Laser-induced vapor nanobubbles improve diffusion in biofilms of antimicrobial agents for wound care.

Being responsible for delayed wound healing, the presence of biofilms in infected wounds leads to chronic, and difficult to treat infections. One of the reasons why antimicrobial treatment often fails to cure biofilm infections is the reduced penetration rate of antibiotics through dense biofilms. Strategies that have the ability to somehow interfere with the integrity of biofilms and allowing a better penetration of drugs are highly sought after. A promising new approach is the use of laser-induced vapor nanobubbles (VNB), of which it was recently demonstrated that it can substantially enhance the penetration of antibiotics into biofilms, resulting in a marked improvement of the killing efficiency. In this study, we examined if treatment of biofilms with laser-induced vapor nanobubbles (VNB) can enhance the potency of antimicrobials which are commonly used to treat wound infections, including povidone-iodine, chlorhexidine, benzalkonium chloride, cetrimonium bromide and mupirocin. Our investigations were performed on Pseudomonas aeruginosa and Staphylococcus aureus biofilms, which are often implicated in chronic wound infections. Pre-treatment of biofilms with laser-induced VNB did enhance the killing efficiency of those antimicrobials which experience a diffusion barrier in the biofilms, while this was not the case for those compounds for which there is no diffusion barrier. The magnitude of the enhanced potency was in most cases similar to the enhancement that was obtained when the biofilms were completely disrupted by vortexing and sonication. These results show that laser-induced VNB are indeed a very efficient way to enhance drug penetration deep into biofilms, and pave the way towards clinical translation of this novel approach for treatment of wound infections.

Biofilm model systems for root canal disinfection: a literature review.

The aim of this review was to present an overview of laboratory root canal biofilm model systems described in the endodontic literature and to critically appraise the various factors that constitute these models. The electronic databases MEDLINE, Web of Science and EMBASE were searched up to and including December 2016 to identify laboratory studies using endodontic biofilm models. The following search terms were used in various combinations: biofilm, root canal, in vitro, endodontic, bacteria, root canal infection model, colony-forming unit. Only English papers from journals with an impact factor were selected. The records were screened by two reviewers, and full-text articles were assessed according to pre-defined criteria. The following data were extracted from the included studies: the microbial composition of the biofilm, the substrate, growth conditions, validation and quantification. Seventy-seven articles met the inclusion criteria. In the majority (86%) of the studies, a monospecies biofilm was cultured. In two studies, a dual-species biofilm was grown; others cultivated a multispecies biofilm, containing at least three species. Enterococcus faecalis was the most frequently used test species (in 79% of all studies, 92% of the monospecies studies). Four studies used an inoculum derived directly from the oral cavity. Human dentine was the most frequently used substratum (88% of the studies). Incubation times differed considerably, ranging from one to seventy days. The most common quantification method (in 87% of the studies) was bacterial culturing, followed by microscopy techniques. The variation in laboratory root canal biofilm model systems is notable. Because of substantial variation in experimental parameters, it is difficult to compare results between studies. This demonstrates the need for a more standardized approach and a validated endodontic biofilm model.

Thermoplastic polyurethane-based intravaginal rings for prophylaxis and treatment of (recurrent) bacterial vaginosis.

The aim of the present study was to develop thermoplastic polyurethane (TPU)-based intravaginal rings (IVRs) for prophylaxis and treatment of bacterial vaginosis via hot melt extrusion/injection molding. Therefore, different TPU grades were processed in combination with lactic acid or metronidazole, targeting a sustained lactic acid release over a 28day-period and sustained metronidazole release over 4-7days. Hot melt extrusion of lactic acid/TPU combinations required a lower extrusion temperature due to the plasticizing properties of lactic acid, evidenced by the lower glass transition temperature (Tg) and cross-over point (Ttanδ=1) values. NIR-chemical imaging data showed a homogenous distribution of lactic acid in TPU matrices at drug loads up to 30% (w/w). The addition of metronidazole did not lower processing temperatures, as the active pharmaceutical ingredient remained crystalline in the TPU matrix. Hydrophobic TPUs with a low ratio between the soft and hard segments (SS/HS ratio) in the polymer structure were suitable carriers for the lactic acid-eluting device over a 28-day period, while hydrophilic TPUs were needed to achieve the required release rate of metronidazole-eluting IVRs. IVRs manufactured with a TPU grade having a higher SS/HS ratio and lactic acid/TPU ratio exhibited a more elastic behavior. The addition of 25% (w/w) metronidazole did not affect the mechanical properties of the IVRs. Hydrophilic TPUs were most prone to biofilm formation by Candida albicans and Staphylococcus aureus, but the incorporation of metronidazole in the device prevented biofilm formation. Based on the drug eluting performance and mechanical tests, a mixture of lactic acid and Tecoflex™ EG-93A (20/80, w/w) and a combination of metronidazole and Tecophilic™ SP-93A-100 (25/75, w/w) were selected to design IVRs for the prophylaxis and treatment of bacterial vaginosis, respectively. Slug mucosal irritation tests predicted low irritation potency for both devices.

Effect of laser-activated irrigation on biofilms in artificial root canals.

AIM: To evaluate the antimicrobial effect of laser-activated irrigation (LAI) on biofilms formed in simulated root canals. METHODOLOGY: A dual-species biofilm of Enterococcus faecalis and Streptococcus mutans was grown in a resin root canal model. Biofilms were formed over 48 h and subsequently subjected to the following treatments, all executed for 20 s: syringe irrigation (SI) with a 27G needle, ultrasonically activated irrigation (UAI) with a size 20 Irrisafe file, and LAI with a 2940 nm Er:YAG laser (20 Hz, 50 µs, 20 or 40 mJ, conical fibre tip at two positions). Tests were performed with both sterile saline as well as NaOCl (2.5%) as the irrigant. Surviving bacteria were harvested and the number of CFU was determined by plate counting and compared across groups (anova, P ≤ 0.05). RESULTS: Using saline as the irrigant, significant reductions in viable counts compared to untreated controls were observed for ultrasonically activated irrigation (0.52 log10 reduction) and for all laser-activated irrigation groups (>1 log10 reduction), but not for syringe irrigation (<0.25 log10 reduction). The reductions in the laser-activated irrigation groups were significantly greater than those of ultrasonically activated irrigation. With NaOCl as the irrigant, significant reductions (>2.2 log10 units) in the number of attached bacteria were observed for all treatment groups with no significant differences between laser-activated and ultrasonically activated irrigation. CONCLUSIONS: Within the limitations of this in vitro set-up, laser-activated irrigation removed more biofilm than ultrasonically activated irrigation when using saline as the irrigant, indicating greater physical biofilm removal. The use of NaOCl resulted in greater biofilm reduction with no significant differences between treatment groups.

ESCMID guideline for the diagnosis and treatment of biofilm infections 2014.

Biofilms cause chronic infections in tissues or by developing on the surfaces of medical devices. Biofilm infections persist despite both antibiotic therapy and the innate and adaptive defence mechanisms of the patient. Biofilm infections are characterized by persisting and progressive pathology due primarily to the inflammatory response surrounding the biofilm. For this reason, many biofilm infections may be difficult to diagnose and treat efficiently. It is the purpose of the guideline to bring the current knowledge of biofilm diagnosis and therapy to the attention of clinical microbiologists and infectious disease specialists. Selected hallmark biofilm infections in tissues (e.g. cystic fibrosis with chronic lung infection, patients with chronic wound infections) or associated with devices (e.g. orthopaedic alloplastic devices, endotracheal tubes, intravenous catheters, indwelling urinary catheters, tissue fillers) are the main focus of the guideline, but experience gained from the biofilm infections included in the guideline may inspire similar work in other biofilm infections. The clinical and laboratory parameters for diagnosing biofilm infections are outlined based on the patient's history, signs and symptoms, microscopic findings, culture-based or culture-independent diagnostic techniques and specific immune responses to identify microorganisms known to cause biofilm infections. First, recommendations are given for the collection of appropriate clinical samples, for reliable methods to specifically detect biofilms, for the evaluation of antibody responses to biofilms, for antibiotic susceptibility testing and for improvement of laboratory reports of biofilm findings in the clinical microbiology laboratory. Second, recommendations are given for the prevention and treatment of biofilm infections and for monitoring treatment effectiveness. Finally, suggestions for future research are given to improve diagnosis and treatment of biofilm infections.

Thiazolidinedione derivatives as novel agents against Propionibacterium acnes biofilms.

AIMS: The aim of the present study was to determine the effect of two thiazolidinedione derivatives on Propionibacterium acnes biofilm formation in vitro and to assess their effect on the susceptibility of P. acnes biofilms towards antimicrobials. METHODS AND RESULTS: The compounds were shown to have a moderate to strong antibiofilm activity when used in subinhibitory concentrations. These compounds do not affect P. acnes attachment but lead to increased dispersal of biofilm cells. This dispersal results in an increased killing of the P. acnes biofilm cells by conventional antimicrobials. CONCLUSION: The antibiofilm effect and the effect on biofilm susceptibility of the thiazolidinedione-derived quorum sensing inhibitors were clearly demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Propionibacterium acnes infections are difficult to treat due to the presence of biofilms at the infection site and the associated resistance towards conventional antimicrobials. Our results indicate that these thiazolidinedione derivatives can be promising leads used for the treatment of P. acnes infections and as anti-acne drugs.

Biofilm inhibitory and eradicating activity of wound care products against Staphylococcus aureus and Staphylococcus epidermidis biofilms in an in vitro chronic wound model.

AIMS: Although several factors contribute to wound healing, bacterial infections and the presence of biofilm can significantly affect healing. Despite that this clearly indicates that therapies should address biofilm in wounds, only few wound care products have been evaluated for their antibiofilm effect. For this reason, we developed a rapid quantification approach to investigate the efficacy of wound care products on wounds infected with Staphylococcus spp. METHODS AND RESULTS: An in vitro chronic wound infection model was used in which a fluorescent Staph. aureus strain was used to allow the rapid quantification of the bacterial burden after treatment. A good correlation was observed between the fluorescence signal and the bacterial counts. When evaluated in this model, several commonly used wound dressings and wound care products inhibited biofilm formation resulting in a decrease between one and seven log CFU per biofilm compared with biofilm formed in the absence of products. In contrast, most dressings only moderately affected mature biofilms. CONCLUSION: Our model allowed the rapid quantification of the bacterial burden after treatment. However, the efficacy of treatment varied between the different types of dressings and/or wound care products. SIGNIFICANCE AND IMPACT OF THE STUDY: Our model can be used to compare the efficacy of wound care products to inhibit biofilm formation and/or eradicate mature biofilms. In addition, the results indicate that treatment of infected wounds should be started as soon as possible and that novel products with more potent antibiofilm activity are needed.

Susceptibility of adult pigeons and hybrid falcons to experimental aspergillosis.

Aspergillosis caused by Aspergillus fumigatus seems to be more prevalent in some avian species than in others. We compared the development of aspergillosis in 8-month-old Gyr-Saker hybrid falcons and 8-month-old pigeons after a single intratracheal inoculation of different dosages of A. fumigatus conidia (10(7), 10(5) and 10(3)). Clinical signs, including vomiting, discoloration of the urates, loss of appetite and dyspnoea, were observed in four out of five falcons and in four out of five pigeons inoculated with 10(7) A. fumigatus conidia. Necropsy revealed the presence of granulomas in the air sacs and/or lungs in four out of five falcons and in four out of five pigeons in the high dosage group. A. fumigatus was isolated from these granulomas in three falcons and in three pigeons. The presence of fungal hyphae was detected with Periodic acid Shiff reagent staining in three out of five falcons and in three out of five pigeons in the high dosage group. Avian respiratory macrophages were clearly present in and around the fungal granulomas. In the other dosage groups, no granulomas, positive A. fumigatus cultures or fungal hyphae were present, except for one falcon in the middle dosage group in which a sterile granuloma without fungal hyphae was noticed. In conclusion, the study shows that adult falcons and pigeons are susceptible to aspergillosis after inoculation of a single dose of conidia intratracheally.

Evaluation of Nd:YAG and Er:YAG irradiation, antibacterial photodynamic therapy and sodium hypochlorite treatment on Enterococcus faecalis biofilms.

AIM: To compare the antimicrobial efficacy of two-high power lasers (Nd:YAG and Er:YAG) and two commercial antimicrobial photodynamic therapy (aPDT) systems with that of sodium hypochlorite (NaOCl) action on Enterococcus faecalis biofilms grown on dentine discs. METHODOLOGY: Enterococcus faecalis biofilms were grown on dentine discs in a microtiter plate, incubated for 24 h and subjected to the following treatments: aPDT (Denfotex and Helbo system), Er:YAG laser irradiation (2940 nm, 50 mJ or 100 mJ, 15 Hz, 40 s), Nd:YAG laser irradiation (1064 nm, 2 W, 15 Hz, 40 s) and immersion in 2.5% (w/v) NaOCl for 1, 5, 10 and 30 min. Surviving bacteria were harvested, and the number of CFU per disc was determined by plate counting. RESULTS: Significant reductions (anova, P ≤ 0.05) in viable counts were observed for aPDT (Helbo) (2 log(10) reduction), Er:YAG irradiation using 100 mJ pulses (4.3 log(10) reduction) and all NaOCl treatments (>6 log(10) reduction). NaOCl (2.5%) for 5 min effectively eliminated all bacteria. aPDT (Denfotex), Er:YAG irradiation using 50 mJ pulses and Nd:YAG treatment caused a reduction in the viable counts of <1 log(10) unit; these results were not significantly different from the untreated controls. CONCLUSION: Within the limitations of this particular laboratory set-up, NaOCl was the most effective in E. faecalis biofilm elimination, while Er:YAG laser treatment (100 mJ pulses) also resulted in high reductions in viable counts. The use of both commercial aPDT systems resulted in a weak reduction in the number of E. faecalis cells. Nd:YAG irradiation was the least effective.

Bio-inspired porous SiC ceramics loaded with vancomycin for preventing MRSA infections.

Implant-related infections are a serious complication in orthopaedic and dental surgery resulting in prolonged hospitalization, high medical costs and patient mortality. The development of porous implants loaded with antibiotics may enable a local delivery for preventing surface colonization and biofilm formation. A new generation of bio-derived porous ceramic material that mimics hierarchical structures from Nature was evaluated. Silicon carbide ceramics derived from Sapelli wood (bioSiC) were obtained by pyrolysis of Entandrophragma cylindricum wood followed by infiltration with molten silicon. This process renders disks that keep the bimodal pore size distribution (3 and 85 µm) of the original material and are highly cytocompatible (BALB/3T3 cell line). The ability of the bio-ceramic to load the antimicrobial agent vancomycin was evaluated by immersion of disks in drug solutions covering a wide range of concentrations. The disks released at pH 7.4 an important amount of drug during the first 2 h (up to 11 mg/g bioSiC) followed by a slower release, which is related to the presence of macro- and mesopores. Finally, the anti-biofilm effect against methicillin resistant Staphylococcus aureus was assessed and a considerable reduction (92%) of the bacterial film was observed. Results highlight the bioSiC potential as component of medicated medical devices.

Quality control of fifteen probiotic products containing Saccharomyces boulardii.

AIMS: The yeast Saccharomyces boulardii is used as a probiotic for the prevention and treatment of diarrhoea. In this study, the quality of 15 probiotic products containing S. boulardii was verified. METHODS AND RESULTS: Using microsatellite typing, the identity of all Saccharomyces strains in the products was confirmed as S. boulardii. Additionally, solid-phase cytometry (SPC) and a plate method were used to enumerate S. boulardii cells. SPC was not only able to produce results more rapidly than plating (4h compared to 48h) but the cell counts obtained with SPC were significantly higher than the plate counts. Finally, we found that <1% of the S. boulardii cells survived 120min in gastric conditions and storage for 3months at 40°C with 75% relative humidity. CONCLUSIONS: We developed a SPC method for the quantification of viable S. boulardii cells in probiotics. Additionally, we demonstrated that gastric conditions and storage have a marked effect on the viability of the yeast cells. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first time SPC is used for the quality control of probiotics with S. boulardii. Additionally, we demonstrated the need for gastric protection and accurate storage.

Comparison of multiple typing methods for Aspergillus fumigatus.

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus. In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus, which requires a high discriminatory power.

Effectiveness of different laser systems to kill Enterococcus faecalis in aqueous suspension and in an infected tooth model.

AIM: To assess the antibacterial action of laser irradiation (Nd:YAG, KTP), photo activated disinfection (PAD) and 2.5% sodium hypochlorite (NaOCl) on Enterococcus faecalis, in an aqueous suspension and in an infected tooth model. METHODOLOGY: Root canals of 60 human teeth with single straight canals were prepared to apical size 50, autoclaved, inoculated with an E. faecalis suspension and incubated for 48 h. They were randomly allocated to four treatment and one control groups. After treatment, the root canals were sampled by flushing with physiological saline, and the number of surviving bacteria in each canal was determined by plate count and solid phase cytometry. The same experimental or control treatments were completed on aqueous suspensions of E. faecalis, and the number of surviving bacteria was determined in the same way. RESULTS: In aqueous suspension, PAD and NaOCl resulted in a significant reduction in the number of E. faecalis cells (P < 0.001), whilst Nd:YAG or KTP had no effect. In the infected tooth model, only the PAD and NaOCl treated teeth yielded significantly different results relative to the untreated controls (P < 0.001). CONCLUSIONS: The laser systems as well as PAD were less effective than NaOCl in reducing E. faecalis, both in aqueous suspension and in the infected tooth model.

Evaluation of the efficacy of disinfection procedures against Burkholderia cenocepacia biofilms.

SUMMARY: In the present study we evaluated the efficacy of various procedures recommended for the disinfection of respiratory equipment and other materials in cystic fibrosis, using both planktonic and sessile Burkholderia cenocepacia cells. A modified European Suspension Test was performed to determine the effects of the disinfection procedures on planktonic cells. The ability of the treatments to kill sessile cells and to remove biofilm biomass was evaluated using two resazurin-based viability assays and a crystal violet staining on biofilms grown and treated in 96-well microtitre plates. The effect of chlorhexidine and hydrogen peroxide treatments on the viability of sessile B. cenocepacia cells was clearly reduced compared to the effects on planktonic cells. Treatments with low concentrations of sodium hypochlorite (0.05%, 5 min) and acetic acid (1.25%, 15 min) also resulted in insufficient reductions in the number of viable sessile cells. There was no relation between the ability of the disinfectants to remove biofilm biomass and their potential to kill biofilm cells. In conclusion, our study indicates that testing of the efficacy of disinfectants should be performed on both planktonic and sessile cells, with particular attention to their effects on cellular viability.

Biofilms in skin infections: Propionibacterium acnes and acne vulgaris.

It is generally accepted that many human infections are biofilm-related and that sessile (biofilm-grown) cells are highly resistant against antimicrobial agents. Propionibacterium acnes plays a role in the pathogenesis of acne vulgaris, a common disorder of the pilosebaceous follicles and it has been suggested that P. acnes cells residing within the follicles grow as a biofilm. Although P. acnes biofilms have not been observed directly in the pilosebaceous unit, the observation that P. acnes readily forms biofilm in vitro as well as on various medical devices in vivo, combined with the high resistance of sessile P. acnes cells and the increased production of particular virulence factors and qourum sensing molecules in sessile cells point in this direction. In addition, in vitro and in vivo biofilm formation has also been demonstrated for other microorganisms involved in skin diseases (including Staphylococcus aureus and Streptococcus pyogenes).

High-resolution genotyping of Aspergillus fumigatus isolates recovered from chronically colonised patients with cystic fibrosis.

A series of 256 Aspergillus fumigatus isolates, recovered from eight patients with cystic fibrosis (CF), were genotyped using microsatellite-based typing. Only a limited number of genotypes were shared between patients and co-colonisation with multiple strains was indicated for all patients. Additionally, some genotypes were isolated recurrently, indicating that they are capable of prolonged colonisation.

Use of the modified Robbins device to study the in vitro biofilm removal efficacy of NitrAdine, a novel disinfecting formula for the maintenance of oral medical devices.

AIMS: To evaluate the use of the modified Robbins device (MRD) to test disinfection strategies against biofilms that form on oral medical devices and to test the biofilm removal efficacy of NitrAdine, a disinfectant for the maintenance of oral medical devices. METHODS AND RESULTS: Biofilms were grown on discs using the MRD and biofilms formed in this system were used to evaluate the efficacy of NitrAdine and to determine the optimal disinfection conditions. Our data indicate that the use of the MRD allows for the rapid and reproducible formation of high-density biofilms. Determination of the efficacy of NitrAdine revealed high activity against biofilms tested (e.g. >3 log reduction for Candida albicans and Staphylococcus aureus) and allowed the determination of the optimal conditions for its use. CONCLUSION: The high reproducibility and flexibility of the MRD make it an excellent candidate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices. Using this system, we were able to demonstrate that NitrAdine exhibits high activity against biofilms formed by the micro-organisms tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that our procedure is appropriate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices.

Factors influencing the trailing endpoint observed in Candida albicans susceptibility testing using the CLSI procedure.

The trailing endpoint phenotype observed during testing of Candida albicans susceptibility to azoles according to the CLSI procedure is defined as a difference in MIC depending on whether the result is obtained after 24 or 48 h. This study investigated whether intrinsic differences between the EUCAST and CLSI methods could explain trailing growth. The glucose concentration in the medium and the shape of the microtitre plate wells were both found to be involved. In order to reduce the incidence of trailing growth according to the CLSI procedure, the use of higher glucose concentrations and flat-bottomed microtitre plates could be valuable improvements.