Micrococcal Nuclease stimulates Staphylococcus aureus Biofilm Formation in a Murine Implant Infection Model

Advancements in contemporary medicine have led to an increasing life expectancy which has broadened the application of biomaterial implants. As each implant procedure has an innate risk of infection, the number of biomaterial-associated infections keeps rising. Staphylococcus aureus causes 34% of such infections and is known as a potent biofilm producer. By secreting micrococcal nuclease S. aureus is able to escape neutrophil extracellular traps by cleaving their DNA-backbone. Also, micrococcal nuclease potentially limits biofilm growth and adhesion by cleaving extracellular DNA, an important constituent of biofilms. This study aimed to evaluate the impact of micrococcal nuclease on infection persistence and biofilm formation in a murine biomaterial-associated infection-model with polyvinylidene-fluoride mesh implants inoculated with bioluminescent S. aureus or its isogenic micrococcal nuclease deficient mutant. Supported by results based on in-vivo bioluminescence imaging, ex-vivo colony forming unit counts, and histological analysis it was found that production of micrococcal nuclease enables S. aureus bacteria to evade the immune response around an implant resulting in a persistent infection. As a novel finding, histological analysis provided clear indications that the production of micrococcal nuclease stimulates S. aureus to form biofilms, the presence of which extended neutrophil extracellular trap formation up to 13 days after mesh implantation. Since micrococcal nuclease production appeared vital for the persistence of S. aureus biomaterial-associated infection, targeting its production could be a novel strategy in preventing biomaterial-associated infection.

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Influence of sub-inhibitory concentrations of antimicrobials on micrococcal nuclease and biofilm formation in Staphylococcus aureus

Scientific Reports ◽

10.1038/s41598-021-92619-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Colin W. K. Rosman ◽

Henny C. van der Mei ◽

Jelmer Sjollema

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Extracellular Polymeric Substances ◽

Extracellular Dna ◽

Micrococcal Nuclease ◽

Host Immune System ◽

Total Biomass ◽

Major Contributor ◽

The Impact

AbstractA major contributor to biomaterial associated infection (BAI) is Staphylococcus aureus. This pathogen produces a protective biofilm, making eradication difficult. Biofilms are composed of bacteria encapsulated in a matrix of extracellular polymeric substances (EPS) comprising polysaccharides, proteins and extracellular DNA (eDNA). S. aureus also produces micrococcal nuclease (MN), an endonuclease which contributes to biofilm composition and dispersion, mainly expressed by nuc1. MN expression can be modulated by sub-minimum inhibitory concentrations of antimicrobials. We investigated the relation between the biofilm and MN expression and the impact of the application of antimicrobial pressure on this relation. Planktonic and biofilm cultures of three S. aureus strains, including a nuc1 deficient strain, were cultured under antimicrobial pressure. Results do not confirm earlier findings that MN directly influences total biomass of the biofilm but indicated that nuc1 deletion stimulates the polysaccharide production per CFU in the biofilm in in vitro biofilms. Though antimicrobial pressure of certain antibiotics resulted in significantly increased quantities of polysaccharides per CFU, this did not coincide with significantly reduced MN activity. Erythromycin and resveratrol significantly reduced MN production per CFU but did not affect total biomass or biomass/CFU. Reduction of MN production may assist in the eradication of biofilms by the host immune system in clinical situations.

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Influence of sub-inhibitory concentrations of antimicrobials on micrococcal nuclease and biofilm formation in Staphylococcus aureus

10.21203/rs.3.rs-115995/v1 ◽

2020 ◽

Author(s):

Jelmer Sjollema ◽

Henny van der Mei ◽

Colin Rosman

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Extracellular Polymeric Substances ◽

Extracellular Dna ◽

Micrococcal Nuclease ◽

Major Contributor ◽

Polysaccharide Production ◽

Minimum Inhibitory Concentrations ◽

The Impact

Abstract A major contributor to biomaterial associated infection (BAI) is Staphylococcus aureus. This pathogen produces a protective biofilm, making eradication difficult. Biofilms are composed of bacteria encapsulated in a matrix of extracellular polymeric substances (EPS) comprising polysaccharides, proteins and extracellular DNA (eDNA). S. aureus also produces micrococcal nuclease (MN), an endonuclease which contributes to biofilm composition and dispersion, mainly expressed by nuc1. MN expression can be modulated by sub-minimum inhibitory concentrations of antimicrobials. We investigated the relation between the biofilm and MN expression and the impact of the application of antimicrobial pressure on this relation. Planktonic and biofilm cultures of three S. aureus strains, including a nuc1 deficient strain, were cultured under antimicrobial pressure. Results indicated that nuc1 deletion stimulates the polysaccharide production per CFU in the biofilm in in vitro biofilms. Also antimicrobial pressure of ciproflocacin, doxycycline and erythromycin resulted in significantly increased quantities of polysaccharides per CFU, but this did only coincide with significantly reduced MN activity in erythromycin. Resveratrol significantly reduced MN production per CFU but did not affect polysaccharides production. In conclusion, various antimicrobials impact the balance of eDNA, polysaccharides and MN production, all in a different way.

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Hyaluronan Modulation Impacts Staphylococcus aureus Biofilm Infection

Infection and Immunity ◽

10.1128/iai.01418-15 ◽

2016 ◽

Vol 84 (6) ◽

pp. 1917-1929 ◽

Cited By ~ 21

Author(s):

Carolyn B. Ibberson ◽

Corey P. Parlet ◽

Jakub Kwiecinski ◽

Heidi A. Crosby ◽

David K. Meyerholz ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Infection Model ◽

Wild Type ◽

Content Type ◽

Joint Infections ◽

The Impact ◽

Biofilm Infection ◽

Biofilm Matrix

Staphylococcus aureusis a leading cause of chronic biofilm infections. Hyaluronic acid (HA) is a large glycosaminoglycan abundant in mammalian tissues that has been shown to enhance biofilm formation in multiple Gram-positive pathogens. We observed that HA accumulated in anS. aureusbiofilm infection using a murine implant-associated infection model and that HA levels increased in a mutant strain lacking hyaluronidase (HysA).S. aureussecretes HysA in order to cleave HA during infection. Throughin vitrobiofilm studies with HA, thehysAmutant was found to accumulate increased biofilm biomass compared to the wild type, and confocal microscopy showed that HA is incorporated into the biofilm matrix. Exogenous addition of purified HysA enzyme dispersed HA-containing biofilms, while catalytically inactive enzyme had no impact. Additionally, induction ofhysAexpression prevented biofilm formation and also dispersed an established biofilm in the presence of HA. These observations were corroborated in the implant model, where there was decreased dissemination from anhysAmutant biofilm infection compared to theS. aureuswild type. Histopathology demonstrated that infection with anhysAmutant caused significantly reduced distribution of tissue inflammation compared to wild-type infection. To extend these studies, the impact of HA andS. aureusHysA on biofilm-like aggregates found in joint infections was examined. We found that HA contributes to the formation of synovial fluid aggregates, and HysA can disrupt aggregate formation. Taken together, these studies demonstrate that HA is a relevant component of theS. aureusbiofilm matrix and HysA is important for dissemination from a biofilm infection.

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A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02588-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2292-2301 ◽

Cited By ~ 23

Author(s):

Angeles Estellés ◽

Anne-Kathrin Woischnig ◽

Keyi Liu ◽

Robert Stephenson ◽

Evelene Lomongsod ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Bacterial Infections ◽

Human Monoclonal Antibody ◽

Integration Host Factor ◽

Extracellular Dna ◽

Infection Model ◽

Antibody Concentration ◽

Human Antibody

ABSTRACTMany serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observedin vitroat an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068in vivowas evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistantStaphylococcus aureus(MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.

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Thymol Inhibits Biofilm Formation, Eliminates Pre-Existing Biofilms, and Enhances Clearance of Methicillin-Resistant Staphylococcus aureus (MRSA) in a Mouse Peritoneal Implant Infection Model

Microorganisms ◽

10.3390/microorganisms8010099 ◽

2020 ◽

Vol 8 (1) ◽

pp. 99 ◽

Cited By ~ 6

Author(s):

Zhongwei Yuan ◽

Yuyun Dai ◽

Ping Ouyang ◽

Tayyab Rehman ◽

Sajjad Hussain ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Methicillin Resistant Staphylococcus Aureus ◽

Inflammatory Responses ◽

Extracellular Dna ◽

Infection Model ◽

Methicillin Resistant ◽

Mouse Infection Model ◽

Cell Counts ◽

First Choice

Methicillin-resistant Staphylococcus aureus (MRSA) is a common human pathogen that causes several difficult-to-treat infections, including biofilm-associated infections. The biofilm-forming ability of S. aureus plays a pivotal role in its resistance to most currently available antibiotics, including vancomycin, which is the first-choice drug for treating MRSA infections. In this study, the ability of thymol (a monoterpenoid phenol isolated from plants) to inhibit biofilm formation and to eliminate mature biofilms, was assessed. We found that thymol could inhibit biofilm formation and remove mature biofilms by inhibiting the production of polysaccharide intracellular adhesin (PIA) and the release of extracellular DNA (eDNA). However, cotreatment with thymol and vancomycin was more effective at eliminating MRSA biofilms, in a mouse infection model, than monotherapy with vancomycin. Comparative histopathological analyses revealed that thymol reduced the pathological changes and inflammatory responses in the wounds. Assessments of white blood cell counts and serum TNF-α and IL-6 levels showed reduced inflammation and an increased immune response following treatment with thymol and vancomycin. These results indicate that combinatorial treatment with thymol and vancomycin has the potential to serve as a more effective therapy for MRSA biofilm-associated infections than vancomycin monotherapy.

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Impact of Extracellular Nuclease Production on the Biofilm Phenotype of Staphylococcus aureus underIn VitroandIn VivoConditions

Infection and Immunity ◽

10.1128/iai.06134-11 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1634-1638 ◽

Cited By ~ 57

Author(s):

Karen E. Beenken ◽

Horace Spencer ◽

Linda M. Griffin ◽

Mark S. Smeltzer

Keyword(s):

Staphylococcus Aureus ◽

Murine Model ◽

Biofilm Formation ◽

Extracellular Dna ◽

Content Type ◽

Extracellular Nuclease ◽

The Impact ◽

Increased Susceptibility

ABSTRACTRecent studies suggest that extracellular DNA promotes biofilm formation inStaphylococcus aureusand, conversely, that extracellular nucleases limit the ability to form a biofilm.S. aureusproduces at least two extracellular nucleases, and in the study described in this report, we examined the impact of each of these nucleases on biofilm formation under bothin vitroandin vivoconditions. Our results demonstrate that both nucleases impact biofilm formation in the clinical isolate UAMS-1. Under certainin vitroconditions, this impact is negative, with mutation of either or both of the nuclease genes (nuc1andnuc2) resulting in an enhanced capacity to form a biofilm. However, this effect was not apparentin vivoin a murine model of catheter-associated biofilm formation. Rather, mutation of either or both nuclease genes appeared to limit biofilm formation to a degree that could be correlated with increased susceptibility to daptomycin.

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RpiRc regulates RsbU to modulate eDNA-dependent biofilm formation and in vivo virulence of Staphylococcus aureus in a mouse model of catheter infection

10.1101/783985 ◽

2019 ◽

Author(s):

Adrien Fischer ◽

Myriam Girard ◽

Floriane Laumay ◽

Anne-Kathrin Woischnig ◽

Nina Khanna ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mouse Model ◽

Biofilm Formation ◽

Ex Vivo ◽

Bacterial Biofilm ◽

Extracellular Dna ◽

Hek293 Cells ◽

Catheter Infection ◽

Biofilm Matrix

AbstractStaphylococcus aureus is a major human pathogen. Despite high incidence and morbidity, molecular mechanisms occurring during infection remain largely unknown. Under defined conditions, biofilm formation contributes to the severity of S. aureus related infections. Extracellular DNA (eDNA), a component of biofilm matrix released from apoptotic bacteria, is involved in biofilm structure and stability. In many bacterial biofilms, eDNA originates from cell lysis although eDNA can also be actively secreted or exported by bacterial membrane vesicles. By screening the Nebraska transposon library, we identified rpiRc as a biofilm regulator involved in eDNA regulation. RpiRc is a transcription factor from the pentose phosphate pathway (PPP) whose product is a polysaccharide intercellular adhesin (PIA) precursor. However, rpiRc mutant strain showed neither susceptibility to DispersinB® (a commercially available enzyme disrupting PIA biofilms) nor alteration of ica transcription (the operon regulating PIA production). Decreased biofilm formation was linked to Sln, an extracellular compound degrading eDNA in an autolysis independent pathway. Biofilm susceptibility to antibiotics in wt and mutant strains was tested using a similar protocol as the Calgary biofilm device. Involvement of RpiRc in S. aureus virulence was assessed ex vivo by internalization experiments into HEK293 cells and in vivo in a mouse model of subcutaneous catheter infection. While minimum inhibitory concentrations (MICs) of planktonic cells were not affected in the mutant strain, we observed increased biofilm susceptibility to almost all tested antibiotics, regardless of their mode of action. More importantly, the rpiRc mutant showed reduced virulence in both ex vivo and in vivo experiments related to decreased fnbpA-B transcription and eDNA production. RpiRc is an important regulator involved in eDNA degradation inside the matrix of mature PIA independent biofilms. These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.Author summaryBiofilm formation contributes to the severity of Staphylococcus aureus related infections. Biofilm matrix is mainly composed by polysaccharide intercellular adhesion (PIA), proteins and extracellular DNA (eDNA). By screening a mutant library of S. aureus, RpiRc was identified as a new regulator of eDNA dependent biofilm formation. How RpiRc regulates biofilm and its role in S. aureus virulence was studied in four different S. aureus strains. Deletion of RpiRc resulted in a pronounced decreased eDNA dependent biofilm formation, but not PIA dependent biofilm formation. Decreased biofilm formation was not related to increased autolysis, but was linked to extracellular compounds found in the supernatant of mutant biofilms. Sln was identified as one of this compound. RpiRc deletion also decreased biofilm recalcitrance (resistance) to selected antibiotics. Involvement of RpiRc in S. aureus pathogenesis was investigated ex vivo by internalization into HEK293 cells and in vivo in a mouse model of catheter infection. RpiRc deletion resulted in decreased virulence related to decreased expression of surface proteins like the fibronectin binding proteins A and B (FnbpA-B). These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.

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Antibiofilm and antivirulence potential of silver nanoparticles against multidrug-resistant Acinetobacter baumannii

Scientific Reports ◽

10.1038/s41598-021-90208-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Helal F. Hetta ◽

Israa M. S. Al-Kadmy ◽

Saba Saadoon Khazaal ◽

Suhad Abbas ◽

Ahmed Suhail ◽

...

Keyword(s):

Silver Nanoparticles ◽

Antibacterial Activity ◽

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Microtiter Plate ◽

Diffusion Method ◽

Resistance Pattern ◽

Infection Model ◽

The Impact

AbstractWe aimed to isolate Acinetobacter baumannii (A. baumannii) from wound infections, determine their resistance and virulence profile, and assess the impact of Silver nanoparticles (AgNPs) on the bacterial growth, virulence and biofilm-related gene expression. AgNPs were synthesized and characterized using TEM, XRD and FTIR spectroscopy. A. baumannii (n = 200) were isolated and identified. Resistance pattern was determined and virulence genes (afa/draBC, cnf1, cnf2, csgA, cvaC, fimH, fyuA, ibeA, iutA, kpsMT II, PAI, papC, PapG II, III, sfa/focDE and traT) were screened using PCR. Biofilm formation was evaluated using Microtiter plate method. Then, the antimicrobial activity of AgNPs was evaluated by the well-diffusion method, growth kinetics and MIC determination. Inhibition of biofilm formation and the ability to disperse biofilms in exposure to AgNPs were evaluated. The effect of AgNPs on the expression of virulence and biofilm-related genes (bap, OmpA, abaI, csuA/B, A1S_2091, A1S_1510, A1S_0690, A1S_0114) were estimated using QRT-PCR. In vitro infection model for analyzing the antibacterial activity of AgNPs was done using a co-culture infection model of A. baumannii with human fibroblast skin cell line HFF-1 or Vero cell lines. A. baumannii had high level of resistance to antibiotics. Most of the isolates harbored the fimH, afa/draBC, cnf1, csgA and cnf2, and the majority of A. baumannii produced strong biofilms. AgNPs inhibited the growth of A. baumannii efficiently with MIC ranging from 4 to 25 µg/ml. A. baumannii showed a reduced growth rate in the presence of AgNPs. The inhibitory activity and the anti-biofilm activity of AgNPs were more pronounced against the weak biofilm producers. Moreover, AgNPs decreased the expression of kpsMII , afa/draBC,bap, OmpA, and csuA/B genes. The in vitro infection model revealed a significant antibacterial activity of AgNPs against extracellular and intracellular A. baumannii. AgNPs highly interrupted bacterial multiplication and biofilm formation. AgNPs downregulated the transcription level of important virulence and biofilm-related genes. Our findings provide an additional step towards understanding the mechanisms by which sliver nanoparticles interfere with the microbial spread and persistence.

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Antimicrobial Activity against Intraosteoblastic Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04359-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2029-2036 ◽

Cited By ~ 36

Author(s):

Florent Valour ◽

Sophie Trouillet-Assant ◽

Natacha Riffard ◽

Jason Tasse ◽

Sacha Flammier ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Ex Vivo ◽

Joint Infection ◽

Treatment Strategies ◽

Bactericidal Effect ◽

Infection Model ◽

Content Type ◽

Intracellular Activity ◽

Mg63 Cells ◽

Choice Of Treatment

ABSTRACTAlthoughStaphylococcus aureuspersistence in osteoblasts, partly as small-colony variants (SCVs), can contribute to bone and joint infection (BJI) relapses, the intracellular activity of antimicrobials is not currently considered in the choice of treatment strategies for BJI. Here, antistaphylococcal antimicrobials were evaluated for their intraosteoblastic activity and their impact on the intracellular emergence of SCVs in anex vivoosteoblast infection model. Osteoblastic MG63 cells were infected for 2 h with HG001S. aureus. After killing the remaining extracellular bacteria with lysostaphin, infected cells were incubated for 24 h with antimicrobials at the intraosseous concentrations reached with standard therapeutic doses. Intracellular bacteria and SCVs were then quantified by plating cell lysates. A bactericidal effect was observed with fosfomycin, linezolid, tigecycline, oxacillin, rifampin, ofloxacin, and clindamycin, with reductions in the intracellular inocula of −2.5, −3.1, −3.9, −4.2, −4.9, −4.9, and −5.2 log10CFU/100,000 cells, respectively (P< 10−4). Conversely, a bacteriostatic effect was observed with ceftaroline and teicoplanin, whereas vancomycin and daptomycin had no significant impact on intracellular bacterial growth. Ofloxacin, daptomycin, and vancomycin significantly limited intracellular SCV emergence. Overall, ofloxacin was the only molecule to combine an excellent intracellular activity while limiting the emergence of SCVs. These data provide a basis for refining the choice of antibiotics to prioritise in the management of BJI, justifying the combination of a fluoroquinolone for its intracellular activity with an anti-biofilm molecule, such as rifampin.

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Antimicrobial and Antibiofilm Activity against Staphylococcus aureus of Opuntia ficus-indica (L.) Mill. Cladode Polyphenolic Extracts

Antioxidants ◽

10.3390/antiox8050117 ◽

2019 ◽

Vol 8 (5) ◽

pp. 117 ◽

Cited By ~ 17

Author(s):

Federica Blando ◽

Rossella Russo ◽

Carmine Negro ◽

Luigi De Bellis ◽

Stefania Frassinetti

Keyword(s):

Staphylococcus Aureus ◽

Antioxidant Activity ◽

Antioxidant Capacity ◽

Biofilm Formation ◽

Ex Vivo ◽

Trolox Equivalent Antioxidant Capacity ◽

Total Phenolic ◽

Antibiofilm Activity ◽

Opuntia Ficus Indica

Plant extracts are a rich source of natural compounds with antimicrobial properties, which are able to prevent, at some extent, the growth of foodborne pathogens. The aim of this study was to investigate the potential of polyphenolic extracts from cladodes of Opuntia ficus-indica (L.) Mill. to inhibit the growth of some enterobacteria and the biofilm formation by Staphylococcus aureus. Opuntia ficus-indica cladodes at two stages of development were analysed for total phenolic content and antioxidant activity by Oxygen Radical Absorbance Capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) (in vitro assays) and by cellular antioxidant activity in red blood cells (CAA-RBC) (ex vivo assay). The Liquid Chromatography Time-of-Flight Mass Spectrometry (LC/MS–TOF) analysis of the polyphenolic extracts revealed high levels of piscidic acid, eucomic acid, isorhamnetin derivatives and rutin, particularly in the immature cladode extracts. Opuntia cladodes extracts showed a remarkable antioxidant activity (in vitro and ex vivo), a selective inhibition of the growth of Gram-positive bacteria, and an inhibition of Staphylococcus aureus biofilm formation. Our results suggest and confirm that Opuntia ficus-indica cladode extracts could be employed as functional food, due to the high polyphenolic content and antioxidant capacity, and used as natural additive for food process control and food safety.

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