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Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases. This paper reports the first example of targeted anti-biofilm therapy in human disease. We have demonstrated that using low-dose nitric oxide as a non-bactericidal signaling molecule to induce biofilm dispersal may be useful as a novel adjunctive therapy to treat chronic pseudomonal biofilm infection in cystic fibrosis.

High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin-antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.

Background. Limitations in treatment of biofilm-associated bacterial infections are often due to subpopulation of persistent bacteria (persisters) tolerant to high concentrations of antibiotics. Based on the increased aminoglycoside efficiency under alkaline conditions, we studied the combination of gentamicin and the clinically compatible basic amino acid L-arginine against planktonic and biofilm bacteria both in vitro and in vivo. Methods. Using Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli bioluminescent strains, we studied the combination of L-arginine and gentamicin against planktonic persisters through time-kill curves of late stationary-phase cultures. In vitro biofilm tolerance towards gentamicin was assessed using PVC 96 well-plates assays. Efficacy of gentamicin as antibiotic lock treatment (ALT) at 5 mg/mL at different pH was evaluated in vivo using a model of totally implantable venous access port (TIVAP) surgically implanted in rats. Results. We demonstrated that a combination of gentamicin and the clinically compatible basic amino acid L-arginine increases in vitro planktonic and biofilm susceptibility to gentamicin, with 99% mortality amongst clinically relevant pathogens, i.e. S. aureus, E. coli and P. aeruginosa persistent bacteria. Moreover, although gentamicin local treatment alone showed poor efficacy in a clinically relevant in vivo model of catheter-related infection, gentamicin supplemented with L-arginine led to complete, long-lasting eradication of S. aureus and E. coli biofilms, when used locally. Conclusion. Given that intravenous administration of L-arginine to human patients is well tolerated, combined use of aminoglycoside and the non-toxic adjuvant L-arginine as catheter lock solution could constitute a new option for the eradication of pathogenic biofilms.

Background Foot infections are a major cause of morbidity in people with diabetes and the most common cause of diabetes-related hospitalization and lower extremity amputation. Staphylococcus aureus is by far the most frequent species isolated from these infections. In particular, methicillin-resistant S. aureus (MRSA) has emerged as a major clinical and epidemiological problem in hospitals. MRSA strains have the ability to be resistant to most β-lactam antibiotics, but also to a wide range of other antimicrobials, making infections difficult to manage and very costly to treat. To date, there are two fifth-generation cephalosporins generally efficacious against MRSA, ceftaroline and ceftobripole, sharing a similar spectrum. Biofilm formation is one of the most important virulence traits of S. aureus. Biofilm growth plays an important role during infection by providing defence against several antagonistic mechanisms. In this study, we analysed the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains isolated from diabetic foot infections. The antibiotic minimum inhibitory concentration (MIC) was determined for ten antimicrobial compounds, along with the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC), followed by PCR identification of genetic determinants of biofilm production and antimicrobial resistance. Results Results demonstrate that very high concentrations of the most used antibiotics in treating diabetic foot infections (DFI) are required to inhibit S. aureus biofilms in vitro, which may explain why monotherapy with these agents frequently fails to eradicate biofilm infections. In fact, biofilms were resistant to antibiotics at concentrations 10–1000 times greater than the ones required to kill free-living or planktonic cells. The only antibiotics able to inhibit biofilm eradication on 50 % of isolates were ceftaroline and gentamicin. Conclusions The results suggest that the antibiotic susceptibility patterns cannot be applied to biofilm established infections. Selection of antimicrobial therapy is a critical step in DFI and should aim at overcoming biofilm disease in order to optimize the outcomes of this complex pathology.

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria co-isolated from chronic infected wounds. Their interactions remain unclear but this coexistence is beneficial for both bacteria and may lead to resistance to antimicrobial treatments. Besides, developing an in vitro model where this coexistence is recreated remains challenging, making difficult their study. The aim of this work was to develop a reliable polymicrobial in vitro model of both species to further understand their interrelationships and the effects of different antimicrobials in coculture. In this work, bioluminescent and fluorescent bacteria were used to evaluate the activity of two antiseptics (chlorhexidine and thymol) against these bacteria planktonically grown, or when forming single and mixed biofilms. At the doses tested (0.4-1,000 mg/L), thymol showed selective antimicrobial action against S. aureus in planktonic and biofilm states, in contrast with chlorhexidine which exerted antimicrobial effects against both bacteria. Furthermore, the initial conditions for both bacteria in the co-culture determined the antimicrobial outcome, showing that P. aeruginosa impaired the proliferation and metabolism of S. aureus . Moreover, S. aureus showed an increased tolerance against antiseptic treatments when co-cultured, attributed to the formation of a thicker mixed biofilm compared to those obtained when monocultured, and also, by the reduction of S. aureus metabolic activity induced by diffusible molecules produced by P. aeruginosa. This work underlines the relevance of polymicrobial populations and their crosstalk and microenvironment in the search of disruptive and effective treatments for polymicrobial biofilms.

Treatments of Mycobacterium marinum , a common non-tuberculous mycobacterium associated with cutaneous infections are very challenging, emphasizing the development of new therapeutic approaches. Here we report the functionalization of mesoporous silica nanoparticles (MSN) with a series of triphenylphosphonium (TPP) substituents, which endowed them with affinity towards the surface of M. marinum in vitro, as well as within infected THP-1 cells. The presence of these nanoparticles at the bacterial surface prevents their uptake by human macrophages and dendritic cells. When loaded with doxycycline, the nanosystem exerts a potent anti-bacterial effect in planktonic cultures, biofilms, and in M. marinum -infected macrophages. Strikingly, in the M. marinum/ zebrafish infection model, the doxycycline-loaded nanoparticles are associated with a pronounced decrease in the bacterial burden and a high embryo survival rate. These results disclose the proposed MSN nanosystems as a promising alternative for the treatment of M. marinum infection and, presumably, against a broader range of mycobacterial infections. Current treatment options for Mycobacterium marinum infections are extensive and challenging. In this work, authors explore the potential of functionalized mesoporous silica nanoparticles as a carrier for delivery of drugs against M. marinum .

The dentin exposure always leads to dentin hypersensitivity and/or caries. Given the dentin's tubular structure and low mineralization degree, reestablishing an effective biobarrier to stably protect dentin remains significantly challenging. This study reports a versatile dentin surface biobarrier consisting of a mesoporous silica-based epigallocatechin-3-gallate (EGCG)/nanohydroxyapatite delivery system and evaluates its stability on the dentinal tubule occlusion and the ( ) biofilm inhibition. The mesoporous delivery system was fabricated and characterized. Sensitive dentin discs were prepared and randomly allocated to three groups: 1, control group; 2, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) group; and 3, the mesoporous delivery system group. The dentin permeability, dentinal tubule occlusion, acid and abrasion resistance, and biofilm inhibition were determined for 1 week and 1 month. The in vitro release profiles of EGCG, Ca, and P were also monitored. The mesoporous delivery system held the ability to sustainably release EGCG, Ca, and P and could persistently occlude dentinal tubules with acid and abrasion resistance, reduce the dentin permeability, and inhibit the biofilm formation for up to 1 month compared with the two other groups. The system provided prolonged stability to combat oral adverse challenges and served as an effective surface biobarrier to protect the exposed dentin. The establishment of the dentin surface biobarrier consisting of a mesoporous delivery system indicates a promising strategy for the prevention and the management of dentin hypersensitivity and caries after enamel loss.

Antimicrobial photodynamic therapy (aPDT) is increasingly being explored for treatment of periodontitis. Here, we investigated the effect of aPDT on human dental plaque bacteria in suspensions and biofilms in vitro using methylene blue (MB)-loaded poly(lactic-co-glycolic) (PLGA) nanoparticles (MB-NP) and red light at 660 nm. The effect of MB-NP-based aPDT was also evaluated in a clinical pilot study with 10 adult human subjects with chronic periodontitis. Dental plaque samples from human subjects were exposed to aPDT—in planktonic and biofilm phases—with MB or MB-NP (25 µg/mL) at 20 J/cm2 in vitro. Patients were treated either with ultrasonic scaling and scaling and root planing (US + SRP) or ultrasonic scaling + SRP + aPDT with MB-NP (25 µg/mL and 20 J/cm2) in a split-mouth design. In biofilms, MB-NP eliminated approximately 25% more bacteria than free MB. The clinical study demonstrated the safety of aPDT. Both groups showed similar improvements of clinical parameters one month following treatments. However, at three months ultrasonic SRP + aPDT showed a greater effect (28.82%) on gingival bleeding index (GBI) compared to ultrasonic SRP. The utilization of PLGA nanoparticles encapsulated with MB may be a promising adjunct in antimicrobial periodontal treatment.

Background Urinary tract catheters, including Double-J or ureteral stents, are prone to bacterial colonization forming biofilms and leading to asymptomatic bacteriuria. In the context of asymptomatic bacteriuria, endourological procedures causing mucosa-inducing lesions can lead to severe infections. Antibiotic prophylaxis is warranted, yet its efficacy is limited by biofilm formation on stents. Biofilms promote antibiotic tolerance, the capacity of genetically susceptible bacteria to survive a normally lethal dose of antimicrobial therapy. The UROPOT study evaluates the effectiveness of a first-in-type metabolism-based aminoglycoside potentiation for (i) preventing infectious complications of asymptomatic bacteriuria during mucosa lesion-inducing endourological procedures and (ii) assessing its anti-tolerance efficacy. Methods The UROPOT trial is a phase I/II single-center (Lausanne University Hospital (CHUV), Switzerland) randomized double-blinded trial. Over 2 years, patients with asymptomatic Escherichia coli and/or Klebsiella pneumoniae bacteriuria, undergoing endourological procedures, will be randomly allocated to one of three treatment arms (1:1:1 randomization ratio, 30 patients per group) to evaluate the efficacy of mannitol-potentiated low-dose amikacin compared to established standard treatments (ceftriaxone or amikacin standard dose). Patients will be recruited at the CHUV Urology Outpatient Clinic. The primary outcome is the comparative incidence of postoperative urinary tract infections (assessed at 48 h) between the investigational amikacin/mannitol therapy and standard (ceftriaxone or amikacin) antibiotic prophylaxis, defined by specific systemic symptoms and/or positive blood and/or urine culture. Secondary outcomes include assessing microbiological eradication through anti-biofilm activity, sustained microbiological eradication, and mannitol and antibiotics pharmacokinetics in blood and urine. Safety outcomes will evaluate the incidence of adverse events following amikacin/mannitol therapy and postoperative surgical complications at postoperative day 14. Discussion UROPOT tests a novel antimicrobial strategy based on “metabolic potentiation” for prophylaxis enabling aminoglycoside dose reduction and targeting biofilm activity. The anti-biofilm effect may prove beneficial, particularly in patients who have a permanent stent in situ needing recurrent endourological manipulations strategies in preventing infections and achieving sustained microbiological eradication in pre-stented patients. Trial registration The protocol is approved by the local ethics committee (CER-VD, 2023–01369, protocole 2.0) and the Swiss Agency for Therapeutic Products (Swissmedic, 701,676) and is registered on the NIH’s ClinicalTrials.gov (trial registration number: NCT05761405). Registered on March 07, 2023.

Cariogenic oral biofilms cause recurrent dental caries around composite restorations, resulting in unprosperous oral health and expensive restorative treatment. Quaternary ammonium monomers that can be copolymerized with dental resin systems have been explored for the modulation of dental plaque biofilm growth over dental composite surfaces. Here, for the first time, we investigated the effect of bis(2-methacryloyloxyethyl) dimethylammonium bromide (QADM) on human overlying mature oral biofilms grown intra-orally in human participants for 7–14 days. Seventeen volunteers wore palatal devices containing composite specimens containing 10% by mass of QADM or a control composite without QADM. After 7 and 14 days, the adherent biofilms were collected to determine bacterial counts via colony-forming unit (CFU) counts. Biofilm viability, chronological changes, and percentage coverage were also determined through live/dead staining. QADM composites caused a significant inhibition of Streptococcus mutans biofilm formation for up to seven days. No difference in the CFU values were found for the 14-day period. Our findings suggest that: (1) QADM composites were successful in inhibiting 1–3-day biofilms in the oral environment in vivo; (2) QADM significantly reduced the portion of the S. mutans group; and (3) stronger antibiofilm activity is required for the control of mature long-term cariogenic biofilms. Contact-killing strategies using dental materials aimed at preventing or at least reducing high numbers of cariogenic bacteria seem to be a promising approach in patients at high risk of the recurrence of dental caries around composites.