A novel micellar formulation based on natural plant extracts enhances the efficacy of hydrogen peroxide against biofilms of Staphylococcus spp. and Pseudomonas aeruginosa.

The antibacterial efficacy of hydrogen peroxide encapsulated in micelles (mH2O2) against biofilms was compared with that of hydrogen peroxide alone and of three commercially available aqueous biocides. The activity of mH2O2 on 24-h biofilms of reference strains of Staphylococcus spp. and Pseudomonas aeruginosa was tested in a static microtiter plate model. The biofilms were incubated with mH2O2 (17% v/v H2O2, 2% lactic acid, 0.3% phytoextract, H2O) and its individual ingredients and compared with three aqueous biocides at different concentrations and times of exposure. After 5-min exposure, 10% mH2O2 (corresponding to 1.7% v/v H2O2) achieved > 8 log10 reductions against all the test strains, while 1.7% H2O2 achieved a maximum of 1.5 log10 reduction. After 5-min exposure, none of the commercially available biocides tested showed themselves to be capable of completely eliminating the test strains embedded in biofilms. Hydrogen peroxide encapsulated in micelles demonstrated enhanced activity against planktonic cells and biofilms of Staphylococcus spp. and P. aeruginosa.

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides, proteins, eDNA and their correlation with biofilm mass.

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R2 = 0.882), while the content of proteins turned out to be weakly (R2 = 0.465) and that of eDNA very weakly associated (R2 = 0.202) to the biofilm mass.

Does Extracellular DNA Production Vary in Staphylococcal Biofilms Isolated From Infected Implants versus Controls?

BACKGROUND: Prosthetic implant infections caused by Staphylococcus aureus and epidermidis are major challenges for early diagnosis and treatment owing to biofilm formation on the implant surface. Extracellular DNA (eDNA) is actively excreted from bacterial cells in biofilms, contributing to biofilm stability, and may offer promise in the detection or treatment of such infections. QUESTIONS/PURPOSES: (1) Does DNA structure change during biofilm formation? (2) Are there time-dependent differences in eDNA production during biofilm formation? (3) Is there differential eDNA production between clinical and control Staphylococcal isolates? (4) Is eDNA production correlated to biofilm thickness? METHODS: We investigated eDNA presence during biofilm formation in 60 clinical and 30 control isolates of S aureus and S epidermidis. The clinical isolates were isolated from patients with infections of orthopaedic prostheses and implants: 30 from infected hip prostheses and 30 from infected knee prostheses. The control isolates were taken from healthy volunteers who had not been exposed to antibiotics and a hospital environment during the previous 3 and 12 months, respectively. Control S epidermidis was isolated from the skin of the antecubital fossa, and control S aureus was isolated from the nares. For the biofilm experiments the following methods were used to detect eDNA: (1) fluorescent staining with 4',6-diamidino-2-phenylindole (DAPI), (2) eDNA extraction using a commercial kit, and (3) confocal laser scanning microscopy for 24-hour biofilm observation using propidium iodide and concanavalin-A staining; TOTO®-1 and SYTO® 60 staining were used for observation and quantification of eDNA after 6 and 24 hours of biofilm formation. Additionally antibiotic resistance was described. RESULTS: eDNA production as observed by confocal laser scanning microscopy was greater in clinical isolates than controls (clinical isolates mean ± SD: 1.84% ± 1.31%; control mean ± SD: 1.17% ± 1.37%; p < 0.005) after 6 hours of biofilm formation. After 24 hours, the amount of eDNA was greater in biofilms of S epidermidis than in biofilms of S aureus (S aureus mean ± SD: 1.35% ± 2.0%; S epidermidis mean ± SD: 6.42% ± 10.6%; p < 0.05). Clinical isolates of S aureus and S epidermidis produced more eDNA than control isolates at 6 hours of biofilm formation. The extraction method also showed that clinical isolates produced substantially greater amounts of eDNA than controls. CONCLUSIONS: S aureus and S epidermidis exhibit a differential production of DNA with time. Clinical isolates associated with implant infections produce greater amounts of eDNA than controls. Future research might focus on the diagnostic value of eDNA as a surrogate laboratory marker for biofilm formation in implant infections. CLINICAL RELEVANCE: eDNA should be considered as a potential future diagnostic tool or even a possible target to modify biofilms for successful treatment of biofilm-associated infections.

Oral microbial colonization in laryngectomized patients as a possible cofactor of biofilm formation on their voice prostheses.

AIM: Biofilm formation on voice prostheses, which are used for voice rehabilitation in laryngectomized patients, is a main cause of device failure. The aim of this study was to assess whether the presence of periodontal pathogens in the biofilm on voice prostheses is related to that in the oral cavity and associated with the periodontal status of the patients. METHODS: Thirty-one laryngectomized patients were invited to participate, 13 of whom met exclusion criteria. The remaining 18 were classified according to the community periodontal index of treatment needs (community periodontal index of treatment needs (CPITN), grades 0-4). Biofilm samples from the oral cavity and voice prostheses were analysed by PCR-based hybridization for 11 pathogens. RESULTS: All dentate patients required periodontal treatment (CPITN-3: n = 4, CPITN-4: n = 8); the remaining six were edentulous. The diversity (i.e. number of bacterial species detected) of pathogens detected on the voice prostheses correlated significantly positively with the diversity of pathogens in the oral cavity and with clinical parameters. Furthermore, the diversity of pathogens differed significantly between dentate and edentulous patients. CONCLUSIONS: Results emphasize the oral cavity as an important source of bacteria for biofilm formation on voice prostheses. Whether these pathogens reduce the lifetime of the device by increased biofilm formation and/or increase the risk of silicone deterioration requires further study.

The effectiveness of surface disinfectants and a micellic H2O2 based water disinfectant on Candida auris.

BACKGROUND: As infection control faces new challenges from emerging, multidrug resistant strains of the yeast Candida auris, this study was conducted in order to examine the efficacy of hospital surface disinfectants and a new water disinfectant against C. auris biofilm forms. METHODS: We tested four reference strains of C. auris (NCPF8971, NCPF8977, NCPF8984, DSM21092) and one C. albicans strain (ATCC10231) against disinfectants based on ethanol (ETH), quaternary ammonium (QAC), a combination of glutaraldehyde, quaternary ammonium and surfactant (ALD) and potassium peroxymonosulfate (PP) as well as 3.4% H2O2 and 4.25% H2O2 alone. In addition, a micellic-based formulation containing 17% v/v hydrogene peroxide disinfectant (mH2O2) was tested. The efficacy of the disinfectants was measured in a 96-well plate using tetrazolium salt reduction (XTT) and the log10 reduction assay. RESULTS: ETH and QAC displayed in clinically recommended concentrations more than 5log10 reduction and more than 80% XTT activity reduction for all of the Candida biofilms and planktonic cells. Only biofilms of C. auris NCPF8984 were additionally sensitive to all remaining disinfectants. All tested C. auris biofilms were sensitive to PP disinfectant and showed more than 5log10 reduction. However, the XTT assay showed a reduction of less than 80% for the PP disinfectant, indicating the presence of active but non-culturable cells. The 25% mH2O2 (corresponding to 4.25% H2O2) killed Candida biofilms after 1 minute.

Infection control and risk factors for acquisition of carbapenemase-producing enterobacteriaceae. A 5 year (2011-2016) case-control study.

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.

Disinfection of contaminated metal implants with an Er:YAG laser.

Infections related to orthopedic procedures are considered particularly severe when implantation materials are used, because effective treatments for biofilm removal are lacking. In this study, the relatively new approach for infection control by using an erbium:yttrium-aluminum-garnet (Er:YAG) laser was tested. This laser vaporizes all water containing cells in a very effective, precise, and predictable manner and results in only minimal thermal damage. For preliminary testing, 42 steel plates and 42 pins were seeded with mixed cultures. First, the minimally necessary laser energy for biofilm removal was determined. Subsequently, the effectiveness of biofilm removal with the Er:YAG laser and the cleansing of the metal implants with octenidine-soaked gauze was compared. Then, we compared the effectiveness of biofilm removal on 207 steel pins from 41 patients directly after explantation. Sonication and scanning electron microscopy were used for analysis. Laser fluences exceeding 2.8 J/cm2 caused a complete extinction of all living cells by a single-laser impulse. Cleansing with octenidine-soaked gauze and irradiation with the Er:YAG laser are both thoroughly effective when applied to seeded pins. In contrast, when explanted pins with fully developed biofilms were analyzed, we found a significant advantage of the laser procedure. The Er:YAG laser offers a secure, complete, and nontoxic eradication of all kinds of pathogens from metal implants without damaging the implant and without the possible development of resistance. The precise noncontact removal of adjacent tissue is a decisive advantage over conventional disinfectants. Therefore, laser irradiation could become a valuable method in every debridement, antibiotics, and implant retention procedure.

Growth Media for Mixed Multispecies Oropharyngeal Biofilm Compositions on Silicone.

AIMS: Microbial colonization of silicone voice prostheses by bacteria and Candida species limits the device lifetime of modern voice prostheses in laryngectomized patients. Thus, research focuses on biofilm inhibitive properties of novel materials, coatings, and surface enhancements. Goal of this in vitro study was the evaluation of seven commonly used growth media to simulate growth of mixed oropharyngeal species as mesoscale biofilms on prosthetic silicone for future research purposes. METHODS AND RESULTS: Yeast Peptone Dextrose medium (YPD), Yeast Nitrogen Base medium (YNB), M199 medium, Spider medium, RPMI 1640 medium, Tryptic Soy Broth (TSB), and Fetal Bovine Serum (FBS) were used to culture combined mixed Candida strains and mixed bacterial-fungal compositions on silicone over the period of 22 days. The biofilm surface spread and the microscopic growth showed variations from in vivo biofilms depending on the microbial composition and growth medium. CONCLUSION: YPD and FBS prove to support continuous in vitro growth of mixed bacterial-fungal oropharyngeal biofilms deposits over weeks as needed for longterm in vitro testing with oropharyngeal biofilm compositions. SIGNIFICANCE AND IMPACT OF STUDY: The study provides data on culture conditions for mixed multispecies biofilm compositions that can be used for future prosthesis designs.

In vitro biofilm growth on modern voice prostheses.

BACKGROUND: Biofilm formation on voice prostheses in laryngectomized patients usually limits the lifetime of the device. The purpose of this study was to compare the biofilm resistance of different valve flaps of modern voice prostheses in an in vitro simulation of an oropharyngeal biofilm. METHODS: Growth of biofilm deposits on valve flaps (n = 12) removed from Provox 2, Provox Vega, Provox ActiValve, Blom Singer Advantage, and Phonax voice prostheses was evaluated and compared to medical-grade silicone (n = 12) in an in vitro biofilm model (22 days) after incubation with a multispecies bacterial-fungal biofilm composition. RESULTS: The Provox ActiValve and the Blom Singer Advantage prostheses showed significantly less surface biofilm formation than the other prostheses and then silicone. CONCLUSION: The use of silver oxide and Teflon as valve flap materials proves to reduce long-term biofilm formation in vitro. The applied model allows rapid screening for novel biofilm-inhibitive materials and durable coatings designated for more biofilm resistant medical devices.

Bacterial Extracellular DNA Production Is Associated with Outcome of Prosthetic Joint Infections.

In a retrospective study the association of the production of extracellular DNA (eDNA) in biofilms of clinical staphylococcal isolates from 60 patients with prosthetic joint infection (PJI) and the clinical outcome were investigated. Data from a previous study on eDNA production determined in 24-hour biofilms of staphylococcal isolates (Staphylococcus aureus n=30, Staphylococcus epidermidis n=30) was correlated with the patients' clinical outcome after 3 and 12 months. Statistical analysis was performed using either the Spearman's rank correlations test or the t-test. eDNA production of S. epidermidis in 24-hour biofilms correlated with the patients' outcome 'not cured' after 12 months. For S. aureus no such correlation was detected. Thus, eDNA may be a virulence factor of S. epidermidis. Quantification of eDNA production as a surrogate marker for biofilm formation might be a potential predictive marker for the management of PJI.

Oral cavity anaerobic pathogens in biofilm formation on voice prostheses.

BACKGROUND: A polymerase chain reaction (PCR)-based method has been used to identify oral anaerobic pathogens in biofilms on voice prostheses. The purpose of the present study was to determine the location of those pathogens inside the biofilms. METHODS: Biofilms of 15 voice prostheses were sampled and used to identify the oral pathogens. Fluorescence in situ hybridization was applied on smears made on glass slides and on sections of intact biofilms visualized by confocal laser scanning microscopy (CLSM). RESULTS: Fusobacterium nucleatum (F. nucleatum) was the most frequently detected pathogen and the only tested species detected in microcolonies. The other microbes (Parvimonas micra [P. micra], Porphyromonas gingivalis [P. gingivalis], Tannerella forsythia [T. forsythia], and Treponema denticola [T. denticola]) were not detected or only detected as single cells. CLSM analysis showed that F. nucleatum resided on the biofilm surface. CONCLUSION: Although detectable, oral anaerobic pathogens seem to be no more than passers-by that adhere without further observed proliferation and apparently play no striking role in biofilm formation on voice prostheses.

Growth kinetics of candida biofilm on medical polymers: a long-term in vitro study.

OBJECTIVES/HYPOTHESIS: Recent in vitro models simulating biofilm formation on medical polymers are restricted to only short-term observation periods of 2 hours to 12 days. STUDY DESIGN: The goal of this study was to develop an in vitro model to generate a long-term polymicrobial biofilm with Candida albicans (C albicans) and Streptococcus salivarius (S salivarius) on thermoplastic medical grade silicone (TPS) and polyurethane (TPU) and continuous documentation of growth kinetics. METHODS: Test platelets of TPS and TPU were incubated in well plates in RPMI agar at 37°C. Both microbial specimen were isolated from explanted voice prostheses and added every second day for 28 days. Afterward, only the nutrition solution has been changed regularly. Biofilm kinetics were monitored using a specially designed image analysis software to calculate the percentual surface covering of each platelet. Biofilm architecture was investigated by scanning electron microscopy (SEM). Microbial infiltration was examined by crystal violet staining and thin section microscopy. RESULTS: On both materials tested, a cover of living candida biofilm could be generated over 140 days. Colonization was permanent with at least 10% surface coverage. Initially, both materials showed coverage of up to 80% followed by biofilm detachment, which could be reduced by adding planktonic microbes. SEM confirmed three-dimensional biofilm architecture with dimorphic candida growth. Microbial material infiltration of nonhypheal types was proved in 2 TPU platelets, but not in TPS. CONCLUSIONS: The in vitro model presented in this study mimics in vivo events of biofilm formation on medical polymers with continuous monitoring of living biofilm kinetics.

Anaerobic and microaerophilic pathogens in the biofilm formation on voice prostheses: A pilot study.

OBJECTIVES/HYPOTHESIS: Voice rehabilitation with voice prostheses is a standard therapy in laryngectomized patients. Biofilm formation on the surface of the voice prostheses causes device failure and requires frequent replacements. Studies analyzing the biofilm of voice prostheses have mainly focused on aerobic bacteria. Anaerobic bacteria as an integral part of the biofilms on voice prostheses have not been investigated yet. STUDY DESIGN: Prospective pilot study on the occurrence of anaerobic and microaerophilic pathogens in biofilm formation on voice prostheses. METHODS: Biofilm samples of 15 voice prostheses were analyzed using a polymerase chain reaction-based hybridization method regarding the presence of 11 selected anaerobic and microaerophilic pathogens. RESULTS: In 80% of the voice prostheses, at least one and up to 10 of the tested bacteria could be identified. Fusobacterium nucleatum was the pathogen most often present (73%). Other frequently occurring pathogens were Treponema denticola (40%), Tannerella forsythia (33%), and Eikenella corrodens (33%). There was no correlation between the number of identified bacteria and the indwelling times (mean, 127 days; maximum, 344 days; minimum, 22 days). CONCLUSIONS: For the first time, anaerobic and microaerophilic pathogens have been identified as part of the biofilm formation on the surface of voice prostheses. Those pathogens might be responsible for accelerated biofilm formation and reduced lifetime of the voice prostheses.