To test the effects of several biocides [N-propanol, a commercially available propanol/ethanol/chlorhexidine mixture, polyvinylpyrolidone (povidone-iodine) and hydrogen peroxide] on established biofilms of Staphylococcus epidermidis isolated from patients with cardiac implant infections and catheter-related bacteraemia. Biofilms were grown in microtitre plates for 24 h, dyed and stained with Crystal Violet. The mean optical density (OD) and the OD ratio (OD_r = OD of the treated biofilm/OD of the untreated biofilm) were used for quantification. Biofilms were incubated with 60% (v/v) N-propanol, the mixture of propanol/ethanol/chlorhexidine, hydrogen peroxide at three concentrations (0.5%, 3% and 5%, v/v) and povidone-iodine for 1, 5, 15, 30 and 60 min. Unstained biofilms were sonicated and plated on Columbia agar for time–kill curves. S. epidermidis skin isolates from healthy volunteers were used as controls. Biofilm ODs of the clinical S. epidermidis isolates and the isolates from the healthy volunteers were significantly different (1.17 ± 0.512 versus 0.559 ± 0.095, respectively; mean ± SD) (P < 0.01). No viable S. epidermidis was detected in biofilms treated with the alcohols, N-propanol or the propanol/ethanol/chlorhexidine mixture. Incubation with povidone-iodine and hydrogen peroxide 3% and 5% led to a log reduction of the viable cells of >5 after incubation for 5 min, however, up to 10³ viable cells were detected in four isolates after 30 min of incubation with povidone-iodine. S. epidermidis obtained from infected implants forms thicker biofilms than that of healthy volunteers. Hydrogen peroxide, at a concentration of 3% and 5%, and alcohols rapidly eradicate S. epidermidis biofilms, whereas povidone-iodine is less effective.

The aim of the present study was to evaluate the in vitro activity and synergism of the combinations of amphotericin B/caspofungin and amphotericin B/posaconazole against Candida albicans, grown either as planktonic cells or in biofilms. Ten C. albicans bloodstream isolates used in this study were collected from intensive care patients admitted to the Vienna University Hospital between 2006 and 2007. Chequerboard tests were employed to determine the efficacy of the antifungal combinations amphotericin B/caspofungin and amphotericin B/posaconazole against both planktonic cells and biofilms. C. albicans biofilms were prepared using the static microtitre plate model. The activity of antifungal combination therapy was determined by visual reading for planktonic cells and using the XTT assay for biofilms. For Candida biofilms the median MIC was 4 mg/L for amphotericin B and caspofungin, and >256 mg/L for posaconazole. The combination amphotericin B/posaconazole yielded synergism [fractional inhibitory concentration index (FICI) <0.26], whereas amphotericin B/caspofungin yielded indifferent interaction only (FICI 0.75–1.25) against all isolates when grown in biofilms. Under planktonic conditions, synergism was demonstrable for the combination amphotericin B/caspofungin against 4 of the 10 isolates, whereas the combination of caspofungin/posaconazole was indifferent against all tested isolates. We showed that MICs for planktonic and biofilm forms of C. albicans were much lower when treated with an antifungal combination than when treated with single agents. The combination of amphotericin B/posaconazole yielded synergism against Candida biofilms, whereas amphotericin B/caspofungin yielded indifferent interaction.

Multidrug resistance may pose a serious problem to antifungal therapy. The Candida albicans Cdr2p is one of two ATP-binding cassette (ABC) transporters mediating antifungal resistance in vivo through increased drug efflux. Echinocandins such as caspofungin represent the newest class of antifungals that target cell wall synthesis. We show here by agar plate resistance assays that cross-resistant clinical isolates of C. albicans display high minimal inhibitory concentrations (MICs) to caspofungin when compared with a sensitive ATCC reference strain. Northern analysis and immunoblotting indicate that these isolates also show high levels of CDR1 and CDR2 expression. To determine a possible contribution of Cdr1p or Cdr2p to caspofungin resistance, we have functionally expressed Cdr1p and Cdr2p in appropriate recipient strains of the yeast Saccharomyces cerevisiae. Yeast cells expressing Cdr1p or Cdr2p exhibit cross-resistance to established antifungal drugs such as azoles and terbinafine. However, Cdr2p and, to a much lesser extent, Cdr1p confer caspofungin hyper-resistance when expressed in yeast. Likewise, Cdr2p confers caspofungin resistance when constitutively overexpressed in a drug-sensitive C. albicans strain. We therefore propose that Cdr2p may contribute to clinical candin resistance. Finally, our data suggest that cross-resistance phenotypes of clinical isolates are the consequence of distinct mechanisms that may operate simultaneously.

Background: Carbapenemase-producing enterobacteriaceae (CPE) are a major threat for severely ill patients. However, only limited data on the epidemiology and on evidence-based infection prevention and control measures are available. The aim of this study was to investigate the epidemiology of patients with CPE, characterizing the CPE isolates by their resistance mechanisms and genetic similarity, to explore risk factors for their acquisition, and to evaluate the effectiveness of the current CPE infection control measures. Methods: A retrospective case-control study was performed using data from 2011 to 2016 in a 1800-bed academic hospital in Central Europe, where risk-based screening at patients´ admission is performed. Carbapenem resistance mechanisms of all carbapenem resistant enterobacteriaceae from patients admitted during this period were investigated. Clinical data of the CPE-positive patients were analysed and compared to a matched control group (case-control ratio of 1:3). We performed univariate and multivariate statistical analysis to identify risk factors for CPE acquisition. Results: Of 621,623 admitted patients in the study period, 75 patients with carriage of carbapenem resistant enterobacteriaceae were included (0.12/1000 admittances). Carbapenemase-encoding genes were detected in 77.3% (58/75) of patients with carbapenem-resistant enterobacteriaceae. The enzyme blaOXA-48 was found in 34.5% (20/58), blaKPC in 29.3% (17/58), blaNDM enzymes in 20.7% (12/58) and blaVIM in 8.6% (5/58) of the isolates. The overall mortality among CPE patients was 25.9% (15/58) and attributable mortality of CPE was 53.3% (8/15). Multivariate analysis revealed four risk factors to be independent predictors of CPE carriage: the length of hospital admission > 20 days (AOR: 4.9, 95% CI: 1.4–15.5; P < 0.001), hospital admission within the previous year (AOR: 22.3, 95% CI: 3.9–88.4; P < 0.001), exposure to a healthcare facility in a country with high or unknown carbapenem-resistant enterobacteriaceae prevalence 3 months before admission (AOR: 11.8, 95% CI: 2.2–63.2; P < 0.01) and the use of antibiotics longer than 10 days (AOR: 5.2, 95% CI: 1.4–35.9; P < 0.05). The current risk-based screening strategy at hospital admission could not identify 37 (63.8%) of the 58 CPE-positive patients. Epidemiological investigation and genotyping revealed that no outbreaks due to CPE occurred during this period. Conclusion: Overall, the CPE carriage rate in patients was very low, the attributable mortality, however, is alarming (53%). BlaOXA-48 and blaKPC were the main cause of carbapenem resistance in enterobacteriaceae. Although the strict application of standard infection control measures was effective for prevention of outbreaks in this setting, an enlarged risk based targeted screening strategy has to be implemented.

Implant-related infections are serious complications of trauma and orthopedic surgery and are most difficult to treat. The bacterial biofilms of 34 clinical Staphylococcus sp. isolates ( Staphylococcus aureus , n = 14; coagulase-negative staphylococci, n = 19) were incubated with daptomycin (DAP; 5, 25, or 100 mg/liter), vancomycin (VAN; 5, 25, or 100 mg/liter), tigecycline (TGC; 1, 5, or 25 mg/liter), fosfomycin (FOM; 100, 250, or 1,000 mg/liter), and cefamandole (FAM; 50, 100, or 500 mg/liter) for 24 h at three different ambient temperatures: 35°C, 40°C, and 45°C. To quantify the reduction of the biomass, the optical density ratio (ODr) of stained biofilms and the number of growing bacteria were determined. Increasing the temperature to 45°C or to 40°C during incubation with FAM, FOM, TGC, VAN, or DAP led to a significant but differential reduction of the thickness of the staphylococcal biofilms compared to that at 35°C ( P < 0.05). Growth reduction was enhanced for DAP at 100 mg/liter at 35°C, 40°C, and 45°C (log count reductions, 4, 3.6, and 3.3, respectively; P < 0.05). A growth reduction by 2 log counts was detected for FAM at a concentration of 500 mg/liter at 40°C and 45°C ( P = 0.01). FOM at 1,000 mg/liter reduced the bacterial growth by 1.2 log counts (not significant). The antibacterial activity of antimicrobial agents is significantly but differentially enhanced by increasing the ambient temperature and using high concentrations. Adjuvant hyperthermia may be of value in the treatment of biofilm-associated implant-related infections.