Biofilm Prevention Research Articles

New Strategies for Inhibition of Listeria monocytogenes and Klebsiella pneumoniae Biofilm Formation and Persistence

Objective: This study aimed to find new strategies for the prevention of bacterial biofilms and investigate the effect of some plant extracts on the biofilm formation by certain pathogenic bacterial strains in-vitro.
 Methods: Fourteen different biofilm forming bacterial isolates were collected and their biofilm were quantitatively measured under different temperature, pH and growth medium using Crystal violet staining method. Exopolysaccharides (EPS) produced by the isolates were estimated and a comparison between the tested isolates was made. The effect of some plant extracts on bacterial growth, biofilm formation and exopolysaccharide quantity was determined.
 Results: The two isolates, Listeria monocytogenes (ATCC13932) and Klebsiella pneumoniae (ATCC700613) were among the most active biofilm forming bacterial isolates. The optimum temperature, pH and media for EPS production and biofilm formation were determined. The effect of some plant extracts of Cranberry; Pomegranate peel and, Arak on growth and formation of biofilm and EPS were recorded. Moreover, minimum inhibition concentration of each plant extract was performed.
 Conclusion: EPS quantity produced from the tested isolates depends on some effective factors such temperature, media contents and pH. Aqueous-Cranberry, aqueous-Pomegranate Peel and methanolic-Arak extracts have antibacterial and antibiofilm activities on L. monocytogenes and K. pneumonia. Thus, application of new natural approaches for inhibiting bacterial biofilm is important to prevent persistent and recurrent biofilm related infections.

Seawater-triggered self-renewable amphiphilic coatings with low water swelling and excellent biofilm prevention properties

Amphiphilic antifouling coatings have the potential to revolutionize the field of eco-friendly antifouling coatings, which are still beset by the drawbacks of instability, complex procedures and water swelling. Herein, we construct seawater-triggered amphiphilic coatings with self-renewable capabilities in seawater via the facile casting of a physical mixture of a hydrolyzable polymer (PTMH), hydroxyl-terminated polydimethoxysilane and an isocyanate crosslinker. The hydrophilic chain is generated in situ from PTMH at the coating/seawater interface and thus ultimately mitigates its seawater swelling. The hydrolyzability of PTMH and the degradation of the urethane crosslinking bond endow the coating with self-renewable properties. Owing to the appropriate hydrolysis rate of PTMH, its water absorption is lower than 3 wt% for 30 days of seawater submersion. Notably, the biofilm prevention capability of the coatings is excellent, outperforming that of polydimethoxysilane-based polyurethane and self-polishing polymers in real sea conditions. We envision that this work will provide a new avenue to design microphase separation-based antifouling coatings for practical applications.

Yersinia enterocolitica-Derived Outer Membrane Vesicles Inhibit Initial Stage of Biofilm Formation.

Yersinia enterocolitica (Y. enterocolitica) is an important food-borne and zoonotic pathogen. It can form biofilm on the surface of food, increasing the risk to food safety. Generally, outer membrane vesicles (OMVs) are spherical nanostructures secreted by Gram-negative bacteria during growth. They play a role in biological processes because they contain biologically active molecules. Several studies have reported that OMVs secreted by various bacteria are associated with the formation of biofilms. However, the interactions between Y. enterocolitica OMVs and biofilm are unknown. This study aims to investigate the effect of Y. enterocolitica OMVs on biofilm formation. Firstly, OMVs were extracted from Y. enterocolitica Y1083, which has a strong biofilm-forming ability, at 15 °C, 28 °C and 37 °C and then characterized. The characterization results showed differences in the yield and protein content of three types of OMVs. Next, by co-culturing the OMVs with Y. enterocolitica, it was observed that the OMVs inhibited the initial stage of Y. enterocolitica biofilm formation but did not affect the growth of Y. enterocolitica. Furthermore, biofilm formation by Salmonella enteritidis and Staphylococcus aureus were also inhibited by OMVs. Subsequently, it was proved that lipopolysaccharides (LPS) in OMVs inhibited biofilm formation., The proteins, DNA or RNA in OMVs could not inhibit biofilm formation. Bacterial motility and the expression of the biofilm-related genes pgaABC, motB and flhBD were inhibited by LPS. LPS demonstrated good anti-biofilm activity against various bacteria. This study provides a new approach to the prevention and control of pathogenic bacterial biofilm.

Phage-Based Control of Methicillin Resistant Staphylococcus aureus in a Galleria mellonella Model of Implant-Associated Infection

Staphylococcus aureus implant-associated infections are difficult to treat because of the ability of bacteria to form biofilm on medical devices. Here, the efficacy of Sb-1 to control or prevent S. aureus colonization on medical foreign bodies was investigated in a Galleria mellonella larval infection model. For colonization control assays, sterile K-wires were implanted into larva prolegs. After 2 days, larvae were infected with methicillin-resistant S. aureus ATCC 43300 and incubated at 37 °C for a further 2 days, when treatments with either daptomycin (4 mg/kg), Sb-1 (107 PFUs) or a combination of them (3 x/day) were started. For biofilm prevention assays, larvae were pre-treated with either vancomycin (10 mg/kg) or Sb-1 (107 PFUs) before the S. aureus infection. In both experimental settings, K-wires were explanted for colony counting two days after treatment. In comparison to the untreated control, more than a 4 log10 CFU and 1 log10 CFU reduction was observed on K-wires recovered from larvae treated with the Sb-1/daptomycin combination and with their singular administration, respectively. Moreover, pre-infection treatment with Sb-1 was found to prevent K-wire colonization, similarly to vancomycin. Taken together, the obtained results demonstrated the strong potential of the Sb-1 antibiotic combinatory administration or the Sb-1 pretreatment to control or prevent S. aureus-associated implant infections.

Adhesion energy, spreading coefficient and interfacial tension as an efficient tool for assessing biocide performance

AbstractIt is estimated that microbiologically influenced corrosion (MIC) contributes to 20%–50% of the total cost of corrosion. Biocides are often used to counter MIC. Normally, the choice of a biocide is based on planktonic kill studies as well as cost of the treatment chemicals. Sessile kill studies have significant advantages over planktonic studies but usually take 8 weeks to complete. Furthermore, both sessile kill and planktonic studies only provide phenomenological results and therefore do not explain the mechanisms. To bridge this gap, we have calculated spreading coefficient and work of adhesion from contact angle measurements as a tool for assessing the performance of a biocide. This novel method provides insights into how biocides interact with a biofilm on a surface and is quicker than sessile kill study. Based on surface energy calculations, we have identified parameters that will aide in the prediction of biofilm removal and prevention. For example, surfactants with a positive spreading coefficient on a substrate and having a stronger affinity for the substrate than the biofilm (i.e., a lower interfacial tension) are effective in biofilm prevention. On the other hand, surfactants with a low interfacial tension with a substrate are effective in biofilm removal. Given the diversity of surfaces to which bacteria attach (carbon steel, near wellbore, membrane, etc.), this rapid technique is a good prediction tool for the effectiveness of a biocide application. This paper will provide an overview of the technique and discuss some examples of surfactants that can be used for biofilm removal and prevention.

Chemorepellent-Loaded Nanocarriers Promote Localized Interference of Escherichia coli Transport to Inhibit Biofilm Formation.

To mitigate antimicrobial resistance, we developed polymeric nanocarrier delivery of the chemorepellent signaling agent, nickel, to interfere with Escherichia coli transport to a surface, an incipient biofilm formation stage. The dynamics of nickel nanocarrier (Ni NC) chemorepellent release and induced chemorepellent response required to effectively modulate bacterial transport for biofilm prevention were characterized in this work. Ni NCs were fabricated with the established Flash NanoPrecipitation method. NC size was characterized with dynamic light scattering. Measured with a zincon monosodium salt colorimetric assay, NC nickel release was pH-dependent, with 62.5% of total encapsulated nickel released at pH 7 within 0-15 min, competitive with rapid E. coli transport to the surface. Confocal laser scanning microscopy of E. coli (GFP-expressing) biofilm growth dynamics on fluorescently labeled Ni NC coated glass coupled with a theoretical dynamical criterion probed the biofilm prevention outcomes of NC design. The Ni NC coating significantly reduced E. coli attachment compared to a soluble nickel coating and reduced E. coli biomass area by 61% compared to uncoated glass. A chemical-in-plug assay revealed Ni NCs induced a chemorepellent response in E. coli. A characteristic E. coli chemorepellent response was observed away from the Ni NC coated glass over 10 μm length scales effective to prevent incipient biofilm surface attachment. The dynamical criterion provided semiquantitative analysis of NC mechanisms to control biofilm and informed optimal chemorepellent release profiles to improve NC biofilm inhibition. This work is fundamental for dynamical informed design of biofilm-inhibiting chemorepellent-loaded NCs promising to mitigate the development of resistance and interfere with the transport of specific pathogens.

Prevention and Eradication of Biofilm by Dendrimers: A Possibility Still Little Explored.

Multidrug resistance (MDR) among pathogens and the associated infections represent an escalating global public health problem that translates into raised mortality and healthcare costs. MDR bacteria, with both intrinsic abilities to resist antibiotics treatments and capabilities to transmit genetic material coding for further resistance to other bacteria, dramatically decrease the number of available effective antibiotics, especially in nosocomial environments. Moreover, the capability of several bacterial species to form biofilms (BFs) is an added alarming mechanism through which resistance develops. BF, made of bacterial communities organized and incorporated into an extracellular polymeric matrix, self-produced by bacteria, provides protection from the antibiotics’ action, resulting in the antibiotic being ineffective. By adhering to living or abiotic surfaces present both in the environment and in the healthcare setting, BF causes the onset of difficult-to-eradicate infections, since it is difficult to prevent its formation and even more difficult to promote its disintegration. Inspired by natural antimicrobial peptides (NAMPs) acting as membrane disruptors, with a low tendency to develop resistance and demonstrated antibiofilm potentialities, cationic polymers and dendrimers, with similar or even higher potency than NAMPs and with low toxicity, have been developed, some of which have shown in vitro antibiofilm activity. Here, aiming to incite further development of new antibacterial agents capable of inhibiting BF formation and dispersing mature BF, we review all dendrimers developed to this end in the last fifteen years. The extension of the knowledge about these still little-explored materials could be a successful approach to find effective weapons for treating chronic infections and biomaterial-associated infections (BAIs) sustained by BF-producing MDR bacteria.

Intensification of Polyphenols Extraction from Eryngium creticum Leaves Using Ired-Irrad® and Evaluation of Antibiofilm and Antibacterial Activities.

(1) Background: Eryngium creticum is a plant medicinally valued, and used in pharmacopeia to treat various diseases. No previous studies have been reported on E. creticum leaf extracts using an IR-assisted technique; thus, this study aimed to intensify polyphenol extraction using Ired-Irrad®, comparing it to the conventional water bath (WB) method. (2) Methods: Optimization of polyphenol extraction from E. creticum leaves was conducted using Response Surface Methodology. Ired-Irrad® was used and compared to the WB method. The biological activities (antiradical, antioxidant, antibacterial, and antibiofilm) of both extracts were assessed. UHPLC analysis was performed to analyze the phytochemical profile of both extracts. (3) Results: Under optimal conditions, IR improved the polyphenol extraction yield by 1.7 times, while lowering ethanol consumption by 1.5 times. Regarding the antibacterial activity, both WB and IR E. creticum leaf extracts exhibited the highest antibacterial activity against Staphylococcus epidermidis. The maximum biofilm prevention capacity was also noticed against S. epidermidis. UHPLC-MS analysis quantified two major phenolic compounds in both extracts: rutin and sinapic acid. (4) Conclusions: Ired-Irrad® technology proved to be an effective technique in intensifying polyphenol recovery, while preserving their quantity and quality.

Biofilms: Formation, Research Models, Potential Targets, and Methods for Prevention and Treatment.

Due to the continuous rise in biofilm‐related infections, biofilms seriously threaten human health. The formation of biofilms makes conventional antibiotics ineffective and dampens immune clearance. Therefore, it is important to understand the mechanisms of biofilm formation and develop novel strategies to treat biofilms more effectively. This review article begins with an introduction to biofilm formation in various clinical scenarios and their corresponding therapy. Established biofilm models used in research are then summarized. The potential targets which may assist in the development of new strategies for combating biofilms are further discussed. The novel technologies developed recently for the prevention and treatment of biofilms including antimicrobial surface coatings, physical removal of biofilms, development of new antimicrobial molecules, and delivery of antimicrobial agents are subsequently presented. Finally, directions for future studies are pointed out.

Antibacterial activity of novel linear polyamines against Staphylococcus aureus.

New therapeutic options are urgently required for the treatment of Staphylococcus aureus infections. Accordingly, we sought to exploit the vulnerability of S. aureus to naturally occurring polyamines. We have developed and tested the anti-staphylococcal activity of three novel linear polyamines based on spermine and norspermine. Using a panel of genetically distinct and clinically relevant multidrug resistant S. aureus isolates, including the polyamine resistant USA300 strain LAC, compound AHA-1394 showed a greater than 128-fold increase in inhibition against specific S. aureus strains compared to the most active natural polyamine. Furthermore, we show that AHA-1394 has superior biofilm prevention and biofilm dispersal properties compared to natural polyamines while maintaining minimal toxicity toward human HepG2 cells. We examined the potential of S. aureus to gain resistance to AHA-1394 following in vitro serial passage. Whole genome sequencing of two stable resistant mutants identified a gain of function mutation (S337L) in the phosphatidylglycerol lysyltransferase mprF gene. Inactivation of mutant mprF confirmed the importance of this allele to AHA-1394 resistance. Importantly, AHA-1394 resistant mutants showed a marked decrease in relative fitness and increased generation time. Intriguingly, mprF::S337L contributed to altered surface charge only in the USA300 background whereas increased cell wall thickness was observed in both USA300 and SH1000. Lastly, we show that AHA-1394 displays a particular proclivity for antibiotic potentiation, restoring sensitivity of MRSA and VRSA isolates to daptomycin, oxacillin and vancomycin. Together this study shows that polyamine derivatives are impressive drug candidates that warrant further investigation.

Optimal loading of iron nanoparticles on reverse osmosis membrane surface to reduce biofouling

Biofouling is one of the major challenges for reverse osmosis (RO) membranes. Commercial RO membranes coated with different loadings (0.1, 0.3, 0.5, 0.7 and 0.9 wt% in solution) of iron nanoparticles (nZVI or FeNPs) were submitted to accelerated biofouling under high-pressure crossflow, using pretreated, sterilized seawater from the Sea of Cortez (Mexico) inoculated with Bacillus halotolerans MCC1 (109 CFU mL−1) as feed. FeNPs were synthesized and characterized (zeta potential, XRD, and SEM) via a reduction method. Coated and uncoated membranes were characterized for roughness, contact angle, surface free energy, XPS, FESEM, water and salt permeance, membrane resistance, fouled flux and fouling resistance. The coated membranes showed a slight increase in roughness and contact angle and a reduction in surface free energy. After accelerated biofouling, the coated membranes showed lower hydraulic resistance and higher permeate flux than the uncoated membrane, albeit with slightly lower salt rejection. Moreover, membrane autopsies revealed a highly significant reduction in biofilm thickness, total cells, TOC, and live and dead cells for the coated membranes relative to the uncoated membrane. The FeNP loading using a 0.3 wt% solution was found to be the most suitable tradeoff between biofilm prevention and flux decline for desalination processes.

Flexible biofilm monitoring device.

Biofilms and their analysis are increasingly attracting the attention of the scientific community due to the immense importance and impact of biofilms in various natural, technical and medical fields. For these purposes, an optimized and extended antibiofilm assay system based on the Calgary Biofilm Device (MBEC Assay® system) consisting of microtiter plate and PCR tubes was established. Its implementation was used to study the growth characteristics of the sessile phenotype of Pseudomonas fluorescens exposed to antimicrobial peptides. Inhibitory effects of an antimicrobial peptide on P. fluorescens biofilm formation could be determined at a concentration of 250μg/ml (biofilm prevention concentration (BPC)) using the modified biofilm assay. Similarly, the biofilm bactericidal concentration (BBC) at 125μg/ml and the minimum biofilm elimination concentration to remove 90% of the total biofilm mass (MBEC90) were measured at a concentration range of 15.625-1.95μg/ml. In conclusion, this optimized system provides a highly variable, simple, and cost-effective alternative to high-throughput screening based on the Calgary Biofilm Device (CBD).

Vancomycin Containing PDLLA and PLGA/β-TCP Inhibit Biofilm Formation but Do Not Stimulate Osteogenic Transformation of Human Mesenchymal Stem Cells.

AimsChronic osteomyelitis, including implant-related prosthetic joint infection, is extremely difficult to cure. We develop vancomycin containing release systems from poly(d,l-lactide) (PDLLA) and poly(d,l-lactide-co-glycolide) (PLGA) composites with beta-tricalcium phosphate (β-TCP) to treat methicillin-resistant Staphylococcus aureus osteomyelitis. We ask whether vancomycin containing PDLLA/β-TCP and PLGA/β-TCP composites will prevent early biofilm formation, allow cell proliferation and osteogenic differentiation, and stimulate osteogenic signaling molecules in the absence of an osteogenic medium.MethodsComposites were produced and characterized with scanning electron microscopy. In vitro vancomycin release was assessed for 6 weeks. Biofilm prevention was calculated by crystal violet staining. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and osteosarcoma cell (SaOS-2) proliferation and differentiation were assessed with water soluble tetrazolium salt and alkaline phosphatase (ALP) staining. Real-time quantitative polymerase chain reaction defined osteogenic signaling molecules for hBM-MSCs.ResultsTotally, 3.1 ± 0.2 mg and 3.4 ± 0.4 mg vancomycin released from PDLLA/β-TCP and the PLGA/β-TCP composites, respectively, and inhibited early biofilm formation. hBM-MSCs and SaOS-2 cells proliferated on the composites and stimulated ALP activity of cells. Runt-related transcription factor 2 (RUNX2) and SRY-Box transcription Factor 9 (SOX9) expressions were, however, lower with composites when compared with control.ConclusionVancomycin containing PDLLA/β-TCP and PLGA/β-TCP composites inhibited early biofilm formation and proliferated and differentiated hBM-MSCs and SaOS-2 cells, but osteogenesis-related RUNX2 and SOX9 transcription factors were not strongly expressed in the absence of an osteogenic medium for 14 days.

Characterization of the Lytic Phage Flora With a Broad Host Range Against Multidrug-Resistant Escherichia coli and Evaluation of Its Efficacy Against E. coli Biofilm Formation.

Escherichia coli is a gram-negative bacterium that is distributed widely throughout the world; it is mainly found in contaminated food, the poultry industry, and animal feces. The emergence of antibiotic-resistant E. coli poses a threat to human and animal health, which has led to renewed interest in phage-based therapy. E. coli biofilm control and prevention are of great importance. In this study, the isolated phages Flora and KM18 were found to belong to the family Myoviridae; the optimal preservation buffer was pH = 6~7, and the phage genome sizes were 168,909 (Flora) and 168,903 (KM18) bp. Phage Flora had a broader lytic spectrum than KM18. Phage Flora had a better antibiofilm effect than kanamycin sulfate in high-concentration E. coli cultures. A combination of the phage Flora and kanamycin sulfate showed better antibiofilm effects than Flora or kanamycin sulfate alone in low-concentration E. coli cultures. These characteristics can serve as a guideline for the selection of effective candidates for phage therapy, in this case antibiotic-resistant E. coli control in the poultry industry.

Cell Cycle Progression Influences Biofilm Formation in Saccharomyces cerevisiae 1308.

ABSTRACTBiofilm-immobilized continuous fermentation is a novel fermentation strategy that has been utilized in ethanol fermentation. Continuous fermentation contributes to the self-proliferation of Saccharomyces cerevisiae biofilms. Previously, we successfully described the cell cycle differences between biofilm-immobilized fermentation and calcium alginate-immobilized fermentation. In the present study, we investigated the relationship between biofilm formation and the cell cycle. We knocked down CLN3, SIC1, and ACE2 and found that Δcln3 and Δsic1 exhibited a predominance of G2/M phase cells, increased biofilm formation, and significantly increased extracellular polysaccharide formation and expression of genes in the FLO gene family during immobilisation fermentation. Δace2 exhibited a contrasting performance. These findings suggest that the increase in the proportion of cells in the G2/M phase of the cell cycle facilitates biofilm formation and that the cell cycle influences biofilm formation by regulating cell adhesion and polysaccharide formation. This opens new avenues for basic research and may also help to provide new ideas for biofilm prevention and optimization.IMPORTANCE Immobilised fermentation can be achieved using biofilm resistance, resulting in improved fermentation efficiency and yield. The link between the cell cycle and biofilms deserves further study since reports are lacking in this area. This study showed that the ability of Saccharomyces cerevisiae to produce biofilm differed when cell cycle progression was altered. Further studies suggested that cell cycle regulatory genes influenced biofilm formation by regulating cell adhesion and polysaccharide formation. Findings related to cell cycle regulation of biofilm formation set the stage for biofilm in Saccharomyces cerevisiae and provide a theoretical basis for the development of a new method to improve biofilm-based industrial fermentation.

SUB GINGIVAL COMPLEX MICROBIAL BIOFILMS AND THEIR ERADICATION

Aim of study: The study was conducted to discuss and explain the ability to use a new methods for removal and even prevention of sub gingival biofilm. Biofilm is a three dimensional structure microbial community that first noted in the oral cavity as plaque on the crown of teeth which is called later supra gingival plaque to play a role in caries development . Un eliminated supra gingival plaque grow sub gingivaly to form more protected biofilm found in the gingival sulcus that is responsible for the conversion of physiological gingival sulcus (0-3 mm) to pathological pocket > 3mm which play a role in the pathogenesis of different periodontal infections. Sub-gingival microbiota include mainly anaerobic proteolytic gram negative rods, these microorganisms considered as late colonizer which required the early colonizer to adhere primarily producing specific receptors, reducing the oxidation reduction potential (Eh ), cooperate metabolically within the biofilm. The study was based on a review submitted by: Cortés, et.al. (2011) titled “ Biofilm formation, control and novel strategies for eradication. The study took place during the period (Oct. 2022 - Feb. 2023) The challenges which faces oral microbiologist were characterizing the composition of sub gingival bacterial biofilm since most of them are un cultivable hence new molecular technique were facilitate these difficulties which in turn open horizons for eradication of these resistant microbial biofilm bacterial cells for novel treatment strategies and to prevent recurrence of periodontal infections.

Assessment of Phage-Mediated Inhibition and Removal of Multidrug-Resistant Pseudomonas aeruginosa Biofilm on Medical Implants.

PurposeDespite the growing interest in bacteriophage (phage) usage for the prevention, control, and removal of bacterial biofilms, few scientific data exist on phage applications on medical implant surfaces, while none exists on multiple implants. In this study, we aimed to isolate, biophysically characterize and assess phages as potential antibiofilm agents to inhibit and remove multidrug-resistant (MDR) Pseudomonas aeruginosa biofilm on catheter and endotracheal tube surfaces.MethodsThe well-identified stored clinical isolates (n = 7) of MDR P. aeruginosa were obtained from Jimma Medical Center. Specific phages were isolated and characterized based on standard protocols. The phages were tested for their antibiofilm effects in preventing colonization and removing preformed biofilms of MDR P. aeruginosa, following phage coating and treatment of catheter and endotracheal tube segments.ResultsTwo P. aeruginosa-specific phages (ΦJHS-PA1139 and ΦSMK-PA1139) were isolated from JMC compound sewage sources. The phages were biophysically characterized as being thermally stable up to 40°C and viable between pH 4.0 and 11.0. The two phages tested against clinical MDR strains of P. aeruginosa showed broad host ranges but not on other tested bacterial species. Both phages reduced MDR bacterial biofilms during the screening step. The phage-coated segments showed 1.2 log10 up to 3.2 log10 inhibition relative to non-coated segments following 6 h coating of segments prior to microbial load exposure. In both phages, 6 h treatment of the segments with 106 PFU/mL yielded 1.0 log10 up to 1.6 log10 reductions for ΦJHS and 1.6 log10 up to 2.4 log10 reductions for ΦSMK.ConclusionOur results suggest that phages have great potential to serve the dual purpose as surface coating agents for preventing MDR bacterial colonization in medical implants and as biofilm removal agents in implant-associated infections.

Clearance of mixed biofilms of Streptococcus pneumoniae and methicillin-susceptible/resistant Staphylococcus aureus by antioxidants N-acetyl-l-cysteine and cysteamine

Biofilm-associated infections are of great concern because they are associated with antibiotic resistance and immune evasion. Co-colonization by Staphylococcus aureus and Streptococcus pneumoniae is possible and a threat in clinical practice. We investigated the interaction between S. aureus and S. pneumoniae in mixed biofilms and tested new antibiofilm therapies with antioxidants N-acetyl-l-cysteine (NAC) and cysteamine (Cys). We developed two in vitro S. aureus–S. pneumoniae mixed biofilms in 96-well polystyrene microtiter plates and we treated in vitro biofilms with Cys and NAC analyzing their effect by CV staining and viable plate counting. S. pneumoniae needed a higher proportion of cells in the inoculum and planktonic culture to reach a similar population rate in the mixed biofilm. We demonstrated the effect of Cys in preventing S. aureus biofilms and S. aureus–S. pneumoniae mixed biofilms. Moreover, administration of 5 mg/ml of NAC nearly eradicated the S. pneumoniae population and killed nearly 94% of MSSA cells and 99% of MRSA cells in the mixed biofilms. The methicillin resistance background did not change the antioxidants effect in S. aureus. These results identify NAC and Cys as promising repurposed drug candidates for the prevention and treatment of mixed biofilms by S. pneumoniae and S. aureus.

Identification and characterization of novel endolysins targeting Gardnerella vaginalis biofilms to treat bacterial vaginosis

Bacterial vaginosis (BV) is a recurrent dysbiosis that is frequently associated with preterm birth, increased risk for acquisition of human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs). The overgrowth of a key pathobiont, Gardnerella vaginalis, as a recalcitrant biofilm is central to the development of this dysbiosis. Overgrowth of vaginal biofilms, seeded by initial G. vaginalis colonization, leads to recurrent symptomatic BV which is poorly resolved by classically used antibiotics. In this light, the use of bacteriophages and/or their proteins, represents a promising alternative. Here we identify 84 diverse anti-Gardnerella endolysins across 7 protein families. A subset of 36 endolysin candidates were refactored and overexpressed in an E. coli BL21 (DE3) system and 5 biochemically and structurally diverse endolysins were fully characterized. Each candidate endolysin showed good lytic activity against planktonic G. vaginalis ATCC14018, as well as G. vaginalis clinical isolates. These endolysin candidates were assayed in biofilm prevention and disruption assays, with biofilm disruption at low microgram concentrations (5 μg/ml) observed. In addition to clonal G. vaginalis biofilms, endolysin candidates could also successfully disrupt polyspecies biofilms. Importantly, none of our candidates showed lytic activity against commensal lactobacilli present in the vaginal microbiota such as L. crispatus, L. jensenii, L. gasseri, and L. iners or against Atopobium vaginae (currently classified as Fannyhessa vaginae). The potency and selectivity of these novel endolysins constitute a promising alternative treatment to combat BV, avoiding problems associated with antibiotic resistance, while retaining beneficial commensal bacteria in the vaginal flora. The diverse library of candidates reported here represents a strong repository of endolysins for further preclinical development.

Inhibitory effects of a water-soluble jujube polysaccharide against biofilm-forming oral pathogenic bacteria

Oral diseases caused by infectious pathogens raises significant concerns in public health. In the light of side effects of current antibiotics therapy and growing drug resistance of pathogenic bacteria, natural products have become attractive alternatives for antibiotics agents in dental practice. This current study investigated the effects of polysaccharides extracted from Zizyphus jujuba Mill. on three major oral biofilm-forming pathogenic bacteria including caries-inducing Streptococcus mutans, lesions-causing MRSA, and periodontitis-related Porphyromonas gingivalis, as well as general oral microbiota. Our results demonstrated that jujube polysaccharide prepared in this study was mainly composed by galacturonic acid with an average molecular weight 242 kDa, which were further characterized for structural features by FT-IR spectra and NMR spectroscopy analysis. This jujube polysaccharide was shown to exhibit remarkable inhibitory effects against all the tested oral bacterial pathogens through various mechanisms including growth inhibition, biofilm prevention and disruption, intervention of bacterial infection (adhesion and invasion), attenuation of cytotoxicity, modulation of excessive inflammatory response of LPS-stimulated and MRSA-infected macrophages as well as positive regulation of oral microbiota. The present study paves the way to explore jujube polysaccharides for the prevention and treatment of oral infectious diseases.Graphic Abstract.