Biofilm-mediated Infection Research Articles

Cellulase exhibited a therapeutic potential to inhibit Salmonella enterica serovar Typhi biofilm by targeting multiple regulatory proteins of biofilm

Biofilm-mediated Salmonella enterica serovar Typhi (Salmonella Ser. Typhi) infections are a growing global health issue due to the formation of antibiotic resistance. The study aimed to discover some of the druggable target proteins of Salmonella Ser. Typhi biofilm and antibiofilm enzyme to prevent Salmonella Ser. Typhi biofilm-mediated infection. Enzymatic therapy has demonstrated effective therapeutic results against bacterial infections due to its specificity and high binding capacity to the target. Therefore, this study focused on the computational interaction between the cellulase enzyme and Salmonella Ser. Typhi biofilm targets proteins with help of the various computational experiments such as ADMET (absorption, distribution, metabolism, excretion, and toxicity), protein-protein interactions, MMGBSA, etc. Further, in vitro validations of the typhoidal biofilm and cellulose presence in Salmonella Ser. Typhi biofilm was conducted using Scanning Electron Microscopy (SEM), Fourier transform infrared spectroscopy, and Raman analysis. Additionally, a minimum biofilm inhibitory concentration assay for cellulase was conducted and find out the optimized cellulase concentration which showed its inhibitory effect on the Salmonella Ser. Typhi. The cellulase antibiofilm effect was analyzed with the help of SEM analysis. Further, the cellulose content in Salmonella Ser. Typhi was quantified before and after treatment of cellulase enzyme. As a result, 58.82 % cellulose content was decreased due to cellulase treatment in Salmonella Ser. Typhi. From the seven selected typhoidal biofilm regulatory proteins of Salmonella Ser. Typhi, we identified only five potential druggable targets: BcsA, CsgE, OmpR, CsgF, and CsgD. The BcsA protein is responsible for cellulose production in Salmonella Ser. Typhi biofilm. Consequently, cellulose worked as a fascinating drug target in Salmonella Ser. Typhi biofilm. Therefore, we used cellulase as a potential antibiofilm enzyme for target-based disruption of biofilm. The cellulase showed a high binding affinity with all five identified target proteins [BcsA(-205.62 kcal/mol) > CsgE(-108.20 kcal/mol) > OmpR(-107.58 kcal/mol) > CsgF(-73.74 kcal/mol) > CsgD(-66.61 kcal/mol)] in the protein-protein interaction analysis. Our computational analysis suggests that the cellulase enzyme may be used as a potential antibiofilm enzyme against Salmonella Ser. Typhi biofilm.

Rational design of a Nano-Antibiotic chitosan hydrogel for the bacterial infection Therapy: In vitro & ex vivo Assessments

In modern times, many antibiotics have become less effective as microorganisms develop resistance. Besides antibiotic resistance, another bacterial strategy that contributes to the capacity to withstand antimicrobials is biofilm formation. Because of these bacterial survival strategies, the desired response cannot be achieved with conventional treatment. Considering the limited discovery of new compounds, the most logical approach is to reconstruct existing antimicrobial molecules with nano-drug delivery systems. With this scientific approach, the aim of the study is to develop a novel nano-antibiotic hydrogel formulation containing silver nanoparticles, chitosan, and amoxicillin. Endodontic disease was used as a model of biofilm-mediated infection, and the antibacterial activity of nano-antibiotic hydrogel was evaluated with the E. faecalis standard bacterial strain. By adopting the Box-Behnken design for the optimisation of formulation variables, an innovative pharmaceutical formulation with antimicrobial and antibiofilm activity was successfully obtained. Further characterisation studies, including nanoparticle characterisation, in vitro cytotoxicity, and ex vivo activity studies, were carried out on dental samples using the optimised formulation. All results were compared with antimicrobial agents routinely used in endodontic treatment. The findings mainly conclude that the optimised nano-antibiotic hydrogel may be an alternative antimicrobial formulation since it is non-cytotoxic and exhibits high antibiofilm activity.

Stimuli-Responsive Release-Active Dressing: A Promising Solution for Eradicating Biofilm-Mediated Wound Infections.

Biofilm-mediated wound infections pose a significant challenge due to the limitations of conventional antibiotics, which often exhibit narrow-spectrum activity, fail to eliminate recurrent bacterial contamination, and are unable to penetrate the biofilm matrix. While the search for alternatives has explored the use of metal nanoparticles and synthetic biocides, these solutions often suffer from unintended toxicity to surrounding tissues and lack controlled administration and release. In this study, we engineered a pH-responsive release-active dressing film based on carboxymethyl cellulose, incorporating a synthetic antibacterial molecule (SAM-17). The dressing film exhibited optimal mechanical stability for easy application and demonstrated excellent fluid absorption properties, allowing for prolonged moisturization at the site of injury. The film exhibited pH-dependent release of cargo, with 78% release within 24 h at acidic pH, enabling targeted antibacterial drug delivery within the wound microenvironment. Furthermore, the release-active film effectively eliminated repeated challenges of bacterial contamination. Remarkably, the film demonstrated a minimal toxicity profile in both in vitro and in vivo models. The film eliminated preformed bacterial biofilms, achieving a reduction of 2.5 log against methicillin-resistant Staphylococcus aureus (MRSA) and 4.1 log against vancomycin-resistant S. aureus (VRSA). In a biofilm-mediated MRSA wound infection model, this release-active film eradicated the biofilm-embedded bacteria by over 99%, resulting in accelerated wound healing. These findings highlight the potential of this film as an effective candidate for tackling biofilm-associated wound infections.

CXCR2 perturbation promotes Staphylococcus aureus implant-associated infection.

Introduction. Staphylococcus aureus is the leading cause of acute medical implant infections, representing a significant modern medical concern. The success of S. aureus as a pathogen in these cases resides in its arsenal of virulence factors, resistance to multiple antimicrobials, mechanisms of immune modulation, and ability to rapidly form biofilms associated with implant surfaces. S. aureus device-associated, biofilm-mediated infections are often persistent and notoriously difficult to treat, skewing innate immune responses to promote chronic reoccurring infections. While relatively little is known of the role neutrophils play in response to acute S. aureus biofilm infections, these effector cells must be efficiently recruited to sites of infection via directed chemotaxis. Here we investigate the effects of modulating CXC chemokine receptor 2 (CXCR2) activity, predominantly expressed on neutrophils, during S. aureus implant-associated infection.Hypothesis. We hypothesize that modulation of CXCR2 expression and/or signalling activities during S. aureus infection, and thus neutrophil recruitment, extravasation and antimicrobial activity, will affect infection control and bacterial burdens in a mouse model of implant-associated infection.Aim. This investigation aims to elucidate the impact of altered CXCR2 activity during S. aureus biofilm-mediated infection that may help develop a framework for an effective novel strategy to prevent morbidity and mortality associated with implant infections.Methodology. To examine the role of CXCR2 during S. aureus implant infection, we employed a mouse model of indwelling subcutaneous catheter infection using a community-associated methicillin-resistant S. aureus (MRSA) strain. To assess the role of CXCR2 induction or inhibition during infection, treatment groups received daily intraperitoneal doses of either Lipocalin-2 (Lcn2) or AZD5069, respectively. At the end of the study, catheters and surrounding soft tissues were analysed for bacterial burdens and dissemination, and Cxcr2 transcription within the implant-associated tissues was quantified.Results. Mice treated with Lcn2 developed higher bacterial burdens within the soft tissue surrounding the implant site, which was associated with increased Cxcr2 expression. AZD5069 treatment also resulted in increased implant- and tissues-associated bacterial titres, as well as enhanced Cxcr2 expression.Conclusion. Our results demonstrate that CXCR2 plays an essential role in regulating the severity of S. aureus implant-associated infections. Interestingly, however, perturbation of CXCR2 expression or signalling both resulted in enhanced Cxcr2 transcription and elevated implant-associated bacterial burdens. Thus, CXCR2 appears finely tuned to efficiently recruit effector cells and mediate control of S. aureus biofilm-mediated infection.

In vitro evaluation of three engineered multispecies endodontic biofilms on a dentinal disk substrate

The aim of this study was the development of a complex multispecies endodontic biofilm using Candida albicans, Proteus mirabilis and Pseudomonas aeruginosa on a biofilm of Enterococcus faecalis in a dentinal substrate design. The endodontic pathology is a biofilm-mediated infection, and the aim of root canal therapy is to reduce, as much as possible, the bacterial population. Thus, it is important to develop a laboratory endodontic biofilm to test the effect of new irrigation and obturation techniques on reduction of bacterial count. The culture of Enterococcus faecalis from ATCC 29212 began with aerobic cultivation on blood agar, followed by transfer to Brain Heart Infusion (BHI) broth with 5% sucrose. Incubation occurred in a shaker at 37 °C for 24 h, followed by an additional 24-h static phase. After 10 d, Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans were introduced sequentially in three distinct groups. Group 1: the order of addition was Candida albicans, Proteus mirabilis, and Pseudomonas aeruginosa; Group 2: the order was Pseudomonas aeruginosa, Candida albicans, and Proteus mirabilis; and Group 3: Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans. After 16 days, the biofilm was carefully extracted, transferred to sterile BHI, and dissected using a sterile needle technique. Subsequently, an optical density test, bacterial counts, and colony enumeration were performed on various agar plates. Group 2 in which Pseudomonas aeruginosa was added directly after Enterococcus faecalis followed by Candida albicans and Proteus mirabilis showed significantly greater total bacterial count than the other two groups.

Bacteriophage entrapped chitosan microgel for the treatment of biofilm-mediated polybacterial infection in burn wounds

Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) bacteria are most commonly present in burn wound infections. Multidrug resistance (MDR) and biofilm formation make it difficult to treat these infections. Bacteriophages (BPs) are proven as an effective therapy against MDR as well as biofilm-associated wound infections. In the present work, a naturally inspired bacteriophage cocktail loaded chitosan microparticles-laden topical gel has been developed for the effective treatment of these infections. Bacteriophages against MDR S. aureus (BPSAФ1) and P. aeruginosa (BPPAФ1) were isolated and loaded separately and in combination into the chitosan microparticles (BPSAФ1-CHMPs, BPPAФ1-CHMPs, and MBP-CHMPs), which were later incorporated into the SEPINEO™ P 600 gel (BPSAФ1-CHMPs-gel, BPPAФ1-CHMPs-gel, and MBP-CHMPs-gel). BPs were characterized for their morphology, lytic activity, burst size, and hemocompatibility, and BPs belongs to Caudoviricetes class. Furthermore, BPSAФ1-CHMPs, BPPAФ1-CHMPs, and MBP-CHMPs had an average particle size of 1.19 ± 0.11, 1.42 ± 0.21, and 2.84 ± 0.28 μm, respectively, and expressed promising in vitro antibiofilm eradication potency. The ultrasound and photoacoustic imaging in infected burn wounds demonstrated improved wound healing reduced inflammation and increased oxygen saturation following treatment with BPs formulations. The obtained results suggested that the incorporation of the BPs in the MP-gel protected the BPs, sustained the BPs release, and improved the antibacterial activity.

Antibiofilm activity of neem leaf (Azadirachta indica A. Juss) ethanolic extracts against Enterococcus faecalis in vitro

Background: Enterococcus faecalis commonly infects root canals by forming a biofilm. Extracts from neem leaves (Azadirachta indica A. Juss) have been shown to have antibacterial properties, indicating their potential in preventing or treating biofilm formation caused by bacteria. Purpose: This study aims to investigate the phytochemical compounds present in neem leaves (Azadirachta indica A. Juss) and establish the concentration of ethanol-based neem leaf extract that can effectively inhibit the in vitro growth of Enterococcus faecalis biofilm. Methods: This study employed the maceration technique for extraction, gas chromatography mass spectroscopy for the analysis of plant chemicals, and a microtiter plate assay for measuring biofilm formation with treatment concentrations of 6.25%, 12.5%, 25%, 50%, and 75%, with a positive control of 0.2% chlorhexidine. Results: A phytochemical analysis revealed that the ethanol extract of neem leaves contained 22 different metabolites, mainly terpenoids and fatty acids. The extract demonstrated antibiofilm activity only at a concentration of 12.5% with an average biofilm inhibition of 36.85%. However, lower concentrations of 6.25%, 25%, 50%, and 75% had the opposite effect, promoting biofilm formation in Enterococcus faecalis. Conclusion: Phytochemical metabolite contained in the ethanolic extracts of neem leaves might contribute a promising agent in treating a biofilm-mediated root canal infection of Enterococcus faecalis.

An Enzymatic Antibiotic Adjuvant Modulates the Infectious Microenvironment to Overcome Antimicrobial Resistance of Pathogens.

The emergence and evolution of antimicrobial resistance (AMR) pose a significant challenge to the current arsenal to fight infection. Antibiotic adjuvants represent an appealing tactic for tackling the AMR of pathogens, however, their practical applications are greatly constrained by the harsh infectious microenvironment. Herein, it is found that silver nanoclusters (Ag NCs) can possess tunable enzymatic activities to modulate infectious microenvironments. Based on this finding, an enzymatic nanoadjuvant (EnzNA) self-assembled from Ag NCs, which is inert under neutral physiological conditions but can readily disassemble into isolated Ag NCs exhibiting biofilm destructive oxidase-mimetic activity in the acidic biofilm microenvironment, is developed. Once internalized into the neutral cytoplasm of bacteria, Ag NCs switch to reveal the thiol oxidase-mimetic activity to suppress ribosomal biogenesis for AMR reversal and evolution inhibition of pathogens. Consequently, EnzNAs revitalize various existing antibiotics against methicillin-resistant Staphylococcus aureus, and potentiate the antibiotic efficacy against biofilm-mediated skin infection and lethal lung infection in mice. These findings highlight the capability of enzyme-mimetic nanomaterials to modulate the infectious microenvironment and potentiate antibiotics, providing a paradigm shift for anti-infection therapy.

Pseudomonas aeruginosa persister cell formation upon antibiotic exposure in planktonic and biofilm state

Persister cell (PC) is dormant, tolerant to antibiotics, and a transient reversible phenotype. These phenotypes are observed in P. aeruginosa and cause bacterial chronic infection as well as recurrence of biofilm-mediated infection. PC formation requires stringent response and toxin-antitoxin (TA) modules. This study shows the P. aeruginosa PC formation in planktonic and biofilm stages on ceftazidime, gentamicin, and ciprofloxacin treatments. The PC formation was studied using persister assay, flow cytometry using Redox Sensor Green, fluorescence as well as Confocal Laser Scanning Microscopy, and gene expression of stringent response and TA genes. In the planktonic stage, ceftazidime showed a high survival fraction, high redox activity, and elongation of cells was observed followed by ciprofloxacin and gentamicin treatment having redox activity and rod-shaped cells. The gene expression of stringent response and TA genes were upregulated on gentamicin followed by ceftazidime treatment and varied among the isolates. In the biofilm stage, gentamicin and ciprofloxacin showed the biphasic killing pattern, redox activity, gene expression level of stringent response and TA varied across the isolates. Ceftazidime treatment showed higher persister cells in planktonic growth while all three antibiotics were able to induce persister cell formation in the biofilm stage.

Investigation of Biofilm Formation Properties of Coagulase Negative Staphylococci Isolated from Catheter-Related Bloodstream Infections

In view of the significant negative impact of biofilm-mediated infection on patient health and the necessity of a reliable phenotypic method to detect biofilm producers, this study aimed to demonstrate phenotypic and molecular biofilm formation in coagulase-negative staphylococci (CoNS) isolated from catheter related infections and to compare the methods used with each other. The study was also aimed to determine the biofilm eradication effect of vancomycin in order to guide for the treatment. For the detection of biofilm formation, a total of 154 CoNS clinical isolates of which 30 being causative agents of catheter related bloodstream infection (CRBSI) (isolated from both the catheter tip and blood cultures of 15 patients), 89 being isolated from peripheral blood cultures of patients without a central venous catheter (CVC) (13 of them were bloodstream infection agents, 76 of them were contaminant), and 35 being isolated as catheter colonizer, were screened by tissue culture plate (TCP), Congo red agar (CRA) method and polymerase chain reaction (icaA, icaD and IS256). Vancomycin minimum inhibitory concentration (MIC) and minimum biofilm eradicating concentration (MBEC) values were determined. The pulsed field gel electrophoresis (PFGE) method was used to show the clonal relationship between CoNS isolated from the catheter tips and peripheral blood of patients with CRBSI. Of the 154 CoNS isolates included in the study, 38.9% were Staphylococcus epidermidis (n= 60), 34.4% were Staphylococcus haemolyticus (n= 53), 20.7% were Staphylococcus hominis (n= 32), and 3.8% were detected as Staphylococcus capitis (n= 6). In our study, biofilm formation was shown in 31.8% with the CRA method and in 68.1% with the TCP method. By TCP method, 22% (n= 34) were determined as weak, 31.2% (n= 48) medium and 14.9% (n= 23) strong biofilm producers. While the sensitivity of the CRA method was found to be low for isolates that were determined as weak positive in the microplate method, the high sensitivity of the CRA method for isolates with medium and strong positivity was found remarkable. The positivity rates of icaA, icaD and IS256 genes in a total of 154 CoNS isolates were found to be 40 (25.9%), 57 (37%) and 77 (50%), respectively. In total, at least one gene positivity was detected in 107 (69.5%) isolates. Single gene positivity was detected in 55 (35.7%), two gene positivity in 35 (22.7%) and three gene positivity was detected in 17 (11%) of the included CoNS. Biofilm formation (four weak, four medium, two strong) was detected by microplate method in 10 of 47 CoNS isolates (five S.epidermidis, three S.hominis, one S.haemolyticus and one S.capitis) in which no genes were detected. Vancomycin MBEC/ MIC values were found to be high and it was observed that as the biofilm forming power of the isolates increased, the MBEC/MIC ratio also increased. The CoNS isolated from the catheter samples and blood of patients diagnosed with CRBSI had a 100% similar profile with PFGE except for one unevaluable isolate. The tissue culture plate (TCP) method was found to be most sensitive, accurate and reproducible screening method for detection of biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci. The presence of the icaAD and IS256 gene is not always associated with in vitro biofilm formation. For this reason, it is more appropriate to use more than one method together for the evaluation of biofilm formation. It was thought that the use of reliable methods to specifically detect biofilms could be helpful in diseases that are difficult to treat. Considering the high rates of biofilm and antimicrobial resistance of biofilm-forming isolates in biomedical device associated infections, it was determined that it would not be sufficient to evaluate only the MIC results for susceptibility results.

Advanced Treatment Modalities For Endodontic Biofilms : A Narrative Review

Endodontic disease is essentially a biofilm-mediated infection with broader bacterial diversity than previously anticipated. Removal of endodontic bacterial biofilms and preventing recontamination of root canal after treatment are the essential elements for successful outcomes of endodontic treatment. Nanoparticles and antibacterial photodynamic therapy corroborate to be emerging tools as they have proved to be effective in eradicating biofilms from the complex root canal system in various in-vitro studies. This article primarily aims to provide an exhaustive overview based on available literature on advanced treatment modalities to eradicate these biofilms.

Anti-biofilm activities of coumarin as quorum sensing inhibitor for Porphyromonas gingivalis

ABSTRACT Porphyromonas gingivalis is a keystone pathogen in periodontitis, a biofilm-mediated infection disease. This research aimed to investigate the effect of coumarin on P. gingivalis biofilm formation. We detected the antimicrobial effect on P. gingivalis planktonic growth, observed membrane structure and morphological change by TEM, and quantified membrane permeability by calcein-AM staining. The cell surface hydrophobicity, aggregation, and attachment were assessed. We also investigated different sub-MIC concentrations of coumarin on biofilm formation, and observed biofilm structureby confocal laser scanning microscopy. The biofilm-related gene expression was evaluated using real-time PCR. The results showed that coumarin inhibited P. gingivalis growth and damaged the cell morphology above 400 μM concentration. Coumarin did not affect cell surface hydrophobicity, aggregation, attachment, and the early stage of biofilm formation at sub-MIC concentrations. Still, it exhibited anti-biofilm effects for the late-stage and pre-formed biofilms dispersion. The biofilms after coumarin treatment became interspersed, and biofilm-related gene expression was downregulated. Coumarin also inhibited AI-2 activity and interacted with the HmuY protein by molecular docking analysis. Our research demonstrated that coumarin inhibited P. gingivalis biofilm formation through a quorum sensing system.

Anthranilate Acts as a Signal to Modulate Biofilm Formation, Virulence, and Antibiotic Tolerance of Pseudomonas aeruginosa and Surrounding Bacteria.

ABSTRACTAnthranilate is a diffusible molecule produced by Pseudomonas aeruginosa and accumulates as P. aeruginosa grows. Anthranilate is an important intermediate for the synthesis of tryptophan and the Pseudomonas quinolone signal (PQS), as well as metabolized by the anthranilate dioxygenase complex (antABC operon products). Here we demonstrate that anthranilate is a key factor that modulates the pathogenicity-related phenotypes of P. aeruginosa and other surrounding bacteria in the environment, such as biofilm formation, antibiotic tolerance, and virulence. We found that the anthranilate levels in P. aeruginosa cultures rapidly increased in the stationary phase and then decreased again, forming an anthranilate peak. Biofilm formation, antibiotic susceptibility, and virulence of P. aeruginosa were significantly altered before and after this anthranilate peak. In addition, these phenotypes were all modified by the mutation of antABC and exogenous addition of anthranilate. Anthranilate also increased the antibiotic susceptibility of other species of bacteria, such as Escherichia coli, Salmonella enterica, Bacillus subtilis, and Staphylococcus aureus. Before the anthranilate peak, the low intracellular anthranilate level was maintained through degradation from the antABC function, in which induction of antABC was also limited to a small extent. The premature degradation of anthranilate, due to its high levels, and antABC expression early in the growth phase, appears to be toxic to the cells. From these results, we propose that by generating an anthranilate peak as a signal, P. aeruginosa may induce some sort of physiological change in surrounding cells.IMPORTANCE Pseudomonas aeruginosa is a notorious pathogen with high antibiotic resistance, strong virulence, and ability to cause biofilm-mediated chronic infection. We found that these characteristics change profoundly before and after the time when anthranilate is produced as an “anthranilate peak”. This peak acts as a signal that induces physiological changes in surrounding cells, decreasing their antibiotic tolerance and biofilm formation. This study is important in that it provides a new insight into how microbial signaling substances can induce changes in the pathogenicity-related phenotypes of cells in the environment. In addition, this study shows that anthranilate can be used as an adjuvant to antibiotics.

Activity of Two Antimicrobial Peptides against Enterococcus faecalis in a Model of Biofilm-Mediated Endodontic Infection.

Enterococcus faecalis is a common cause of biofilm-associated opportunistic infections, which are often difficult to treat. The formation of E. faecalis biofilms on the dentinal walls of the root canal is frequently the cause of endodontic treatment failure and secondary apical periodontitis. In a preliminary work, two recognized antifungal peptides, KP and L18R, showed antibacterial activity against planktonic E. faecalis cells at micromolar concentrations. Moreover, L18R proved to reduce the biomass in the early stage of E. faecalis biofilm development on polystyrene plates, while a qualitative biofilm inhibition was demonstrated on hydroxyapatite disks by confocal laser scanning microscopy (CLSM). The aim of this study was to better characterize the effect of both peptides on E. faecalis biofilm. A reduction in metabolic activity after peptide treatment was detected by Alamar Blue assay, while a remarkable impairment in the architecture of E. faecalis biofilms on hydroxyapatite disks, along with a significant reduction in viable bacteria, was caused mostly by L18R, as assessed by CLSM and scanning electron microscopy. The lack of cytotoxicity of the investigated peptides against L929 murine fibroblasts was also determined. Obtained results suggest L18R as a promising candidate for the development of new strategies for endodontic infection control.

Contribution of Essential Oils to the Fight against Microbial Biofilms—A Review

The increasing clinical use of artificial medical devices raises the issue of microbial contamination, which is a risk factor for the occurrence of biofilm-associated infections. A huge amount of scientific data highlights the promising potential of essential oils (EOs) to be used for the development of novel antibiofilm strategies. We aimed to review the relevant literature indexed in PubMed and Embase and to identify the recent directions in the field of EOs, as a new modality to eradicate microbial biofilms. We paid special attention to studies that explain the mechanisms of the microbicidal and antibiofilm activity of EOs, as well as their synergism with other antimicrobials. The EOs are difficult to test for their antimicrobial activity due to lipophilicity and volatility, so we have presented recent methods that facilitate these tests. There are presented the applications of EOs in chronic wounds and biofilm-mediated infection treatment, in the food industry and as air disinfectants. This analysis concludes that EOs are a source of antimicrobial agents that should not be neglected and that will probably provide new anti-infective therapeutic agents.

Intravital Multiphoton Examination of Implant-Associated Staphylococcus aureus Biofilm Infection.

Bacterial infections associated with implanted medical devices represents a healthcare crisis due to their persistence, antibiotic tolerance, and immune avoidance. Indwelling devices are rapidly coated with host plasma and extracellular matrix proteins which can then be exploited by bacterial pathogens for adherence and subsequent biofilm development. Our understanding of the host-pathogen interface that determines the fate of biofilm-mediated infections is limited to the experimental models employed by laboratories studying these organisms. Current in vivo models of biofilm-mediated infection, while certainly useful, are typically limited to end-point analyses of bacterial burden enumeration, immune cell profiling, and cytokine/chemokine analysis. Thus, with these models, the complex, real-time assessment of biofilm development and innate immune cell activity remains imperceptible. Here, we describe a novel murine biofilm infection model employing time-lapse intravital multiphoton microscopy which permits concurrent and real-time visualization of Staphylococcus aureus biofilm formation and immune cell activity. Using cell tracking, we found that S. aureus biofilms impede neutrophil chemotaxis, redirecting their migration patterns to prevent biofilm invasion. This approach is the first to directly examine device-associated biofilm development and host-pathogen interactions and will serve to both further our understanding of infection development and help reveal the effects of future antibiofilm treatment strategies.

Electrochemical communication in biofilm of bacterial community.

Electrochemical communication during biofilm formation has recently been identified. Bacteria within biofilm-adopt different strategies for electrochemical communication such as direct contact via membrane-bound molecules, diffusive electron transfer via soluble redox-active molecules, and ion channel-mediated long-range electrochemical signaling. Long-range electrical signals are important to communicate with distant members within the biofilm, which function through spatially propagating waves of potassium ion (K+ ) that depolarizes neighboring cells. During propagation, these waves coordinate between the metabolic states of interior and peripheral cells of the biofilm. The understanding of electrochemical communication within the biofilm may provide new strategies to control biofilm-mediated drug resistance. Here, we summarized the different mechanisms of electrochemical communication among bacterial populations and suggested its possible role in the development of high level of antibiotic resistance. Thus, electrochemical signaling opens a new avenue concerning the electrophysiology of bacterial biofilm and may help to control the biofilm-mediated infection by developing future antimicrobials.

In Memory of Mark Shirtliff, PhD: Modern Concepts in Diagnosis, Treatment, Prevention, and Cost of Infection After Fracture Surgery

This issue in Techniques in Orthopaedics is dedicated to our friend and colleague Mark Shirtliff, PhD. Dr Shirtliff had a remarkable career that was cut short tragically at the age of 49 during a rafting accident. Mark’s professional accomplishments were many (see details of his life and career at: https://markshirtliffbiofilmfoundation.org), and he certainly will have a long-lasting effect on our field. His contributions included important discoveries and developments in the field of biofilm-mediated infection, as well as the selfless role he played as a mentor to so many other researchers. The sheer size and breadth of support for him shown at his memorial service speak to what an important influence he had on the many who had the pleasure of working with him. Although Mark’s professional accomplishments were impressive, in many ways he was even more memorable for the type of person he was. He was one of the most charismatic, entertaining, and fascinating people that you could ever meet. Mark was the rare person who was able to give a basic science talk to clinicians that was simultaneously hilarious and thought-provoking. Most importantly he was a talented communicator who could convey complex information in a manner that even a clinician could understand. Any small meeting with Mark inevitably involved a lot of laughter and many outrageous comments—all part of the fun we had in working with him. His tremendous intelligence, creativity, and passion for science were wrapped up with his charm, lightning-quick wit, and self-deprecating modesty. As Mark dedicated much of his professional life to a better understanding of biofilm-related infections, such as those observed after fracture surgery, we hope that this issue serves as an appropriate tribute to his career. Many of the authors of these articles worked closely with Dr Shirtliff for many years and his influence on our lives has been important to us. The 6 articles in this symposium discuss many of the important issues pertaining to current approaches for prevention, diagnosis, and treatment of infections after orthopedic trauma surgery. Clinicians are well aware of the societal importance of the all too common deep infections after fracture surgery. Unfortunately, this is nowhere close to a solved problem,1 and much progress is needed. Dr Johnson and colleagues lead off the symposium with a detailed discussion of the problems that are endemic to timely and accurate diagnosis in the clinical management of patients with infection after fracture fixation. Dr Natoli and colleagues next lay out modern diagnostic techniques, such as those that were of particular interest to Dr Shirtliff, and their potential to help us with the problems the first article identified. The third article, led by Dr Wise, details the current evidence for risk factors associated with infection, followed by Dr Joshi who provides a comprehensive look at clinical measures that can be used for infection prevention. Dr Costales and colleagues detail the approach to treating infections after tibia fracture fixation, an issue that continues to plague fracture surgeons. The issue ends with Dr Levy and colleagues’ sophisticated look at the costs of infection after fracture-related surgeries. Taken together this body of work summarizes important issues and identifies areas for future work. The authors are grateful of the journal for giving us the opportunity to present this information to the readers, and we hope that it serves as a small tribute to Mark Shirtliff.

Streptomyces diastaticus isolated from the marine crustacean Portunus sanguinolentus with potential antibiofilm activity against Candida albicans.

Biofilm-mediated multidrug resistance has turned into major challenge for the treatment of C. albicans infections. In the present study, actinomycetes (SS5) isolated from marine crustacean were investigated for their ability to inhibit C. albicans biofilm formation. Cultural, morphological and 16S rRNA analysis revealed that the isolated strain was Streptomyces diastaticus. Ethyl acetate bioactive fractions (6µgmL-1) from SS5 showed potent antibiofilm activity against C. albicans. Light microscopic and CLSM analysis further substantiated the antibiofilm activity of the bioactive fraction against C. albicans. The bioactive fraction was subjected to FTIR and GC-MS for characterization. From GC-MS analysis, the presence of 31 compounds were revealed, among which the alkanes are predominantly present. Hence, further investigation for the potential of these bioactive compounds against C. albicans biofilm will help in the identification of promising candidate for the prevention of biofilm-mediated infection.

Strategies to Prevent Biofilm Infections on Biomaterials: Effect of Novel Naturally-Derived Biofilm Inhibitors on a Competitive Colonization Model of Titanium by Staphylococcus aureus and SaOS-2 Cells

Biofilm-mediated infection is a major cause of bone prosthesis failure. The lack of molecules able to act in biofilms has driven research aimed at identifying new anti-biofilm agents via chemical screens. However, to be able to accommodate a large number of compounds, the testing conditions of these screenings end up being typically far from the clinical scenario. In this study, we assess the potential applicability of three previously discovered anti-biofilm compounds to be part of implanted medical devices by testing them on in vitro systems that more closely resemble the clinical scenario. To that end, we used a competition model based on the co-culture of SaOS-2 mammalian cells and Staphylococcus aureus (collection and clinical strains) on a titanium surface, as well as titanium pre-conditioned with high serum protein concentration. Additionally, we studied whether these compounds enhance the previously proven protective effect of pre-incubating titanium with SaOS-2 cells. Out of the three, DHA1 was the one with the highest potential, showing a preventive effect on bacterial adherence in all tested conditions, making it the most promising agent for incorporation into bone implants. This study emphasizes and demonstrates the importance of using meaningful experimental models, where potential antimicrobials ought to be tested for the protection of biomaterials in translational applications.