Reduced Biofilm Biomass Research Articles

Proteomic analysis of the food spoiler Pseudomonas fluorescens ITEM 17298 reveals the antibiofilm activity of the pepsin-digested bovine lactoferrin

Pseudomonas fluorescens is implicated in food spoilage especially under cold storage. Due to its ability to form biofilm P. fluorescens resists to common disinfection strategies increasing its persistance especially across fresh food chain. Biofilm formation is promoted by several environmental stimuli, but gene expression and protein changes involved in this lifestyle are poorly investigated in this species.In this work a comparative proteomic analysis was performed to investigate metabolic pathways of underlying biofilm formation of the blue cheese pigmenting P. fluorescens ITEM 17298 after incubation at 15 and 30 °C; the same methodology was also applied to reveal the effects of the bovine lactoferrin hydrolysate (HLF) used as antibiofilm agent.At 15 °C biofilm biomass and motility increased, putatively sustained by the induction of regulators (PleD, AlgB, CsrA/RsmA) involved in these phenotypic traits. In addition, for the first time, TycC and GbrS, correlated to indigoidine synthesis (blue pigment), were detected and identified. An increase of virulence factors amounts (leukotoxin and PROKKA_04561) were instead found at 30 °C. HLF caused a significant reduction in biofilm biomass; indeed, at 15 °C HLF repressed PleD, TycC and GbrS and induced the negative regulators of alginate biosynthesis; at both temperatures induced the cyclic-di-GMP-binding biofilm dispersal mediator (PROKKA_02061).In conclusion, in this work protein determinats of biofilm formation were revelead in ITEM 17298 under the low temperature; the synthesis of these latter were inhibited by HLF confirming its possible exploitation as antibiofilm agent for biotechnological applications in cold stored foods.

Antimicrobial activity of rhodomyrtone isolated from Rhodomyrtus tomentosa (Aiton) Hassk

Rhodomyrtone was isolated from the leaves of Rhodomyrtus tomentosa (Aiton) Hassk grown in Vietnam using chromatographic methods. Its chemical structure was confirmed by means of spectroscopic data analysis. The pH drop measurement, enzyme activity assays and fluorescence stain were used to examine rhodomyrtone anticaries activity. It was found that rhodomyrtone suppressed acid production by Streptococcus mutans, a major cariogenic agent in human by inhibiting enzyme activities responsible for acid production and tolerance, including membrane bound enzymes F-ATPase and phosphotransferase system (PTS), as well as glycolysis enzymes glyceraldehyphosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) in cytoplasm with the IC50 values of 24 μM, 19 μM, 23 μM and 28 μM, respectively. Moreover, 50 μM rhodomyrtone reduced biofilm biomass formed by S. mutans up to 59% (p < 0.05). Fluorescent images indicated that cells on the biofilms were significantly killed. Thus, rhodomyrtone is a new and potential anticaries agent against S. mutans.

Safety and efficacy of a bacteriophage cocktail in an in vivo model of Pseudomonas aeruginosa sinusitis

Pseudomonas aeruginosa (PA) is a bacterial pathogen that frequently displays antibiotic resistance. Its presence within the sinuses of chronic rhinosinusitis sufferers is associated with poorer quality of life. Obligately lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death. The aims of this study were to assess the safety and efficacy of a PA phage cocktail (CT-PA) in a sheep model of rhinosinusitis. The sheep rhinosinusitis model was adapted to simulate PA infection in sheep frontal sinuses. To assess efficacy, after a 7-day biofilm formation period, sheep received twice-daily frontal trephine flushes of CT-PA or saline for 1 week. Biofilm quantitation on frontal sinus mucosa was performed using LIVE/DEAD BacLight staining. To assess safety, sheep received twice-daily frontal trephine flushes of CT-PA or vehicle control for 3 weeks. Blood and fecal samples were collected throughout treatment. Histopathology of frontal sinus, lung, heart, liver, spleen, and kidney tissue was performed. Sinus cilia were visualized using scanning electron microscopy (SEM). The Efficacy arm showed a statistically significant reduction in biofilm biomass with all concentrations of CT-PA tested (P < 0.05). Phage presence in sinuses was maintained for at least 16hours after the final flush. All Safety arm sheep completed 3 weeks of treatment. Phage was detected consistently in feces and sporadically in blood and organ samples. Histology and SEM of tissues revealed no treatment-related damage. In conclusion, CT-PA was able to decrease sinus PA biofilm at concentrations of 108-1010 PFU/mL. No safety concerns were noted.

Antimicrobial and Antibiofilm Activities of Citrus Water-Extracts Obtained by Microwave-Assisted and Conventional Methods.

Citrus pomace is a huge agro-food industrial waste mostly composed of peels and traditionally used as compost or animal feed. Owing to its high content of compounds beneficial to humans (e.g., flavonoids, phenol-like acids, and terpenoids), citrus waste is increasingly used to produce valuable supplements, fragrance, or antimicrobials. However, such processes require sustainable and efficient extraction strategies by solvent-free techniques for environmentally-friendly good practices. In this work, we evaluated the antimicrobial and antibiofilm activity of water extracts of three citrus peels (orange, lemon, and citron) against ten different sanitary relevant bacteria. Both conventional extraction methods using hot water (HWE) and microwave-assisted extraction (MAE) were used. Even though no extract fully inhibited the growth of the target bacteria, these latter (mostly pseudomonads) showed a significant reduction in biofilm biomass. The most active extracts were obtained from orange and lemon peel by using MAE at 100 °C for 8 min. These results showed that citrus peel water infusions by MAE may reduce biofilm formation possibly enhancing the susceptibility of sanitary-related bacteria to disinfection procedures.

Antibacterial and antibiofilm activity of carvacrol against Salmonella enterica serotype Typhimurium

The present study evaluated the antibacterial and antibiofilm activity of carvacrol against Salmonella Typhimurium. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined and the time-kill curve and scanning electron microscopy (SEM) were performed to evaluate antibacterial activity. Antibiofilm activity was evaluated by quantifying total biomass using crystal violet assay, and metabolic activity was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The action of carvacrol against preformed biofilm on polypropylene and stainless steel was also evaluated by colony counting and SEM. The MIC and MBC was 312 µg mL-1. Carvacrol at MIC and 2 x MIC eliminated cells after 6 and 1 h of treatment, respectively, as exhibited in the time-kill curve. The greatest reduction in biofilm biomass and metabolic activity was 1,719 OD550 and 0,089 OD550 respectively, both at 4 x MIC of carvacrol. In carvacrol treated biofilms of S. Typhimurium on polypropylene, a reduction of 5.12 log was observed with 4 x MIC, while on stainless steel, carvacrol at 4 x MIC reduced bacterial counts by 5 log. The results showed that carvacrol exhibits antibacterial activity and can be used as an alternative for the control of S. Typhimurium biofilms.

Nitric Oxide-Loaded Antimicrobial Polymer for the Synergistic Eradication of Bacterial Biofilm.

Bacterial biofilms are often difficult to treat and represent the main cause of chronic and recurrent infections. In this study, we report the synthesis of a novel antimicrobial/antibiofilm polymer that consists of biocompatible oligoethylene glycol, hydrophobic ethylhexyl, cationic primary amine, and nitric oxide (NO)-releasing functional groups. The NO-loaded polymer has dual-action capability as it can release NO which triggers the dispersion of biofilm, whereas the polymer can induce bacteria cell death via membrane wall disruption. By functionalizing the polymers with NO, we observed a synergistic effect in biofilm dispersal, planktonic and biofilm killing activities against Pseudomonas aeruginosa. The NO-loaded polymer results in 80% reduction in biofilm biomass and kills >99.999% of planktonic and biofilm P. aeruginosa cells within 1 h of treatment at a polymer concentration of 64 μg mL-1. To achieve this synergistic effect, it is imperative that the NO donors and antimicrobial polymer exist as a single chemical entity, instead of a cocktail physical mixture of two individual components. The excellent antimicrobial/antibiofilm activity of this dual-action polymer suggests the advantages of combination therapy in combating bacterial biofilms.

Efficacy of sodium dodecyl sulphate and natural extracts against E. coli biofilm

The aim of this study was to determine and compare the efficacy of a standard cleaning agent, sodium dodecyl sulphate, and natural extracts from pomegranate peel grape skin and bay laurel leaf against E. coli biofilm. The biofilm was exposed for 10 minutes to three different concentrations of each tested compound. The results show that bay laurel leaf extract is the most efficient with 43% biofilm biomass reduction, followed by pomegranate peel extract (35%); sodium dodecyl sulphate and grape skin extract each have 30% efficacy. Our study demonstrated that natural extracts from selected plants have the same or even better efficacy against E. coli biofilm removal from surfaces than the tested classical cleaning agent do. All this indicates that natural plant extracts, which are acceptable from the health and environment points of view, can be potential substitutes for classical cleaning agents.

Novel bioactive peptides from PD-L1/2, a type 1 ribosome inactivating protein from Phytolacca dioica L. Evaluation of their antimicrobial properties and anti-biofilm activities

Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis.

Effect of dental monomers and initiators on Streptococcus mutans oral biofilms

ObjectiveResin-based composites are known to elute leachables that include unincorporated starting materials. The objective of this work was to determine the effect of common dental monomers and initiators on Streptococcus mutans biofilm metabolic activity and biomass. MethodsS. mutans biofilms were inoculated in the presence of bisphenol A glycerolate dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA), camphorquinone (CQ), and ethyl 4-(dimethylamino)benzoate (4E) at 0.01μg/mL up to 500μg/mL, depending on the aqueous solubility of each chemical. Biofilms were evaluated at 4h and 24h for pH (n=3–8), biomass via crystal violet (n=12), metabolic activity via tetrazolium salt (n=12), and membrane permeability for selected concentrations via confocal microscopy (n=6). Parametric and non-parametric statistics were applied. Results500μg/mL TEGDMA reduced 24h metabolic activity but not biomass, similar to prior results with leachables from undercured BisGMA-TEGDMA polymers. 50μg/mL BisGMA reduced biofilm biomass and activity, slightly delayed the pH drop, and decreased the number of cells with intact membranes. 100μg/mL CQ delayed the pH drop and metabolic activity at 4h but then significantly increased the 24h metabolic activity. 4E had no effect up to 10μg/mL. SignificanceMonomers and initiators that leach from resin composites affect oral bacterial biofilm growth in opposite ways. Leachables, which can be released for extended periods of time, have the potential to alter oral biofilm biomass and activity and should be considered in developing and evaluating new dental materials.

Identification and functional characterization of type II toxin/antitoxin systems in Aggregatibacter actinomycetemcomitans.

SummaryType II toxin/antitoxin (TA) systems contribute to the formation of persister cells and biofilm formation for many organisms. Aggregatibacter actinomycetemcomitans thrives in the complex oral microbial community subjected to continual environmental flux. Little is known regarding the presence and function of type II TA systems in this organism or their contribution to adaptation and persistence in the biofilm. We identified 11 TA systems that are conserved across all seven serotypes of A. actinomycetemcomitans and represent the RelBE, MazEF and HipAB families of type II TA systems. The systems selectively responded to various environmental conditions that exist in the oral cavity. Two putative RelBE‐like TA systems, D11S_1194‐1195 and D11S_1718‐1719 were induced in response to low pH and deletion of D11S_1718‐1719 significantly reduced metabolic activity of stationary phase A. actinomycetemcomitans cells upon prolonged exposure to acidic conditions. The deletion mutant also exhibited reduced biofilm biomass when cultured under acidic conditions. The D11S_1194 and D11S_1718 toxin proteins inhibited in vitro translation of dihydrofolate reductase (DHFR) and degraded ribosome‐associated, but not free, MS2 virus RNA. In contrast, the corresponding antitoxins (D11S_1195 and D11S_1719), or equimolar mixtures of toxin and antitoxin, had no effect on DHFR production or RNA degradation. Together, these results suggest that D11S_1194‐1195 and D11S_1718‐1719 are RelBE‐like type II TA systems that are activated under acidic conditions and may function to cleave ribosome‐associated mRNA to inhibit translation in A. actinomycetemcomitans. In vivo, these systems may facilitate A. actinomycetemcomitans adaptation and persistence in acidic local environments in the dental biofilm.

Enhancing the antimicrobial and antibiofilm effectiveness of silver nanoparticles prepared by green synthesis.

The use of polyphenol-rich plant extracts is well established for the green synthesis of silver nanoparticles (AgNPs). However, the size of the AgNPs varies substantially depending on the extract used and many researchers report sizes above 20 nm, which are not optimal for antimicrobial activity. Herein, using catechin as a model polyphenol, we have explored two techniques to improve its stabilising capacity and therefore decrease the subsequent AgNP size: cross-linking catechin with sodium tetraborate (borax); and preparation of a water soluble oligomer from catechin (polycat). The prepared AgNPs from the three stabilising systems, cat@AgNPs, cat-borax@AgNPs and polycat@AgNPs, were characterised by UV-Vis spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS). Cat-borax produced smaller AgNPs (18.4 nm) than catechin (42.3 nm) but the smallest particles were prepared with polycat (8.5 nm). Antimicrobial efficacy was assessed against Gram positive and Gram negative bacteria and was compared with 10 nm sodium citrate capped AgNPs (citrate@AgNPs). Polycat@AgNPs showed superior antimicrobial activity to cat@AgNPs and cat-borax@AgNPs as well as citrate@AgNPs, exhibiting MICs of only 1.25 μg mL-1 (Ag) for Pseudomonas aeruginosa and Acinetobacter baumannii. Polycat@AgNPs also demonstrated substantially enhanced antibiofilm activity. An Ag concentration of only 5 μg mL-1, was sufficient for a 99.9% reduction in biofilm cell viability and a 99.1% reduction in biofilm biomass with polycat@AgNPs. Uptake of polycat@AgNPs by bacteria was determined to be significantly higher than for citrate@AgNPs and tomographic and SEM images showed evidence of destruction of bacteria cells by polycat@AgNPs.

Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability.

Pyocyanin secreted by Pseudomonas aeruginosa is a virulence factor that damages epithelial cells during infection through the action of reactive oxygen species, however, little is known about its direct effect on biofilms. We demonstrated that pyocyanin-producing P. aeruginosa strains (PA14WT, DKN370, AES-1R, and AES-2) formed robust biofilms in contrast to the poorly formed biofilms of the pyocyanin mutant PA14ΔphzA-G and the low pyocyanin producer AES-1M. Addition of DNase I and reduced glutathione (GSH) significantly reduced biofilm biomass of pyocyanin-producing strains (P < 0.05) compared to non-pyocyanin producers. Subsequently we showed that a combined treatment comprising: GSH + DNase I + antibiotic, disrupted and reduced biofilm biomass up to 90% in cystic fibrosis isolates AES-1R, AES-2, LESB58, and LES431 and promoted lung epithelial cell (A549) recovery and growth. We also showed that exogenously added GSH restored A549 epithelial cell glutathione reductase activity in the presence of pyocyanin through recycling of GSSG to GSH and consequently increased total intracellular GSH levels, inhibiting oxidative stress, and facilitating cell growth and confluence. These outcomes indicate that GSH has multiple roles in facilitating a return to normal epithelial cell growth after insult by pyocyanin. With increased antibiotic resistance in many bacterial species, there is an urgency to establish novel antimicrobial agents. GSH is able to rapidly and comprehensively destroy P. aeruginosa associated biofilms while at a same time assisting in the recovery of host cells and re-growth of damaged tissue.

Antibacterial activity of a new lectin isolated from the marine sponge Chondrilla caribensis

A new lectin from the marine sponge Chondrilla caribensis (CCL) was isolated by affinity chromatography in Sepharose 6B media. CCL is a homotetrameric protein formed by subunits of 15,445 ±2Da. The lectin showed affinity for disaccharides containing galactose and mucin. Mass spectrometric analysis revealed about 50% of amino acid sequence of CCL, which showed similarity with a lectin isolated from Aplysina lactuca. Secondary structure consisted of 10% α-helix, 74% β-sheet/β-turn and 16% coil, and this profile was unaltered in a broad range of pH and temperatures. CCL agglutinated Staphylococcus aureus, S epidermidis and Escherichia coli, and it was able to reduce biofilm biomass, but showed no inhibition of planktonic growth of these bacteria. CCL activity was inhibited by α-lactose, indicating that Carbohydrate Recognition Domain (CRD) of the lectin was involved in antibiofilm activity.

Antibacterial activity of a novel antimicrobial peptide [W7]KR12-KAEK derived from KR-12 against Streptococcus mutans planktonic cells and biofilms

The aims of this study were to describe the synthesis of a novel synthetic peptide based on the primary structure of the KR-12 peptide and to evaluate its antimicrobial and anti-biofilm activities against Streptococcus mutans. The antimicrobial effect of KR-12 and [W7]KR12-KAEK was assessed by determining the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. The evaluation of anti-biofilm activity was assessed through total biomass quantification, colony forming unit counting and scanning electron microscopy. [W7]KR12-KAEK showed MIC and MBC values ranging from 31.25 to 7.8 and 62.5 to 15.6 μg ml−1, respectively. Furthermore, [W7]KR12-KAEK significantly reduced biofilm biomass (50–100%). Regarding cell viability, [W7]KR12-KAEK showed reductions in the number of CFUs at concentrations ranging from 62.5 to 7.8 μg ml−1 and 500 to 62.5 μg ml−1 with respect to biofilm formation and preformed biofilms, respectively. SEM micrographs of S. mutans treated with [W7]KR12-KAEK suggested damage to the bacterial surface. [W7]KR12-KAEK is demonstrated to be an antimicrobial agent to control microbial biofilms.

Activity of Bacteriophages in Removing Biofilms of Pseudomonas aeruginosa Isolates from Chronic Rhinosinusitis Patients.

Introduction: Pseudomonas aeruginosa infections are prevalent amongst chronic rhinosinusitis (CRS) sufferers. Many P. aeruginosa strains form biofilms, leading to treatment failure. Lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death.Aim: To assess the activity of a phage cocktail in eradicating biofilms of ex vivo P.aeruginosa isolates from CRS patients.Methods: P. aeruginosa isolates from CRS patients with and without cystic fibrosis (CF) across three continents were multi-locus sequence typed and tested for antibiotic resistance. Biofilms grown in vitro were treated with a cocktail of four phages (CT-PA). Biofilm biomass was measured after 24 and 48 h, using a crystal violet assay. Phage titrations were performed to confirm replication of the phages. A linear mixed effects model was applied to assess the effects of treatment, time, CF status, and multidrug resistance on the biomass of the biofilm.Results: The isolates included 44 strain types. CT-PA treatment significantly reduced biofilm biomass at both 24 and 48 h post-treatment (p < 0.0001), regardless of CF status or antibiotic resistance. Biomass was decreased by a median of 76% at 48 h. Decrease in biofilm was accompanied by a rise in phage titres for all except one strain.Conclusion: A single dose of phages is able to significantly reduce biofilms formed in vitro by a range of P.aeruginosa isolates from CRS patients. This represents an exciting potential and novel targeted treatment for P. aeruginosa biofilm infections and multidrug resistant bacteria.

Low density polyethylene functionalized with antibiofilm compounds inhibits Escherichia coli cell adhesion.

The present work concerns an efficient strategy to obtain novel medical devices materials able to inhibit biofilm formation. The new materials were achieved by covalent grafting of p-aminocinnamic or p-aminosalicylic acids on low density polyethylene coupons. The polyethylene surface, previously activated by oxygen plasma treatment, was functionalized using 2-hydroxymethylmetacrylate as linker. The latter was reacted with succinic anhydride affording the carboxylic end useful for the immobilization of the antibiofilm molecules. The modified surface was characterized by scanning electron microscope, X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared and fluorescence analyses. The antibiofilm activity of the modified materials were tested against Escherichia coli biofilm grown in the Center of Disease Control biofilm reactor. The results revealed that the grafted cinnamic and salicylic acid derivatives reduced biofilm biomass, in comparison with the control, by 73.7 ± 10.7% and 63.4 ± 7.1%, respectively. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3251-3261, 2017.

Antibiofilm activity and mode of action of DMSO alone and its combination with afatinib against Gram-negative pathogens.

Biofilms are complex microbial communities that tend to attach to either biotic or abiotic surface. Enclosed in a self-produced extracellular polymeric substance (EPS) matrix, the biofilms often cause persistent infections. The objective of this study was to investigate the antibiofilm activity of dimethyl sulfoxide (DMSO) and afatinib against Gram-negative pathogens. Test microorganisms used in this study were Escherichia coli ATCC 1299, Pseudomonas aeruginosa ATCC 10145, and Salmonella typhimurium ATCC 14028. Biofilms were developed in 96-well microplate at 37°C for 24h. Following removal of non-adherent cells, analysis of biofilm viability, biofilm biomass, and extracellular polymeric substances (EPS) matrix were performed using resazurin assay, crystal violet assay, and attenuated total reflectance fourier transform infrared (ATR-FTIR) spectroscopy, respectively. Bradford protein assay was conducted to determine the total amount of EPS proteins. The results demonstrated that both 32% DMSO alone and its combination with 3.2μg/mL afatinib were effective in killing biofilm cells and reducing biofilm biomass. IR spectral variations of EPS matrix of biofilms in the range between 1700 and 900cm-1 were also observed. Reduction in EPS proteins verified the chemical modifications of EPS matrix. In conclusion, 32% DMSO alone and its combination with 3.2μg/mL afatinib showed remarkable antibiofilm activities against Gram-negative pathogens. It was suggested that the biofilm inhibition was mediated by the chemical modification of EPS matrix.

Effect of different antibiotics on biofilm produced by uropathogenic Escherichia coli isolated from children with urinary tract infection.

Recurrent urinary tract infections (UTIs) occur frequently in children and women. Intracellular bacterial communities (IBCs) and biofilm formation by Escherichia coli are risk factors for recurrence. The aim of this study was to evaluate the effect of different antibiotics on biofilms by E. coli strains isolated from children with UTI and to correlate virulence factors and IBCs with biofilm formation. A total of 116 E. coli strains were tested for biofilm formation using the crystal violet microplate technique. 58.6% of the strains did not produce biofilm, while 16.4%, 18.1% and 6.8% formed weak, moderate and strong biofilms, respectively. No correlation was found between the ability to form biofilms and the presence of IBCs. Biofilm formation was significantly associated with pili P codifying genes, whereas other virulence factors were not statistically associated. Antibiotics, including ampicillin, cephalothin, ceftriaxone, ceftazidime, amikacin and ciprofloxacin, were evaluated at different concentrations after 48 h of biofilm formation. Except ampicillin, the other antibiotics tested induced a significant reduction of biofilm biomass. In the case of recurrent UTIs potentially associated with the presence of biofilm, the use of third-generation cephalosporin, fluoroquinolones and aminoglycosides could be recommended. These antibiotics demonstrated to reduce biofilm biomass produced even by resistant strains.

High-purity Nisin Alone or in Combination with Sodium Hypochlorite Is Effective against Planktonic and Biofilm Populations of Enterococcus faecalis

IntroductionNisin, a broad-spectrum bacteriocin, has recently been highlighted for its biomedical applications. To date, no studies have examined the antimicrobial and antibiofilm properties of high-purity (>95%) nisin (nisin ZP) on Enterococcus faecalis and biofilms formed by this species. We hypothesize that nisin can inhibit E. faecalis and reduce biofilm biomass, and combinations of nisin and sodium hypochlorite (NaOCl) will enhance the antibiofilm properties against E. faecalis biofilms. MethodsUsing broth cultures, disc diffusion assays, and biofilm assays, we examined the effects of nisin on various E. faecalis growth parameters and biofilm properties (biovolume, thickness, and roughness). Confocal microscopy was used in conjunction with Imaris and Comstat2 software (Kongens Lyngby, Copenhagen, Denmark) to measure and analyze the biofilm properties. ResultsNisin significantly decreased the growth of planktonic E. faecalis dose dependently. The minimum inhibitory concentrations against E. faecalis strains OG-1 and ATCC 29212 were 15 and 50 μg/mL, and the minimum bactericidal concentrations were 150 and 200 μg/mL, respectively. A reduction in biofilm biovolume and thickness was observed for biofilms treated with nisin at ≥10 μg/mL for 10 minutes. In addition, the combination of nisin with low doses of NaOCl enhanced the antibiofilm properties of both antimicrobial agents. ConclusionsNisin alone or in combination with low concentrations of NaOCl reduces the planktonic growth of E. faecalis and disrupts E. faecalis biofilm structure. Our results suggest that nisin has potential as an adjunctive endodontic therapeutic agent and as an alternative to conventional NaOCl irrigation.

Eugenol inhibits quorum sensing and biofilm of toxigenic MRSA strains isolated from food handlers employed in Saudi Arabia

ABSTRACTFood handlers are important component in assessment and maintenance of food quality as they are carriers of food pathogens causing spoilage. Food spoilage is attributed to quorum sensing (QS) controlled development of biofilms. Therefore, there is an urgent need to develop novel QS and biofilm inhibitors to prevent spoilage of food products. In the present study, toxin producing biofilm forming methicillin-resistant Staphylococcus aureus (MRSA) were isolated from food handlers. Further, eugenol was screened for its QS and anti-biofilm properties. Analysis of nasal and hand swabs revealed the presence of seven toxigenic and biofilm forming MRSA strains. Eugenol demonstrated significant anti-QS activity in CVO26 and also reduced the QS-regulated production of elastase, protease, chitinase, pyocyanin and exopolysaccharide (EPS) in PAO1 considerably. Eugenol demonstrated 17%–86%, 24%–69%, 30%–91%, 9%–94% and 4%–89% reduction in biofilm biomass of S. aureus ATCC 25923 and MRSA strains FSA3, FSA11, FSA13 and FSA32, respectively. Sub-inhibitory concentrations of eugenol also decreased the metabolic activity in biofilm cells. Molecular docking analysis showed high binding affinity of eugenol that represents its biofilm inhibitory activity. This is the first report on the carriage of toxigenic drug-resistant biofilm forming S. aureus by food handlers and inhibition of their biofilms in the Kingdom of Saudi Arabia. The findings give a clear insight into the food safety hazards associated with the carriage of S. aureus and present eugenol as a broad-spectrum anti-QS and anti-biofilm agent.