In Vitro Evaluation of Resistance Development to Silver Sulfadiazine and Subsequent Cross-Resistance to Antibiotics.

Treatment of chronic wounds that are at risk of infection, or that are infected, require the use of antimicrobial dressings, most often those that contain silver. Silver exerts its antimicrobial effects by binding to multiple cellular components and, as such, bacterial resistance to it is low; however, molecular silver resistance has been documented and is attributed to the presence of the sil operon or changes in genes encoding porin and efflux pump expression. The aim of this study was to evaluate spontaneous silver resistance development in common opportunistic pathogens, Staphylococcus, Pseudomonas and Enterococcus cloacae, as well as resistance development when exposed to subtherapeutic concentrations over a prolonged period. Furthermore, following silver resistance development, cross-resistance to several classes of antibiotics was evaluated. Following exposure of the strains to silver sulfadiazine (SSD) at two times and four times minimum inhibitory concentration (MIC), the mutation rate was <1010 colony forming unit (CFU)/mL. Serial passage of S. aureus and P. aeruginosa in subinhibitory concentrations of SSD selected for no resistant mutants. The SSD MIC of E. cloacae increased past the solubility limit of SSD at serial passage 17. MIC testing of this isolate showed a >2048-fold increase in MIC to silver in comparison to the parent strain. MIC testing of the serial passage isolates demonstrated no cross-resistance to antibiotics from six different classes. Overall, the results of this study show resistance development to silver is low and, if it does occur, it does not confer resistance to several antibiotic classes. However, as this study was carried out with a small number of strains, a study with a larger panel of strains and sequencing of the strains to determine the exact mechanism of resistance would be needed to investigate the threat of silver resistance further.

Controlled-release iodine foam dressings demonstrate broad-spectrum biofilm management in several in vitro models.

Multiple in vitro models were utilised to evaluate the biofilm management capabilities of seven commercially-available wound dressings, varying in composition and antibacterial ingredients, to reduce common aerobic, anaerobic, and multispecies biofilms. The Center for Disease Control bioreactor was used to evaluate single species Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) 24 and 48 hours biofilms, as well as a multispecies biofilm consisting of these two organisms in addition to Enterococcus faecalis (E. faecalis). As wound biofilms often exist in hypoxic wound environments, a direct contact anaerobic model system was used to evaluate efficacy on Bacteroides fragilis (B. fragilis). Biofilm control was evaluated against P. aeruginosa in the drip flow bioreactor model, where a constant flow of proteinaceous media is used to create a more challenging and wound-like model. The results demonstrated that biofilm management capabilities varied amongst wound dressings. Two dressings, a controlled-release iodine foam dressing and a silver nanocrystalline dressing, showed potent >4 log reductions in recovered organisms compared with untreated controls in all biofilm models evaluated. The effectiveness of other dressings to manage bioburden varied between dressing, test organism, and model system. A silver foam dressing showed moderate biofilm control in some models. However, biofilm exposure to methylene blue and gentian violet-containing foam dressings showed negligible log reductions in all in vitro biofilm methods examined. The data outlined in this in vitro study support the use of the iodine foam dressing for wounds with infection and biofilm.

Antibiofilm Efficacy of Polihexanide, Octenidine and Sodium Hypochlorite/Hypochlorous Acid Based Wound Irrigation Solutions against Staphylococcus aureus, Pseudomonas aeruginosa and a Multispecies Biofilm.

Infection and the formation of biofilms have been shown to have a significant role in increased inflammation and delayed wound healing. Wound irrigation solutions are used to debride wounds, removing cell debris and infecting microorganisms, therefore preventing infection. The aim of this study was to evaluate a Polihexanide (PHMB) based wound irrigation solution, Octenidine HCl based wound irrigation solution and electrolysed water based wound care solution for antibiofilm efficacy against Staphylococcus aureus, Pseudomonas aeruginosa and a multispecies biofilm in several models to gain a broad understanding of ability. The PHMB based wound irrigation solution demonstrated broad range antibiofilm efficacy against P. aeruginosa, S. aureus and the multispecies biofilm. The Octenidine HCl based wound irrigation solution and the electrolysed water based wound care solution demonstrated potent antibiofilm efficacy against S. aureus and to a lesser extent P. aeruginosa. Overall, less efficacy was observed in the drip flow bioreactor model for all 3 test solutions, which may be attributed to the continuous flow of nutrients during treatment, which may have diluted or washed away the solution. The data presented also highlights the importance of testing antibiofilm activity in a range of biofilm models and against different bacterial strains to get an overall representation of efficacy.

The Ability of a Concentrated Surfactant Gel to Reduce an Aerobic, Anaerobic and Multispecies Bacterial Biofilm In Vitro.

Biofilm formation in wounds can lead to increased inflammation, infection and delayed wound healing. Additionally, biofilms show increased recalcitrance to antimicrobials compared to their planktonic counterparts making them difficult to manage and treat. Biofilms are frequently polymicrobial, consisting of aerobic and anaerobic bacteria, as well as fungi and yeasts. The aim of this study was to evaluate the effects of a concentrated surfactant gel with antibacterial preservative agents (CSG) against wound relevant opportunistic pathogens, including an aerobic biofilm, anaerobic biofilm and multispecies biofilm. The CSG was added to a 48 h anaerobic biofilm of Bacteroides fragilis, a 24 h multispecies biofilm of Acinetobacter baumannii, Staphylococcus aureus and Staphylococcus epidermidis and a 24 h biofilm of Pseudomonas aeruginosa grown in an in vitro wound relevant environment. Following a contact time of 24 h with the CSG, the bacterial cell density of the biofilms was reduced by 2-4 log in comparison to an untreated control. The results demonstrate the ability of the CSG to disrupt wound relevant biofilms and support the use of the CSG in the clinic to treat wounds caused by biofilm related infections.

The Effects of a Concentrated Surfactant Gel on Biofilm EPS.

INTRODUCTION: The aim of this study was to evaluate if a poloxamer-based concentrated surfactant gel (CSG), containing antibacterial preservative agents, had the ability to reduce the levels of biofilm extracellular polymeric substances (EPS), specifically proteins and extracellular DNA (eDNA), as these are found to be the most immunogenic, within an in vitro biofilm. MATERIALS AND METHODS: A 24-hour biofilm of P. aeruginosa ATCC 15442 was grown in a 12-well plate and treated for 24 hours with a CSG coated onto Multisorb® (BSN Medical Limited, Hull, United Kingdom). EPS were extracted from each sample using 1M sodium chloride. Protein and DNA in EPS extractions was determined quantitatively using the Pierce™ Coomassie (Bradford) protein assay kit and a microplate SYTO 9™ (ThermoFisher Scientific, Paisley, United Kingdom) fluorescent assay, respectively. Protein and DNA was also determined qualitatively using confocal laser scanning microscopy (CLSM). RESULTS: Following 24-hour growth of P. aeruginosa ATCC 15442 biofilm, 7.38mg/mL protein was isolated from the extracted EPS in the untreated control. In comparison, the protein concentration found in the extracted EPS from biofilms treated with a CSG was 6.39mg/mL, showing a 13.4% reduction. Following 24-hour growth of P. aeruginosa ATCC 15442 biofilm, 11.71mg/mL eDNA was isolated from the extracted EPS in the untreated control. In comparison, the eDNA concentration found in the extracted EPS from biofilms treated with a CSG was 0.65mg/mL, showing a 94.5% reduction. Following statistical analysis of the data, the decrease in protein isolated following CSG treatment was within error; however, the decrease in eDNA isolated was statistically significant, showing the ability of the CSG to break up biofilm EPS in vitro. Using confocal laser microscopy and staining techniques, a large quantity of protein and eDNA could be observed in samples from the untreated control. In comparison, a reduction in EPS protein and eDNA was observed in samples that had been treated with a CSG. CONCLUSION: The data presented here potentially shows the ability of a CSG to reduce components of the P. aeruginosa biofilm EPS. The reduction in eDNA following CSG treatment may contribute to the dispersal of the biofilm, potentially increasing the susceptibility of it to antimicrobials, and should be explored further.

Efficacy of Poloxamer-Based Wound Dressings on Acinetobacter baumanni Biofilms.

Objective: This study evaluated the antimicrobial and antibiofilm activity of a concentrated surfactant gel preserved with antimicrobials and a concentrated surfactant gel with 1% silver sulfadiazine (SSD) against 12 clinical strains of Acinetobacter baumannii and the type strain A. baumannii ATCC 19606. Approach: The biofilm-forming potential of the A. baumannii isolates was investigated using a crystal violet assay and classifying the isolates as "non-adherent," "weak," "moderate," or "strong" biofilm formers. The antimicrobial activity was determined using the zone of inhibition (ZOI) method. The antibiofilm activity was evaluated against A. baumannii ATCC 19606 using the Center for Disease Control bioreactor model. Results: A. baumannii readily forms biofilms with 8 out of the 12 clinical isolates being classified as strong biofilm formers (OD570 > 0.4). The concentrated surfactant gel with 1% SSD demonstrated antimicrobial activity against all isolates with a ZOI of 7.2-14 mm. Antibiofilm activity against a 24 h biofilm of A. baumannii ATCC 19606 was found, with a ≥7 log decrease in bacterial cell density following 24 h treatment with the concentrated surfactant gel with 1% SSD. The concentrated surfactant gel preserved with antimicrobials also showed some biofilm disruption with ∼3 log decrease in bacterial cell density being found. Innovation: The concentrated surfactant gel with 1% SSD used in this study showed antimicrobial and antibiofilm activity against A. baumannii. Conclusion: The concentrated surfactant gel with 1% SSD used in this study showed efficacy against A. baumannii, a common cause of wound infections, and should be considered for treatment of wounds infected with A. baumannii in health care settings.