Impact of Mycobacteroides abscessus colony morphology on biofilm formation and antimicrobial resistance.

Mycobacteroides abscessus is one of the most resistant bacteria so far known and causes severe and hard to treat lung infections in predisposed patients such as those with Cystic Fibrosis (CF). Further, it causes nosocomial infections by forming biofilms on medical devices or water reservoirs. An eye-catching feature of M. abscessus is the growth in two colony morphotypes. Depending on the presence or absence of glycopeptidolipids on the cell surface, it forms smooth or rough colonies. In this study, a porous glass bead biofilm model was used to compare biofilm formation, biofilm organization and biofilm matrix composition in addition to the antimicrobial susceptibility of M. abscessus biofilms versus suspensions of isogenic (smooth and rough) patient isolates. Both morphotypes reached the same cell densities in biofilms. The biofilm architecture, however, was dramatically different with evenly distributed oligo-layered biofilms in smooth isolates, compared to tightly packed, voluminous biofilm clusters in rough morphotypes. Biofilms of both morphotypes contained more total biomass of the matrix components protein, lipid plus DNA than was seen in corresponding suspensions. The biofilm mode of growth of M. abscessus substantially increased resistance to the antibiotics amikacin and tigecycline. Tolerance to the disinfectant peracetic acid of both morphotypes was increased when grown as biofilm, while tolerance to glutaraldehyde was significantly increased in biofilm of smooth isolates only. Overall, smooth colony morphotypes had more pronounced antimicrobial resistance benefit when growing as biofilm than M. abscessus showing rough colony morphotypes.

SxsA, a novel surface protein mediating cell aggregation and adhesive biofilm formation of Staphylococcus xylosus.

Biofilm formation of staphylococci has been an emerging field of research for many years. However, the underlying molecular mechanisms are still not fully understood and vary widely between species and strains. The aim of this study was to identify new effectors impacting biofilm formation of two Staphylococcus xylosus strains. We identified a novel surface protein conferring cell aggregation, adherence to abiotic surfaces, and biofilm formation. The S. xylosus surface protein A (SxsA) is a large protein occurring in variable sizes. It lacks sequence similarity to other staphylococcal surface proteins but shows similar structural domain organization and functional features. Upon deletion of sxsA, adherence of S. xylosus strain TMW 2.1523 to abiotic surfaces was completely abolished and significantly reduced in TMW 2.1023. Macro- and microscopic aggregation assays further showed that TMW 2.1523 sxsA mutants exhibit reduced cell aggregation compared with the wildtype. Comparative genomic analysis revealed that sxsA is part of the core genome of S. xylosus, Staphylococcus paraxylosus, and Staphylococcus nepalensis and additionally encoded in a small group of Staphylococcus cohnii and Staphylococcus saprophyticus strains. This study provides insights into protein-mediated biofilm formation of S. xylosus and identifies a new cell wall-associated protein influencing cell aggregation and biofilm formation.

The Tol-Pal system of Acinetobacter baumannii is important for cell morphology, antibiotic resistance and virulence.

Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions. This Gram-negative bacterium is one of the most successful human pathogens worldwide and responsible for hospital-acquired infections. This is due to its outstanding potential to adapt to very different environments, to persist in the human host and most important, its ability to develop multidrug resistance. Our combined approach of genomic and phenotypic analyses led to the identification of the envelope spanning Tol-Pal system in A. baumannii. We found that the deletion of the tolQ, tolR, tolA, tolB, and pal genes affects cell morphology and increases antibiotic sensitivity, such as the ∆tol-pal mutant exhibits a significantly increased gentamicin and bacitracin sensitivity. Furthermore, Galleria mellonella caterpillar killing assays revealed that the ∆tol-pal mutant exhibits a decreased killing phenotype. Taken together, our findings suggest that the Tol-Pal system is important for cell morphology, antibiotic resistance, and virulence of A. baumannii.

Proteome Analysis of a M. avium Mutant Exposes a Novel Role of the Bifunctional Protein LysX in the Regulation of Metabolic Activity.

Lysyl-phosphatidylglycerol is one of the components of the mycobacterial membrane that contributes to the resistance to cationic antimicrobial peptides, a host-induced frontline defense against invading pathogens. Its production is catalyzed by LysX, a bifunctional protein with lysyl transferase and lysyl transfer RNA synthetase activity. Comparative proteome analysis of a lysX mutant of Mycobacterium avium strain 104 and the wild type indicated that the lysX mutant strain undergoes a transition in phenotype by switching the carbon metabolism to ß-oxidation of fatty acids, along with accumulation of lipid inclusions. Surprisingly, proteins associated with intracellular survival were upregulated in the lysX mutant, even during extracellular growth, preparing bacteria for the conditions occurring inside host cells. In line with this, the lysX mutant exhibited enhanced intracellular growth in human-blood-derived monocytes. Thus, our study exposes the significance of lysX in the metabolism and virulence of the environmental pathogen M. avium hominissuis.

The VBNC state: a fundamental survival strategy of Acinetobacter baumannii.

IMPORTANCE: Currently, the viable but non-culturable (VBNC) state is an underappreciated niche for pathogenic bacteria which provides a continuous source for recurrent infections and transmission. We propose the VBNC state to be a global persistence mechanism used by various A. baumannii strains to cope with many stresses it is confronted with in the clinical environment and in the host. This requires a novel strategy to detect viable cells of this pathogen that is not only based on plating assays.

Ciprofloxacin-Loaded Polyvinylpyrrolidone Foils for the Topical Treatment of Wound Infections with Methicillin-Resistant Staphylococcus aureus (MRSA).

Bacterial infections are a constant challenge in the management of acute and chronic wounds. Chronic wounds, such as diabetic foot ulcers, have increased significantly in the last few years due to the rise of an aging population. A better understanding of the infectious pathophysiological mechanisms is urgently needed along with new options for the treatment of wound infections and wound-healing disorders. New advances in the preparation of biocompatible dressing materials that can be loaded with antimicrobial drugs may improve the topical treatment of infected wounds. In this study, we investigated the antimicrobial activity of polyvinylpyrrolidone (PVP) foils loaded with ciprofloxacin (Cipro-foils) in the presence of acetic acid as a co-solvent. We used ex vivo human wounds that were infected with two bacterial strains: methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa (PAO1). The effectiveness of the treatment was demonstrated by the quantification of the living bacteria extracted from the wound and the detection of released immunological mediators in skin extracts and in the skin culture media. We found that Cipro-foils effectively treated the infection with both PAO1 and MRSA. Other than PAO1, MRSA had no lytic activity toward skin proteins. MRSA infections increased cytokines' expression and release. Interestingly, treatment with Cipro-foils could partially counteract these effects.

Effects of Adipose-Derived Stem Cells and Their Conditioned Medium in a Human Ex Vivo Wound Model.

Adult stem cells have been extensively investigated for tissue repair therapies. Adipose-derived stem cells (ASCs) were shown to improve wound healing by promoting re-epithelialization and vascularization as well as modulating the inflammatory immune response. In this study, we used ex vivo human skin cultured in a six-well plate with trans-well inserts as a model for superficial wounds. Standardized wounds were created and treated with allogeneic ASCs, ASCs conditioned medium (ASC-CM), or cell culture medium (DMEM) supplemented with fetal calf serum (FCS). Skin viability (XTT test), histology (hematoxylin and eosin, H and E), ß-catenin expression as well as inflammatory mediators and growth factors were monitored over 12 days of skin culture. We observed only a moderate time-dependent decrease in skin metabolic activity while skin morphology was preserved, and re-epithelialization occurred at the wound edges. An increase in ß-catenin expression was observed in the newly formed epithelia, especially in the samples treated with ASC-CM. In general, increased growth factors and inflammatory mediators, e.g., hepatocytes growth factor (HGF), platelet-derived growth factor subunit AA (PDGF-AA), IL-1α, IL-7, TNF-α, and IL-10, were observed over the incubation time. Interestingly, different expression profiles were observed for the different treatments. Samples treated with ASC-CM significantly increased the levels of inflammatory cytokines and PDGF-AA with respect to control, whereas the treatment with ASCs in DMEM with 10% FCS resulted in significantly increased levels of fibroblast growth factor-basic (FGF-basic) and moderate increases of immunomodulatory cytokines. These results confirm that the wound microenvironment can influence the type of mediators secreted by ASCs and the mode as to how they improve the wound healing process. Comparative investigations with pre-activated ASCs will elucidate further aspects of the wound healing mechanism and improve the protocols of ACS application.

Tolerance of biofilm of a carbapenem-resistant Klebsiella pneumoniae involved in a duodenoscopy-associated outbreak to the disinfectant used in reprocessing.

BACKGROUND: One possible transmission route for nosocomial pathogens is contaminated medical devices. Formation of biofilms can exacerbate the problem. We report on a carbapenemase-producing Klebsiella pneumoniae that had caused an outbreak linked to contaminated duodenoscopes. To determine whether increased tolerance to disinfectants may have contributed to the outbreak, we investigated the susceptibility of the outbreak strain to disinfectants commonly used for duodenoscope reprocessing. Disinfection efficacy was tested on planktonic bacteria and on biofilm. METHODS: Disinfectant efficacy testing was performed for planktonic bacteria according to EN standards 13727 and 14561 and for biofilm using the Bead Assay for Biofilms. Disinfection was defined as ≥ 5log10 reduction in recoverable colony forming units (CFU). RESULTS: The outbreak strain was an OXA-48 carbapenemase-producing K. pneumoniae of sequence type 101. We found a slightly increased tolerance of the outbreak strain in planktonic form to peracetic acid (PAA), but not to other disinfectants tested. Since PAA was the disinfectant used for duodenoscope reprocessing, we investigated the effect of PAA on biofilm of the outbreak strain. Remarkably, disinfection of biofilm of the outbreak strain could not be achieved by the standard PAA concentration used for duodenoscope reprocessing at the time of outbreak. An increased tolerance to PAA was not observed in a K. pneumoniae type strain tested in parallel. CONCLUSIONS: Biofilm of the K. pneumoniae outbreak strain was tolerant to standard disinfection during duodenoscope reprocessing. This study establishes for the first time a direct link between biofilm formation, increased tolerance to disinfectants, reprocessing failure of duodenoscopes and nosocomial transmission of carbapenem-resistant K. pneumoniae.

Subgingival delivery of oral debriding agents: a proof of concept.

OBJECTIVE: This study is a proof of concept to determine the efficacy of a custom-fabricated tray in placing antimicrobial and debriding agents in the periodontal pockets of persons with active gingival infections. Localized subgingival delivery of antimicrobial and antibiotic agents is routinely employed as adjunctive therapy for the treatment and management ofperiopathogens associated with periodontal disease. Because these delivery techniques often face time constraints and impose temporary restrictions on patient brushing and flossing, a custom-formed prescription dental tray can be used to deliver and maintain medications in periodontal pockets between office visits and without brushing or flossing restrictions. The ability of this tray to maintain sufficient concentrations of medication in the periodontal pockets to have a therapeutic effect is evaluated here with theoretical modeling and practical application. METHODS: Hydrogen peroxide is an oral debriding agent and oral wound cleanser with antimicrobial properties. The debriding effect of 1.7% hydrogen peroxide gel was tested in vitro on Streptococcus mutans biofilm using glass carriers for collection. Diffusion modeling tested the potential of the customized tray to place hydrogen peroxide gel into the sulcus in the presence of crevicular fluid flow. Changes in periodontal microflora with scanning electron microscopy analysis of in vivo paper point site sampling were analyzed before and after a thin ribbon of 1.7% hydrogen peroxide gel (approximately 0.7 gm) and a subtherapeutic dose (three drops) of Vibramycin (50 mg/5 ml) were placed via Perio Trays into periodontal pockets, ranging from 4-8 mm at daily prescribed intervals for two to five weeks. RESULTS: In vitro results indicate that 1.7% hydrogen peroxide gel breaks down the exopolysaccharide slime and cell walls ofS. mutans, and begins to debride the cells from glass carriers within 10 minutes. Diffusion modeling indicates that hydrogen peroxide can penetrate into the deeper pockets (9 mm), but also its concentration in these deep pockets will increase over wearing time in the absence of degradation by peroxidases and catalase. Site sampling data confirm diffusion modeling results, with evidence that medication delivered with the prescription tray reduced subgingival bacterial loads and enhanced healing of corresponding oral tissues. CONCLUSION: The prescription Perio Tray effectively placed medication in the gingival sulcus. Mathematical modeling indicated Perio Tray placement of hydrogen peroxide gel in periodontal pockets with depths up to 9 mm over 15 minutes treatment time was theoretically possible. Pathology reports reveal reductions in subgingival bacterial loads and improvements in pretreatment pocket depths of up to 8 mm after 1.7% hydrogen peroxide and Vibramycin Syrup were prescribed for use with the Perio Tray. The in vitro analysis indicating that hydrogen peroxide is the active and effective oral debriding agent needs to be confirmed with additional studies.

Screening of Surfactants for Improved Delivery of Antimicrobials and Poly-Lactic-co-Glycolic Acid Particles in Wound Tissue.

Topical wound management is often a challenge due to the poor penetration of antimicrobials in wound tissue and across the biofilm matrix where bacteria are embedded. Surfactants have been used for decades to improve the stability of formulations, increase drug solubility, and enhance penetration. In this study, we screened different detergents with respect to their cytotoxicity and their ability to improve the penetration of poly-lactic-co-glycolic acid (PLGA) particles in wound tissue. Among the tested surfactants, Kolliphor SLS and Tween 80 increased the penetration of PLGA particles and had a limited cytotoxicity. Then, these surfactants were used to formulate PLGA particles loaded with the poorly water-soluble antibiotic ciprofloxacin. The antimicrobial efficacy of the formulations was tested in a wound infection model based on human ex vivo skin. We found that even though PLGA particles had the same antimicrobial efficiency than the particle-free drug formulation, thanks to their solubilizing and anti-biofilm properties, the surfactants remarkably improved the antimicrobial activity of ciprofloxacin with respect to the drug formulation in water. We conclude that the use of Tween 80 in antimicrobial formulations might be a safe and efficient option to improve the topical antimicrobial management of chronic wound infections.

Mutations in the gdpP gene are a clinically relevant mechanism for ß-lactam resistance in meticillin-resistant Staphylococcus aureus lacking mec determinants.

In Staphylococcus aureus, resistance to ß-lactamase stable ß-lactam antibiotics is mediated by the penicillinbinding protein 2a, encoded by mecA or by its homologues mecB or mecC. However, a substantial number of meticillin-resistant isolates lack known mec genes and, thus, are called meticillin resistant lacking mec (MRLM). This study aims to identify the genetic mechanisms underlying the MRLM phenotype. A total of 141 MRLM isolates and 142 meticillin-susceptible controls were included in this study. Oxacillin and cefoxitin minimum inhibitory concentrations were determined by broth microdilution and the presence of mec genes was excluded by PCR. Comparative genomics and a genome-wide association study (GWAS) approach were applied to identify genetic polymorphisms associated with the MRLM phenotype. The potential impact of such mutations on the expression of PBP4, as well as on cell morphology and biofilm formation, was investigated. GWAS revealed that mutations in gdpP were significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme involved in the degradation of the second messenger cyclic-di-AMP in S. aureus. A total of 131 MRLM isolates carried truncations, insertions or deletions as well as amino acid substitutions, mainly located in the functional DHH-domain of GdpP. We experimentally verified the contribution of these gdpP mutations to the MRLM phenotype by heterologous complementation experiments. The mutations in gdpP had no effect on transcription levels of pbp4; however, cell sizes of MRLM strains were reduced. The impact on biofilm formation was highly strain dependent. We report mutations in gdpP as a clinically relevant mechanism for ß-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, since MRLM are easily misclassified as MSSA (meticillin-susceptible S. aureus), which may lead to unnoticed spread of ß-lactam-resistant isolates and subsequent treatment failure.

The role of microbial biofilms in osteonecrosis of the jaw associated with bisphosphonate therapy.

Microbial biofilms have been observed and described in bone specimens of patients with bisphosphonate (BP)-associated osteonecrosis of the jaw (BONJ) and investigators are more recently suggesting that this condition essentially represents an osteomyelitis of the jaw clinically, with greater susceptibility in some patients on BP therapy. This article explains the role of microbial biofilms in BONJ and also discusses associated factors in the disease pathogenesis, which include BP effects on bone remodeling, anti-angiogenesis, matrix necrosis, microcracks, soft tissue toxicity, and inflammation and wound healing. Recent findings suggest a key role for microbial biofilms in the pathogenesis of BONJ; this has important therapeutic implications because biofilm organisms represent a clinical target for prevention and treatment efforts aimed at reducing the significant morbidity and costs associated with this condition.

Molecular epidemiology and spatial distribution of Selenomonas spp. in subgingival biofilms.

The aetiology of periodontal disease has been a field of intensive research in the past decades. Along with a variety of other putative pathogens, different members of the genus Selenomonas have repeatedly been associated with both generalized aggressive periodontitis and chronic periodontitis. For the present study, a specific oligonucleotide probe targeting the majority of all oral Selenomonas spp. was designed. Their prevalence was determined, using dot-blot hybridization, in a total of 742 subgingival samples collected from patients with generalized aggressive (n=62) and chronic periodontitis (n=82), and from periodontitis-resistant subjects (n=19). In addition, fluorescence in situ hybridization (FISH) and electron microscopy were performed to analyze the spatial arrangement of Selenomonas in subgingival biofilms collected from patients with generalized aggressive periodontitis. In the samples from patients, Selenomonas spp. showed a lower prevalence in both diseased groups compared with other putative pathogens, and a relatively high prevalence in the periodontitis-resistant group. Consequently, Selenomonas spp. do not seem to be suitable diagnostic marker organisms for periodontal disease. By contrast, FISH and electron microscopic analysis of periodontal carriers revealed that Selenomonas spp. appeared in large numbers in all parts of the collected biofilms and seemed, if present in a site from patients, to make a relevant contribution to their structural organization.

Demonstration of bacterial biofilms in culture-negative silicone stent and jones tube.

PURPOSE: To demonstrate the presence of bacterial biofilms on a dacryocystorhinostomy silicone stent and a Jones tube. METHODS: One dacryocystorhinostomy silicone stent and one Jones tube were removed from 2 patients who presented with an infection of their respective nasolacrimal system. Cultures were obtained, and the implants were processed for scanning electron microscopy and confocal laser scanning microscopy, advanced microscopic methods that are applicable for detection of uncultivable biofilm organisms. RESULTS: Routine bacterial cultures revealed no growth, but bacterial biofilms on outer and inner surfaces of both implants were confirmed by advanced microscopic techniques. CONCLUSIONS: To the authors' knowledge, this is the first article that documents the presence of biofilms on a Crawford stent or a Jones tube on patients who presented with infections involving the nasolacrimal system. Although initial cultures revealed absence of any bacterial growth, confocal laser scanning microscopy and scanning electron microscopy documented bacterial colonization. Clinicians should consider the role of biofilms and the limitation of our standard culturing techniques while treating patients with device- or implant-related infections.

CpALS4770 and CpALS4780 contribution to the virulence of Candida parapsilosis.

The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated. The present project was aimed at investigating the contribution of C. parapsilosis Als proteins by generating edited strains lacking functional Als proteins. CPAR2_404770 and CPAR2_404780, further indicated as CpALS4770 and CpALS4780, were selected for the generation of single and double edited strains using an episomal CRISPR/Cas9 technology. Phenotypic characterization of mutant strains revealed that editing of both genes had no impact on the in vitro growth of C. parapsilosis or on morphogenesis. Notably, CpALS4770-edited strain showed a reduction of biofilm formation and adhesive properties to human buccal cells (HBECs). Conversely, single CpALS4780-edited strain did not show any difference compared to the wild-type strain in all the assays performed, while the double CpALS4770-CpALS4780 mutant revealed an increased ability to produce biofilm, a hyper-adhesive phenotype to HBECs, and a marked tendency to form cellular aggregates. Murine vaginal infection experiments indicated a significant reduction in CFUs recovered from BALC/c mice infected with single and double edited strains, compared to those infected with the wild-type strain. These finding clearly indicate that CpAls4770 plays a role in adhesion to biotic and abiotic surfaces, while both CpALS4770 and CpALS4780 genes are required for C. parapsilosis ability to colonize and persist in the vaginal mucosa.

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes.

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I-IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches.

Three-dimensional macromolecular organization of cryofixed Myxococcus xanthus biofilms as revealed by electron microscopic tomography.

Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.

Biofilm removal with a dental water jet.

OBJECTIVE: The objective of this study was to evaluate the effect of a dental water jet on plaque biofilm removal using scanning electron microscopy (SEM). METHODOLOGY: Eight teeth with advanced aggressive periodontal disease were extracted. Ten thin slices were cut from four teeth. Two slices were used as the control. Eight were inoculated with saliva and incubated for 4 days. Four slices were treated using a standard jet tip, and four slices were treated using an orthodontic jet tip. The remaining four teeth were treated with the orthodontic jet tip but were not inoculated with saliva to grow new plaque biofilm. All experimental teeth were treated using a dental water jet for 3 seconds on medium pressure. RESULTS: The standard jet tip removed 99.99% of the salivary (ex vivo) biofilm, and the orthodontic jet tip removed 99.84% of the salivary biofilm. Observation of the remaining four teeth by the naked eye indicated that the orthodontic jet tip removed significant amounts of calcified (in vivo) plaque biofilm. This was confirmed by SEM evaluations. CONCLUSION: The Waterpik dental water jet (Water Pik, Inc, Fort Collins, CO) can remove both ex vivo and in vivo plaque biofilm significantly.

Evaluation of Drug Delivery and Efficacy of Ciprofloxacin-Loaded Povidone Foils and Nanofiber Mats in a Wound-Infection Model Based on Ex Vivo Human Skin.

Topical treatment of wound infections is often a challenge due to limited drug availability at the site of infection. Topical drug delivery is an attractive option for reducing systemic side effects, provided that a more selective and sustained local drug delivery is achieved. In this study, a poorly water-soluble antibiotic, ciprofloxacin, was loaded on polyvinylpyrrolidone (PVP)-based foils and nanofiber mats using acetic acid as a solubilizer. Drug delivery kinetics, local toxicity, and antimicrobial activity were tested on an ex vivo wound model based on full-thickness human skin. Wounds of 5 mm in diameter were created on 1.5 × 1.5 cm skin blocks and treated with the investigated materials. While nanofiber mats reached the highest amount of delivered drug after 6 h, foils rapidly achieved a maximum drug concentration and maintained it over 24 h. The treatment had no effect on the overall skin metabolic activity but influenced the wound healing process, as observed using histological analysis. Both delivery systems were efficient in preventing the growth of Pseudomonas aeruginosa biofilms in ex vivo human skin. Interestingly, foils loaded with 500 µg of ciprofloxacin accomplished the complete eradication of biofilm infections with 1 × 109 bacteria/wound. We conclude that antimicrobial-loaded resorbable PVP foils and nanofiber mats are promising delivery systems for the prevention or topical treatment of infected wounds.

Detection of a microbial biofilm in intraamniotic infection.

OBJECTIVE: Microbial biofilms are communities of sessile microorganisms formed by cells that are attached irreversibly to a substratum or interface or to each other and embedded in a hydrated matrix of extracellular polymeric substances. Microbial biofilms have been implicated in >80% of human infections such as periodontitis, urethritis, endocarditis, and device-associated infections. Thus far, intraamniotic infection has been attributed to planktonic (free-floating) bacteria. A case is presented in which "amniotic fluid sludge" was found to contain microbial biofilms. This represents the first report of a microbial biofilm in the amniotic cavity. STUDY DESIGN: "Amniotic fluid sludge" was detected by transvaginal sonography and retrieved by transvaginal amniotomy. Bacteria were identified with scanning electron microscopy and fluorescence in situ hybridization for conserved regions of the microbial genome; the exopolymeric matrix was identified by histochemistry by the wheat germ agglutinin lectin method. The structure of the biofilm was imaged with confocal laser scanning microscopy. RESULTS: "Amniotic fluid sludge" was imaged with scanning electron microscopy, which allowed the identification of bacteria embedded in an amorphous material and inflammatory cells. Bacteria were demonstrated with fluorescent in situ hybridization using a eubacteria probe. Extracellular matrix was identified with the wheat germ agglutinin lectin stain. Confocal microscopy allowed 3-dimensional visualization of the microbial biofilm. CONCLUSION: Microbial biofilms have been identified in a case of intraamniotic infection with "amniotic fluid sludge."