Next-generation sequencing (NGS) may offer a more comprehensive assessment of penile implant biofilm compared to conventional culture. Improved characterization of biofilms using NGS may provide insight as to how infections may be reduced by potentially guiding tailored antibiotic prophylaxis, antibiotic irrigation, or implant antibiotic coatings. We hypothesize that penile implant biofilm differs between diabetic (DM) and non-DM patients because of the potentially immunocompromised nature of DM patients.

Hypergranulation, bacterial infection, and device dislodgment are common complications associated with percutaneous gastronomy (PG) tube placement for enteral feeding largely attributable to delayed stoma tract maturation around the device. Stoma tract maturation is a wound-healing process that requires collective and complete migration of an advancing epithelial layer. While it is widely accepted that micropatterned surfaces enhance cell migration when cells are cultured directly on the substrate, few studies have investigated the influence of apical contact guidance from micropatterned surfaces on cell migration, as occurs during stoma tract formation. Here, we developed 2D and 3D in vitro epithelial cell migration assays to test the effect of various Sharklet micropatterns on apically-guided cell migration. The 2D modified scratch wound assay identified a Sharklet micropattern (+10SK50x50) that enhanced apical cell migration by 4-fold (p = 0.0105) compared to smooth controls over the course of seven days. The best-performing micropattern was then applied to cylindrical prototypes with the same outer diameter as a pediatric PG tube. These prototypes were evaluated in the novel 3D migration assay where magnetic levitation aggregated cells around prototypes to create an artificial stoma. Results indicated a 50% increase (p < 0.0001) in cell migration after seven days along Sharklet-micropatterned prototypes compared to smooth controls. The Sharklet micropattern enhanced apically-guided epithelial cell migration in both 2D and 3D in vitro assays. These data suggest that the incorporation of a Sharklet micropattern onto the surface of a PG tube may accelerate cell migration via apical contact, improve stoma tract maturation, and reduce skin-associated complications.

Purpose. Surface microtopography offers a promising approach for infection control. The goal of this study was to provide evidence that micropatterned surfaces significantly reduce the potential risk of medical device‐associated infections. Methodology. Micropatterned and smooth surfaces were challenged in vitro against the colonization and transference of two representative bacterial pathogens ‐ Staphylococcus aureus and Pseudomonas aeruginosa. A percutaneous rat model was used to assess the effectiveness of the micropattern against device‐associated S. aureus infections. After the percutaneous insertion of silicone rods into (healthy or immunocompromised) rats, their backs were inoculated with S. aureus. The bacterial burdens were determined in tissues under the rods and in the spleens. Results. The micropatterns reduced adherence by S. aureus (92.3 and 90.5% reduction for flat and cylindrical surfaces, respectively), while P. aeruginosa colonization was limited by 99.9% (flat) and 95.5% (cylindrical). The micropatterned surfaces restricted transference by 95.1% for S. aureus and 94.9% for P. aeruginosa, compared to smooth surfaces. Rats with micropatterned devices had substantially fewer S. aureus in subcutaneous tissues (91%) and spleens (88%) compared to those with smooth ones. In a follow‐up study, immunocompromised rats with micropatterned devices had significantly lower bacterial burdens on devices (99.5 and 99.9% reduction on external and internal segments, respectively), as well as in subcutaneous tissues (97.8%) and spleens (90.7%) compared to those with smooth devices. Conclusion. Micropatterned surfaces exhibited significantly reduced colonization and transference in vitro, as well as lower bacterial burdens in animal models. These results indicate that introducing this micropattern onto surfaces has high potential to reduce medical device‐associated infections.

Tracheal intubation disrupts physiological homeostasis of secretion production and clearance, resulting in secretion accumulation within endotracheal tubes (ETTs). Novel in vitro and in vivo models were developed to specifically recapitulate the clinical manifestations of ETT occlusion. The novel Sharklet™ micropatterned ETT was evaluated, using these models, for the ability to reduce the accumulation of both bacterial biofilm and airway mucus compared to a standard care ETT. Novel ETTs with micropattern on the inner and outer surfaces were placed adjacent to standard care ETTs in in vitro biofilm and airway patency (AP) models. The primary outcome for the biofilm model was to compare commercially-available ETTs (standard care and silver-coated) to micropatterned for quantity of biofilm accumulation. The AP model’s primary outcome was to evaluate accumulation of artificial airway mucus. A 24-h ovine mechanical ventilation model evaluated the primary outcome of relative quantity of airway secretion accumulation in the ETTs tested. The secondary outcome was measuring the effect of secretion accumulation in the ETTs on airway resistance. Micropatterned ETTs significantly reduced biofilm by 71% (p = 0.016) compared to smooth ETTs. Moreover, micropatterned ETTs reduced lumen occlusion, in the AP model, as measured by cross-sectional area, in distal (85%, p = 0.005), middle (84%, p = 0.001) and proximal (81%, p = 0.002) sections compared to standard care ETTs. Micropatterned ETTs reduced the volume of secretion accumulation in a sheep model of occlusion by 61% (p < 0.001) after 24 h of mechanical ventilation. Importantly, micropatterned ETTs reduced the rise in ventilation peak inspiratory pressures over time by as much as 49% (p = 0.005) compared to standard care ETTs. Micropatterned ETTs, demonstrated here to reduce bacterial contamination and mucus occlusion, will have the capacity to limit complications occurring during mechanical ventilation and ultimately improve patient care.

Abstract BackgroundCatheter‐related bloodstream infections (CRBSIs) and catheter‐related thrombosis (CRT) are common complications of central venous catheters (CVC), which are used to monitor patient health and deliver medications. CVCs are subject to protein adsorption and platelet adhesion as well as colonization by the natural skin flora (i.e. Staphylococcus aureus and Staphylococcus epidermidis). Antimicrobial and antithrombotic drugs can prevent infections and thrombosis‐related complications, but have associated resistance and safety risks. Surface topographies have shown promise in limiting platelet and bacterial adhesion, so it was hypothesized that an engineered Sharklet micropattern, inspired by shark‐skin, may provide a combined approach as it has wide reaching anti‐fouling capabilities. To assess the feasibility for this micropattern to improve CVC‐related healthcare outcomes, bacterial colonization and platelet interactions were analyzed in vitro on a material common for vascular access devices. MethodsTo evaluate bacterial inhibition after simulated vascular exposure, micropatterned thermoplastic polyurethane surfaces were preconditioned with blood proteins in vitro then subjected to a bacterial challenge for 1 and 18 h. Platelet adhesion was assessed with fluorescent microscopy after incubation of the surfaces with platelet‐rich plasma (PRP) supplemented with calcium. Platelet activation was further assessed by monitoring fibrin formation with fluorescent microscopy after exposure of the surfaces to platelet‐rich plasma (PRP) supplemented with calcium in a flow‐cell. Results are reported as percent reductions and significance is based on t‐tests and ANOVA models of log reductions. All experiments were replicated at least three times. ResultsBlood and serum conditioned micropatterned surfaces reduced 18 h S. aureus and S. epidermidis colonization by 70% (p ≤ 0.05) and 71% (p < 0.01), respectively, when compared to preconditioned unpatterned controls. Additionally, platelet adhesion and fibrin sheath formation were reduced by 86% and 80% (p < 0.05), respectively, on the micropattern, when compared to controls. ConclusionsThe Sharklet micropattern, in a CVC‐relevant thermoplastic polyurethane, significantly reduced bacterial colonization and relevant platelet interactions after simulated vascular exposure. These results suggest that the incorporation of the Sharklet micropattern on the surface of a CVC may inhibit the initial events that lead to CRBSI and CRT.

Background Bacterial surface contamination contributes to transmission of nosocomial infections. Chemical cleansers used to control surface contamination are often toxic and incorrectly implemented. Additional non-toxic strategies should be combined with regular cleanings to mitigate risks of human error and further decrease rates of nosocomial infections. The Sharklet micropattern (MP), inspired by shark skin, is an effective tool for reducing bacterial load on surfaces without toxic additives. The studies presented here were carried out to investigate the MP surfaces capability to reduce colonization of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) compared to smooth control surfaces. Methods The MP and smooth surfaces produced in acrylic film were compared for remaining bacterial contamination and colonization following inoculation. Direct sampling of surfaces was carried out after inoculation by immersion, spray, and/or touch methods. Ultimately, a combination assay was developed to assess bacterial contamination after touch transfer inoculation combined with drying (persistence) to mimic common environmental contamination scenarios in the clinic or hospital environment. The combination transfer and persistence assay was then used to test antimicrobial copper beside the MP for the ability to reduce MSSA and MRSA challenge. Results The MP reduced bacterial contamination with log reductions ranging from 87-99% (LR = 0.90-2.18; p < 0.05) compared to smooth control surfaces. The MP was more effective than the 99.9% pure copper alloy C11000 at reducing surface contamination of S. aureus (MSSA and MRSA) through transfer and persistence of bacteria. The MP reduced MSSA by as much as 97% (LR = 1.54; p < 0.01) and MRSA by as much as 94% (LR = 1.26; p < 0.005) compared to smooth controls. Antimicrobial copper had no significant effect on MSSA contamination, but reduced MRSA contamination by 80% (LR = 0.70; p < 0.005). Conclusion The assays developed in this study mimic hospital environmental contamination events to demonstrate the performance of a MP to limit contamination under multiple conditions. Antimicrobial copper has been implemented in hospital room studies to evaluate its impact on nosocomial infections and a decrease in HAI rate was shown. Similar implementation of the MP has potential to reduce the incidence of HAIs although future clinical studies will be necessary to validate the MP’s true impact.

In chronic wounds, biofilm infects host tissue for extended periods of time. This work establishes the first chronic preclinical model of wound biofilm infection aimed at addressing the long‐term host response. Although biofilm‐infected wounds did not show marked differences in wound closure, the repaired skin demonstrated compromised barrier function. This observation is clinically significant, because it leads to the notion that even if a biofilm infected wound is closed, as observed visually, it may be complicated by the presence of failed skin, which is likely to be infected and/or further complicated postclosure. Study of the underlying mechanisms recognized for the first time biofilm‐inducible miR‐146a and miR‐106b in the host skin wound‐edge tissue. These miRs silenced ZO‐1 and ZO‐2 to compromise tight junction function, resulting in leaky skin as measured by transepidermal water loss (TEWL). Intervention strategies aimed at inhibiting biofilm‐inducible miRNAs may be productive in restoring the barrier function of host skin. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ABSTRACT Pseudomonas aeruginosa strains recovered from chronic pulmonary infections in cystic fibrosis patients are frequently mucoid. Such strains express elevated levels of alginate but reduced levels of the aggregative polysaccharide Psl; however, the mechanistic basis for this regulation is not completely understood. Elevated pslA expression was observed in an amrZ null mutant and in strains expressing a DNA-binding-deficient AmrZ. AmrZ is a transcription factor that positively regulates twitching motility and alginate synthesis, two phenotypes involved in P. aeruginosa biofilm development. AmrZ bound directly to the pslA promoter in vitro , and molecular analyses indicate that AmrZ represses psl expression by binding to a site overlapping the promoter. Altered expression of amrZ in nonmucoid strains impacted biofilm structure and architecture, as structured microcolonies were observed with low AmrZ production and flat biofilms with amrZ overexpression. These biofilm phenotypes correlated with Psl levels, since we observed elevated Psl production in amrZ mutants and lower Psl production in amrZ -overexpressing strains. These observations support the hypothesis that AmrZ is a multifunctional regulator mediating transition of P. aeruginosa biofilm infections from colonizing to chronic biofilms through repression of the psl operon while activating the algD operon.

Management of deep sternal wound infection (SWI), a serious complication after cardiac surgery with high morbidity and mortality incidence, requires invasive procedures such as, debridement with primary closure or myocutaneous flap reconstruction along with use of broad spectrum antibiotics. The purpose of this clinical series is to investigate the presence of biofilm in patients with deep SWI. A biofilm is a complex microbial community in which bacteria attach to a biological or non-biological surface and are embedded in a self-produced extracellular polymeric substance. Biofilm related infections represent a major clinical challenge due to their resistance to both host immune defenses and standard antimicrobial therapies. Candidates for this clinical series were patients scheduled for a debridement procedure of an infected sternal wound after a cardiac surgery. Six patients with SWI were recruited in the study. All cases had marked dehiscence of all layers of the wound down to the sternum with no signs of healing after receiving broad spectrum antibiotics post-surgery. After consenting patients, tissue and/or extracted stainless steel wires were collected during the debridement procedure. Debrided tissues examined by Gram stain showed large aggregations of Gram positive cocci. Immuno-fluorescent staining of the debrided tissues using a specific antibody against staphylococci demonstrated the presence of thick clumps of staphylococci colonizing the wound bed. Evaluation of tissue samples with scanning electron microscope (SEM) imaging showed three-dimensional aggregates of these cocci attached to the wound surface. More interestingly, SEM imaging of the extracted wires showed attachment of cocci aggregations to the wire metal surface. These observations along with the clinical presentation of the patients provide the first evidence that supports the presence of biofilm in such cases. Clinical introduction of the biofilm infection concept in deep SWI may advance the current management strategies from standard antimicrobial therapy to anti-biofilm strategy.

The Staphylococcus aureus cid and lrg operons play significant roles in the control of autolysis and accumulation of extracellular genomic DNA (eDNA) during biofilm development. Although the molecular mechanisms mediating this control are only beginning to be revealed, it is clear that cell death must be limited to a subfraction of the biofilm population. In the present study, we tested the hypothesis that cid and lrg expression varies during biofilm development as a function of changes in the availability of oxygen. To examine cid and lrg promoter activity during biofilm development, fluorescent reporter fusion strains were constructed and grown in a BioFlux microfluidic system, generating time-lapse epifluorescence images of biofilm formation, which allows the spatial and temporal localization of gene expression. Consistent with cid induction under hypoxic conditions, the cid:: gfp fusion strain expressed green fluorescent protein predominantly within the interior of the tower structures, similar to the pattern of expression observed with a strain carrying a gfp fusion to the hypoxia-induced promoter controlling the expression of the lactose dehydrogenase gene. The lrg promoter was also expressed within towers but appeared more diffuse throughout the tower structures, indicating that it was oxygen independent. Unexpectedly, the results also demonstrated the existence of tower structures with different expression phenotypes and physical characteristics, suggesting that these towers exhibit different metabolic activities. Overall, the findings presented here support a model in which oxygen is important in the spatial and temporal control of cid expression within a biofilm and that tower structures formed during biofilm development exhibit metabolically distinct niches.

Biofilms are a predominant form of growth for bacteria in the environment and in the clinic. Critical for biofilm development are adherence, proliferation, and dispersion phases. Each of these stages includes reinforcement by, or modulation of, the extracellular matrix. Pseudomonas aeruginosa has been a model organism for the study of biofilm formation. Additionally, other Pseudomonas species utilize biofilm formation during plant colonization and environmental persistence. Pseudomonads produce several biofilm matrix molecules, including polysaccharides, nucleic acids, and proteins. Accessory matrix components shown to aid biofilm formation and adaptability under varying conditions are also produced by pseudomonads. Adaptation facilitated by biofilm formation allows for selection of genetic variants with unique and distinguishable colony morphology. Examples include rugose small-colony variants and wrinkly spreaders (WS), which over produce Psl/Pel or cellulose, respectively, and mucoid bacteria that over produce alginate. The well-documented emergence of these variants suggests that pseudomonads take advantage of matrix-building subpopulations conferring specific benefits for the entire population. This review will focus on various polysaccharides as well as additional Pseudomonas biofilm matrix components. Discussions will center on structure-function relationships, regulation, and the role of individual matrix molecules in niche biology.

Biofilms contribute to Pseudomonas aeruginosa persistence in a variety of diseases, including cystic fibrosis, burn wounds, and chronic suppurative otitis media. However, few studies have directly addressed P. aeruginosa biofilms in vivo. We used a chinchilla model of otitis media, which has previously been used to study persistent Streptococcus pneumoniae and Haemophilus influenzae infections, to show that structures formed in vivo are biofilms of bacterial and host origin within a matrix that includes Psl, a P. aeruginosa biofilm polysaccharide. We evaluated three biofilm and/or virulence mediators of P. aeruginosa known to affect biofilm formation in vitro and pathogenesis in vivo-bis-(3',5')-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing-in a chinchilla model. We show that c-di-GMP overproduction has a positive impact on bacterial persistence, while quorum sensing increases virulence. We found no difference in persistence attributed to flagella. We conclude from these studies that a chinchilla otitis media model provides a means to evaluate pathogenic mediators of P. aeruginosa and that in vitro phenotypes should be examined in multiple infection systems to fully understand their role in disease.