A multisite validation of a two hours antibiotic susceptibility flow cytometry assay directly from positive blood cultures.

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.

Evaluation of rapid colistin susceptibility directly from positive blood cultures using a flow cytometry assay.

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.

Anti-Candida activity of antimicrobial impregnated central venous catheters.

BACKGROUND: Whenever the rate of central line-associated bloodstream infections (CLABSIs) remains high even after the implementation of preventive strategies, the use of chlorhexidine/silver sulfadiazine (CSS) or minocycline/rifampin (MR)-impregnated central venous catheters (CVCs) is currently recommended. Nevertheless, the efficacy of such CVCs against Candida albicans and other emerging non-albicans spp. has been insufficiently studied. This study aims to compare the activity of CSS and MR-impregnated CVCs against the yeasts most frequently isolated from CLABSIs. METHODS: For biofilm formation assays, type strains and clinical isolates of C. albicans, C. glabrata and C. parapsilosis sensu stricto were used. Segments of standard polyurethane, MR and second-generation CSS-CVCs were tested. The biofilm metabolic activity was measured by a semi-quantitative XTT reduction assay. RESULTS: CSS catheter segments significantly reduced the biofilm metabolic activity by all tested Candida spp., with inhibition ranging from 60% to 100%. The MR catheter segments promoted C. albicans and C. parapsilosis biofilm formation and exhibited an inconspicuous effect against C. glabrata. CONCLUSIONS: Among the recommended antimicrobial CVCs, CSS-CVCs proved to be superior in the inhibition of biofilm formation by the most frequent yeasts causing CLABSIs. Data from this in vitro study may suggest that patients at high risk for invasive candidosis could benefit from the use of CSS-CVCs.

High-touch surfaces: microbial neighbours at hand.

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.

Impact of ERG3 mutations and expression of ergosterol genes controlled by UPC2 and NDT80 in Candida parapsilosis azole resistance.

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.

Anti-biofilm activity of low-molecular weight chitosan hydrogel against Candida species.

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.

In vivo antibiofilm effect of cerium, chitosan and hamamelitannin against usual agents of catheter-related bloodstream infections.

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.

Cerium, chitosan and hamamelitannin as novel biofilm inhibitors?

OBJECTIVES: The colonization of indwelling medical devices and subsequent biofilm formation represents a global challenge since it promotes the persistence of infection and contributes to antimicrobial resistance. The aim of this study was to determine the antimicrobial activity of cerium, chitosan and hamamelitannin against usual microbial colonizers and to assess their efficacy regarding biofilm formation on polyurethane (PUR)-like catheters. METHODS: The antimicrobial and anti-biofilm effect of cerium nitrate, low molecular weight chitosan (LMWC) and hamamelitannin was tested against Staphylococcus epidermidis, Staphylococcus aureus, Acinetobacter baumannii and Candida albicans strains. Biofilm formation was assessed with PUR-like catheter segments and the metabolic activity was quantified by colorimetry with a tetrazolium reduction assay. RESULTS: Cerium nitrate and LMWC inhibited the microbial growth of all microbial strains tested; hamamelitannin showed no inhibition. Regarding biofilm formation on PUR-like catheters, with subinhibitory concentrations: cerium nitrate significantly inhibited the metabolic activity of C. albicans; LMWC reduced the metabolic activity of S. epidermidis and C. albicans; and hamamelitannin decreased the metabolic activity of all tested bacteria, but not of yeasts. CONCLUSIONS: The microbicidal activity of cerium nitrate and LMWC was clearly demonstrated in this study, as was their fungistatic effect at lower concentrations. Hamamelitannin significantly reduced biofilm metabolic activity of all tested bacteria. These microbial inhibitors may play a promising role regarding different biomedical applications.

The anti-Candida activity of Thymbra capitata essential oil: effect upon pre-formed biofilm.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected] Thymbra capitata essential oil is traditionally considered to exhibit powerful antiseptic properties, thus being used to treat cutaneous infections. The aim of the present study was to evaluate the effect of Thymbra capitata essential oil upon pre-formed biofilm of different Candida strains while comparing it with the activity against planktonic cells. MATERIALS AND METHODS: Fifteen Candida isolates were included, corresponding to clinical and collection type strains. Essential oil was obtained by hydrodistillation and its composition analysed by GC/MS. Activity upon planktonic cells was evaluated according to M27-A3 macromethod. Its effect upon 24h preformed biofilm biomass was determined using the crystal violet procedure and the metabolic activity was studied applying the XTT/menadione technique. RESULTS: Biofilm biomass and metabolic activity of all tested species were reduced up to 50% at MIC values. The effect was more pronounced at double MIC values, achieving >80% reduction, except for Candida albicans that presented a more resistant profile (62%). CONCLUSION: Thymbra capitata essential oil presented an important effect upon Candida biofilms. It is proposed as a valuable antifungal product to be used in an appropriate pharmaceutical formulation for the management of resistant mucocutaneous candidosis.

A novel flow cytometric protocol for assessment of yeast cell adhesion.

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner.