE | ID: mdl-22889741

ABSTRACT

BACKGROUND/AIM: Recurrent vulvovaginal candidosis (RVVC) needs alternative therapeutic approaches. Gentian violet (GeV) has been traditionally used to treat mucocutaneous candidosis. The aim of the present study was to evaluate the in vitro activity of GeV against Candida spp. and contribute to clarify the mechanism of action, supporting its clinical therapeutic use. METHODS: Seventeen clinical Candida isolates from RVVC and one C. albicans type collection (ATCC 10231) were studied; the antifungal activity of GeV was evaluated according to the CLSI M27-A3 protocol. To elucidate its mechanism of action, cells were stained with propidium iodide and afterwards analyzed by flow cytometer. RESULTS: GeV showed a fungicidal activity against most Candida spp. C. albicans and C. tropicalis were the most susceptible species. Minimal lethal concentrations were similar to minimal inhibitory concentrations for most tested strains. The fungicidal effect was not related to a primary lesion of the cytoplasmic membrane. CONCLUSION: In accordance with our findings, GeV is a valuable potent fungicidal drug to be used topically, isolated or in combination with oral antifungal drugs, particularly in RVVC cases.


Subject(s)
Antifungal Agents/pharmacology , Candida/drug effects , Gentian Violet/pharmacology , Candida albicans/drug effects , Candida tropicalis/drug effects , Candidiasis, Vulvovaginal/drug therapy , Candidiasis, Vulvovaginal/microbiology , Female , Fluconazole/pharmacology , Gentian Violet/administration & dosage , Humans , Microbial Sensitivity Tests
20.
Eur J Clin Microbiol Infect Dis ; 31(12): 3351-7, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22843284

ABSTRACT

Culture in selective media represents the standard diagnostic method to confirm Legionella pneumophila infection, despite requiring a prolonged incubation period; antigen detection by immunofluorescence (IFS) and molecular techniques are also available, but they do not allow antimicrobial susceptibility evaluation. Our objective was to optimise flow cytometry (FC) protocols for the detection of L. pneumophila in respiratory samples and for susceptibility evaluation to first-line drugs. In order to optimise the FC protocol, a specific monoclonal antibody, conjugated with fluorescein isothiocyanate (FITC), was incubated with type strain L. pneumophila ATCC 33152. The limit of detection was established by analysing serial dilutions of bacterial suspension; specificity was assayed using mixtures of prokaryotic and eukaryotic microorganisms. The optimised FC protocol was used to assess 50 respiratory samples and compared with IFS evaluation. The susceptibility profile to erythromycin, ciprofloxacin and levofloxacin was evaluated by FC using propidium iodide and SYBR Green fluorescent dyes; the results were compared with the Etest afterwards. The optimal specific antibody concentration was 20 µg/ml; 10(2)/ml Legionella organisms were detected by this protocol and no cross-reactions with other microorganisms were detected. The five positive respiratory samples (10 %) determined by IFS were also detected by FC, showing 100 % correlation. After 1 h of incubation at 37 °C with different antimicrobials, SYBR Green staining could discriminate between treated and non-treated cells. A novel flow cytometric approach for the detection of L. pneumophila from clinical samples and susceptibility evaluation is now available, representing an important step forward for the diagnosis of this very relevant agent.


Subject(s)
Anti-Bacterial Agents/pharmacology , Flow Cytometry/methods , Legionella pneumophila/drug effects , Legionella pneumophila/isolation & purification , Legionnaires' Disease/diagnosis , Antibodies, Bacterial , Antibodies, Monoclonal , Cross Reactions , Fluorescence , Fluorescent Dyes/metabolism , Humans , Legionnaires' Disease/microbiology , Microbial Sensitivity Tests/methods , Sensitivity and Specificity , Staining and Labeling/methods
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