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Although needleless connectors (NCs) are widely used in clinical practice, they carry significant risk of bloodstream infection (BSI). In this study, we quantified differences in bacterial transfer and biofilm formation between various NCs. Prospective, clinically simulated in vitro experimental study. We tested 20 NCs in a 5-day clinical simulation of inoculations onto NC septum surfaces, which were then flushed with saline and cultured for bacterial transfer. Biofilm formation was measured through destructive sampling of the connector-catheter system. Moreover, 8 NC design factors were evaluated for their influence on bacterial transfer and biofilm formation. This study was designed without a disinfection protocol to ascertain the intrinsic risk of each NC. Clave Neutron and MicroClave had the lowest overall mean log density of bacteria in the flush compared to other NCs ( < .05), except there were no statistically significant differences between Clave Neutron, Microclave, SafeTouch, and SafeAccess ( ≥ .05). The amount of biofilm in the NC was positively associated with bacteria in the flush ( .0005). Among 8 design factors, flow path was most important, with the internal cannula associated with a statistically significant 1 log reduction (LR) in bacteria in the flush ( = 49%) and 0.5-2 LR in the connector ( = 34%). All factors together best explained bacteria in the flush ( = 65%) and biofilm in the connector ( = 48%). Bacterial transfer and biofilm formation in the connector-catheter system varied statistically significantly between the 20 NCs, suggesting that NC choice can lower the risk of developing catheter-related BSIs.

Chronic, non-healing wounds contribute significantly to the suffering of patients with co-morbidities in the clinical population with mild to severely compromised immune systems. Normal wound healing proceeds through a well-described process. However, in chronic wounds this process seems to become dysregulated at the transition between resolution of inflammation and re-epithelialization. Bioburden in the form of colonizing bacteria is a major contributor to the delayed headlining in chronic wounds such as pressure ulcers. However how the microbiome influences the wound metabolic landscape is unknown. Here, we have used a Systems Biology approach to determine the biochemical associations between the taxonomic and metabolomic profiles of wounds colonized by bacteria. Pressure ulcer biopsies were harvested from primary chronic wounds and bisected into top and bottom sections prior to analysis of microbiome by pyrosequencing and analysis of metabolome using 1H nuclear magnetic resonance (NMR) spectroscopy. Bacterial taxonomy revealed that wounds were colonized predominantly by three main phyla, but differed significantly at the genus level. While taxonomic profiles demonstrated significant variability between wounds, metabolic profiles shared significant similarity based on the depth of the wound biopsy. Biochemical association between taxonomy and metabolic landscape indicated significant wound-to-wound similarity in metabolite enrichment sets and metabolic pathway impacts, especially with regard to amino acid metabolism. To our knowledge, this is the first demonstration of a statistically robust correlation between bacterial colonization and metabolic landscape within the chronic wound environment.

Background Many chronic diseases, such as non-healing wounds are characterized by prolonged inflammation and respond poorly to conventional treatment. Bacterial biofilms are a major impediment to wound healing. Persistent infection of the skin allows the formation of complex bacterial communities termed biofilm. Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts and are orders of magnitude more resistant to antibiotics, host immune response, and environmental stress. Staphylococcus aureus is prevalent in cutaneous infections such as chronic wounds and is an important human pathogen. Results The impact of S. aureus soluble products in biofilm-conditioned medium (BCM) or in planktonic-conditioned medium (PCM) on human keratinocytes was investigated. Proteomic analysis of BCM and PCM revealed differential protein compositions with PCM containing several enzymes involved in glycolysis. Global gene expression of keratinocytes exposed to biofilm and planktonic S. aureus was analyzed after four hours of exposure. Gene ontology terms associated with responses to bacteria, inflammation, apoptosis, chemotaxis, and signal transduction were enriched in BCM treated keratinocytes. Several transcripts encoding cytokines were also upregulated by BCM after four hours. ELISA analysis of cytokines confirmed microarray results at four hours and revealed that after 24 hours of exposure, S. aureus biofilm induced sustained low level cytokine production compared to near exponential increases of cytokines in planktonic treated keratinocytes. The reduction in cytokines produced by keratinocytes exposed to biofilm was accompanied by suppressed phosphorylation of MAPKs. Chemical inhibition of MAPKs did not drastically reduce cytokine production in BCM-treated keratinocytes suggesting that the majority of cytokine production is mediated through MAPK-independent mechanisms. Conclusions Collectively the results indicate that S. aureus biofilms induce a distinct inflammatory response compared to their planktonic counterparts. The differential gene expression and production of inflammatory cytokines by biofilm and planktonic cultures in keratinocytes could have implications for the formation and persistence of chronic wounds. The formation of a biofilm should be considered in any study investigating host response to bacteria.

Considering the prevalence and pathogenicity of biofilms in wounds, this study was designed to evaluate the anti-biofilm capabilities of eight commercially available wound care products using established in vitro assays for biofilms. The products evaluated included dressings with multiple delivery formats for ionic silver including nanocrystalline, gelling fibers, polyurethane (PU) foam, and polymer matrix. Additionally, non-silver-based products including an extracellular polymeric substance (EPS)-dissolving antimicrobial wound gel (BDWG), a collagenase-based debriding ointment and a fish skin-based skin substitute were also evaluated. The products were evaluated on Staphylococcus aureus and Pseudomonas aeruginosa mixed-species biofilms grown using colony drip flow reactor (CDFR) and standard drip flow reactor (DFR) methodologies. Anti-biofilm efficacy was measured by viable plate counts and confocal scanning laser microscopy (CSLM). Four of the eight wound care products tested were efficacious in inhibiting growth of new biofilm when compared with untreated controls. These four products were further evaluated against mature biofilms. BDWG was the only product that achieved greater than 2-log growth reduction (5.88 and 6.58 for S. aureus and P. aeruginosa, respectively) of a mature biofilm. Evaluating both biofilm prevention and mature biofilm disruption capacity is important to a comprehensive understanding of the anti-biofilm efficacy of wound care products.

Bacterial biofilm formation is a complex developmental process involving cellular differentiation and the formation of intricate 3D structures. Here we demonstrate that exposure to ferric chloride triggers rugose biofilm formation by the uropathogenic Escherichia coli strain UTI89 and by enteric bacteria Citrobacter koseri and Salmonella enterica serovar typhimurium . Two unique and separable cellular populations emerge in iron-triggered, rugose biofilms. Bacteria at the air–biofilm interface express high levels of the biofilm regulator csgD , the cellulose activator adrA , and the curli subunit operon csgBAC . Bacteria in the interior of rugose biofilms express low levels of csgD and undetectable levels of matrix components curli and cellulose. Iron activation of rugose biofilms is linked to oxidative stress. Superoxide generation, either through addition of phenazine methosulfate or by deletion of sodA and sodB , stimulates rugose biofilm formation in the absence of high iron. Additionally, overexpression of Mn-superoxide dismutase, which can mitigate iron-derived reactive oxygen stress, decreases biofilm formation in a WT strain upon iron exposure. Not only does reactive oxygen stress promote rugose biofilm formation, but bacteria in the rugose biofilms display increased resistance to H ₂O ₂ toxicity. Altogether, we demonstrate that iron and superoxide stress trigger rugose biofilm formation in UTI89. Rugose biofilm development involves the elaboration of two distinct bacterial populations and increased resistance to oxidative stress.

BackgroundBacterial biofilms have been implicated with breast implant complications including capsular contracture and anaplastic large-cell lymphoma. The actual mechanisms for either are still under active investigation and are not clear. Due to their increased surface area, implants with textured surfaces may harbor greater biofilm loads than those with smooth surfaces.MethodsBiofilm formation on the outer surface material was compared using implants with various surface areas and roughness, including Natrelle® (Smooth), SmoothSilk®/SilkSurface® (Silk), VelvetSurface ® (Velvet), Siltex®, and Biocell®. The roughness and surface area of each material were assessed using non-contact profilometry. Bacterial attachment (2 h) and biofilm formation (24 h) were evaluated for Staphylococcus epidermidis, Pseudomonas aeruginosa, and Ralstonia pickettii over nine independent experiments using a CDC biofilm reactor and viable plate counts (VPCs) as well as confocal scanning laser microscopy. VPCs of the textured implants were compared relative to the Smooth implant.ResultsSurface areas increased with roughness and were similar among the three least rough implants (Smooth, Silk, and Velvet) and among the roughest implants (Siltex and Biocell). Overall, VPC indicated there was significantly more bacterial attachment and biofilm formation on the Siltex and Biocell implants than the Silk or Velvet implants, although there were differences between species and time points. CSLM confirmed the formation of thicker biofilms on the implants with rougher surface textures.ConclusionThis in vitro study confirmed that implant surfaces with rougher texture, resulting in more surface area, harbored greater biofilm loads than those with smoother surfaces.No Level AssignedThis journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

The study of bacterial interaction between Streptococcus mutans and Actinomyces naeslundii may disclose important features of biofilm interspecies relationships. The aim of this study was to characterize—with an emphasis on biofilm formation and composition and metabolic activity—single- and dual-species biofilms of S. mutans or A. naeslundii, and to use a drip flow reactor (DFR) to evaluate biofilm stress responses to 0.2% chlorhexidine diacetate (CHX). Single- and dual-species biofilms were grown for 24 h. The following factors were evaluated: cell viability, biomass and total proteins in the extracellular matrix, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide—“XTT”—reduction and lactic acid production. To evaluate stress response, biofilms were grown in DFR. Biofilms were treated with CHX or 0.9% sodium chloride (NaCl; control). Biofilms were plated for viability assessment. Confocal laser-scanning microscopy (CLSM) was also performed. Data analysis was carried out at 5% significance level. S. mutans viability and lactic acid production in dual-species biofilms were significantly reduced. S. mutans showed a higher resistance to CHX in dual-species biofilms. Total protein content, biomass and XTT reduction showed no significant differences between single- and dual-species biofilms. CLSM images showed the formation of large clusters in dual-species biofilms. In conclusion, dual-species biofilms reduced S. mutans viability and lactic acid production and increased S. mutans’ resistance to chlorhexidine.

Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.

Prosthetic joint infections (PJIs) are among the most devastating complications after joint replacement. There is limited evidence regarding the efficacy of different antiseptic solutions in reducing planktonic microorganism burden. The purpose of this study was to test the efficacy of different antiseptic solutions against clinically relevant planktonic microorganisms. We designed an experiment examining the efficacy of several antiseptic solutions against clinically relevant planktonic microorganisms in vitro. Regarding planktonic microorganisms, povidone-iodine had 99.9% or greater reduction for all microorganisms tested except for methicillin-resistant Staphylococcus aureus, which was reduced by 60.44%. Irrisept (Irrimax Corp) had 99.9% or greater reduction for all microorganisms except Staphylococcus epidermidis (98.31%) and Enterococcus faecalis (48.61%). Bactisure (Zimmer Surgical Inc) had 99.9% or greater reduction for all microorganisms tested. Various measures exist for PJI prevention, one of which is intraoperative irrigation. We tested irrigants against clinically relevant planktonic microorganisms in vitro and found significant differences in efficacy among them. Further clinical outcome data are necessary to determine whether these solutions can impact PJI in vivo. [Orthopedics. 2022;45(2):122–127.]

The ability to generate two-dimensional images of a wound that contains information about the distribution of bacteria overlaid with the distribution of drugs and metabolites could enhance our understanding of wound healing processes. Advances in technology are leading to a rapid expansion in mass spectrometry-based imaging. When combined with the ability of matrix assisted laser desorption ionization to ionize a wide range of molecules, imaging mass spectrometry is a powerful biomedical research tool. However, this technique has yet to be used to investigate bacterial colonization of wounds or the distribution of antimicrobial agents on tissue. To address this, distribution and persistence of the antimicrobial agent chlorhexidine on a model human tissue was investigated. The ability to detect and localize Staphylococcus aureus on the same tissue model was also addressed. Sub-millimeter resolution ion images from these experiments show the promise of using mass spectrometry imaging to investigate the growth and treatment of bacteria on skin. This methodology will be of value in the development of wound dressings with improved antimicrobial properties and a more careful analysis of the concentration of antimicrobial agents required to prevent biofilm formation and persistence.