Screening of Antimicrobial, Antibiofilm, and Cytotoxic Activities of Some Medicinal Plants from Balikesir Province, Türkiye: Potential Effects of Allium paniculatum Flower.

Objectives: Plant extracts are important natural resources that may have antimicrobial and antibiofilm effects against pathogens. This study was conducted to investigate the in vitro antimicrobial activities of methanol extracts of some medicinal plants (Achillea nobilis subspecies neilreichii (A. Kern.) Velen., Aetheorhiza bulbosa (L.) Cass, Allium paniculatum L, Asphodelus aestivus Brot., Ballota nigra L., Cistus laurifolius L., Cistus salviifolius L., Dioscorea communis (L.) Caddick and Wilkin, Galium verum L., Hypericum triquetrifolium Turra, Paliurus spina-christi Mill., Primula vulgaris Huds. subspecies rubra (Sm.) Arcang., Ranunculus arvensis L. and Teucrium polium L.) from Balikesir province in Türkiye. Materials and Methods: Preliminary antimicrobial activity screening was conducted for all extracts. Antibiofilm activity studies were conducted on mature Candida albicans biofilms. Moreover, the cytotoxicities of A. paniculatum flower extract on A549 and Vero cell lines were determined using a colorimetric tetrazolium-based assay. Results: A. paniculatum flower, P. vulgaris root, C. laurifolius, C. salviifolius, and A. nobilis displayed good activity [minimum inhibitory concentrations (MIC): 9.75, 156, 312, 312 and 312 µg/mL, respectively] against C. albicans American Type Culture Collection 10231. Biofilm studies were conducted on these plant extracts. The methanol extract of A. paniculatum flower decreased the number of C. albicans [colony-forming unit (CFU)/mL] in mature biofilm statistically at 32 x MIC and higher concentrations (p < 0.01). A. paniculatum flower extract had a cytotoxic effect (killing more than 50% of cells) at high concentrations, and its effect on Vero cells was similar to that on A549 cells. Conclusion: This study demonstrated the importance of the methanol extract of A. paniculatum flower as a natural alternative against C. albicans infections, including biofilms.

Antimicrobial Activity of Ceragenins against Vancomycin-Susceptible and -Resistant Enterococcus spp.

Ceragenins (CSAs) are a new class of antimicrobial agents designed to mimic the activities of endogenous antimicrobial peptides. In this study, the antibacterial activities of various ceragenins (CSA-13, CSA-44, CSA-90, CSA-131, CSA-138, CSA-142, and CSA-192), linezolid, and daptomycin were assessed against 50 non-repeated Enterococcus spp. (17 of them vancomycin-resistant Enterococcus-VRE) isolated from various clinical specimens. Among the ceragenins evaluated, the MIC50 and MIC90 values of CSA-44 and CSA-192 were the lowest (2 and 4 µg/mL, respectively), and further studies were continued with these two ceragenins. Potential interactions between CSA-44 or CSA-192 and linezolid were tested and synergistic interactions were seen with the CSA-192-linezolid combination against three Enterococcus spp., one of them VRE. The effects of CSA-44 and CSA-192 on the MIC values of vancomycin were also investigated, and the largest MIC change was seen in the vancomycin-CSA-192 combination. The in vivo effects of CSA-44 and CSA-192 were evaluated in a Caenorhabditis elegans model system. Compared to no treatment, increased survival was observed with C. elegans when treated with ceragenins. In conclusion, CSA-44 and CSA-192 appear to be good candidates (alone or in combination) for the treatment of enterococcal infections, including those from VRE.

Anti-microbial, anti-oxidant and wound healing capabilities of Aloe vera-incorporated hybrid nanoflowers.

The active ingredients of Aloe vera have attracted attention for their potential use in nanotechnology-based medical applications and biomaterial production. It has many therapeutic applications in modern world. This study used Aloe vera extract in different concentrations to synthesize Aloe vera-incorporated hybrid nanoflowers (AV-Nfs). The most uniform morphology in the nanoflowers obtained was at a concentration of 2 mL. The AV-Nfs were well characterized by scanning electron microscopy, X-ray spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction (XRD). The highest peroxidase-mimicking activity of the components was 1.488 EU/mg at 60°C and pH 6. The DPPH assay determined the antioxidant activity of the components and the MTT assay tested on CCD-1072Sk fibroblast cell line determined the effect of AV-Nfs on cell proliferation. Separate treatment of AV-Nfs with Cu3(PO4)2·3H2O significantly increased cell proliferation according to free Aloe vera and CuSO4. In vitro wound healing results showed that AV-Nfs could significantly close wounds compared to free Aloe vera. In this study, AV-Nfs showed antimicrobial activity against Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli and Klebsiella pneumoniae at minimum inhibitory concentration of 625 µg/mL, suggesting that AV-Nfs may be used in wound healing applications with enhanced biological properties. AV-Nfs showed no activity against the yeast Candida albicans.

Impact of N-Acetylcysteine and Antibiotics Against Single and Dual Species Biofilms of Pseudomonas aeruginosa and Achromobacter xylosoxidans.

Lungs of cystic fibrosis patients are often colonized or infected with organisms, such as Pseudomonas aeruginosa and other emerging pathogenic bacteria such as Achromobacter xylosoxidans. Further, it is well established that infections of the cystic fibrosis lung airways are caused by polymicrobial infections, although its composition and diversity may change throughout the patient's life. In the present study, we investigated the effects of N-acetylcysteine (NAC) and amikacin, aztreonam, ciprofloxacin, and tobramycin alone and in combination against single- and dual-species biofilms of P. aeruginosa and A. xylosoxidans, in vitro and in the Caenorhabditis elegans infection model. Results showed that tobramycin and ciprofloxacin were the most effective antibiotics, while aztreonam was the least effective antibiotic against both single- and dual-species biofilms of P. aeruginosa and A. xylosoxidans. However, NAC showed little effect on both single- and dual-species, even with a combination of antibiotics. Increased survival was observed in C. elegans when treated with NAC in combination with tobramycin or ciprofloxacin, compared to no treatment or NAC alone. Tobramycin and ciprofloxacin were found effective in biofilms, but more research is needed to better understand the effects of NAC and antibiotics against single- and dual-species biofilms.

Comparative Fungicidal Activities of N-Chlorotaurine and Conventional Antiseptics against Candida spp. Isolated from Vulvovaginal Candidiasis.

N-chlorotaurine (NCT), the N-chloro derivative of the amino acid taurine, is a long-lived oxidant produced by stimulated human leucocytes. NCT has antimicrobial activities which are generally enhanced in the presence of organic material. The aim of this study was to investigate fungicidal effects of NCT and conventional antiseptics against Candida isolated from vulvovaginal candidiasis (VVC). Chlorhexidine (CHX, 1.6%), octenidine dihydrochloride (OCT, 0.08%), povidone iodine (PVP-I, 8%), boric acid (8%), and NCT (0.1% (5.5 mM)) were evaluated against forty-four Candida isolates, according to European Standard methods, at 30, 60, 90, and 120 min and 24 h in the presence of skim milk as an organic material. CHX, OCT, and PVP-I showed rapid fungicidal activity against all Candida isolates with 5-6 log10 reduction of viable counts after 30 min, whereas boric acid and NCT needed 1 h against Candida albicans and 2 h against non-albicans Candida for a significant 3 log10 reduction. NCT showed fungicidal activity (defined as ≥4 log10 reduction) against C. albicans within 90 min and C. non-albicans within 24 h. Based upon all presently available data, including our results, NCT could be used as a new agent for treatment of local fungal infections such as VVC.

Optimization of the Micellar-Based In Situ Gelling Systems Posaconazole with Quality by Design (QbD) Approach and Characterization by In Vitro Studies.

BACKGROUND: Fungal ocular infections can cause serious consequences, despite their low incidence. It has been reported that Posaconazole (PSC) is used in the treatment of fungal infections in different ocular tissues by diluting the oral suspension, and successful results were obtained despite low ocular permeation. Therefore, we optimized PSC-loaded ocular micelles and demonstrated that the permeation/penetration of PSC in ocular tissues was enhanced. METHODS: The micellar-based in situ gels based on the QbD approach to increase the ocular bioavailability of PSC were developed. Different ratios of Poloxamer 407 and Poloxamer 188 were chosen as CMAs. Tsol/gel, gelling capacity and rheological behavior were chosen as CQA parameters. The data were evaluated by Minitab 18, and the formulations were optimized with the QbD approach. The in vitro release study, ocular toxicity, and anti-fungal activity of the optimized formulation were performed. RESULTS: Optimized in situ gel shows viscoelastic property and becomes gel form at physiological temperatures even when diluted with the tear film. In addition, it has been shown that the formulation had high anti-fungal activity and did not have any ocular toxicity. CONCLUSIONS: In our previous studies, PSC-loaded ocular micelles were developed and optimized for the first time in the literature. With this study, the in situ gels of PSC for ocular application were developed and optimized for the first time. The optimized micellar-based in situ gel is a promising drug delivery system that may increase the ocular permeation and bioavailability of PSC.

Posaconazole micelles for ocular delivery: in vitro permeation, ocular irritation and antifungal activity studies.

Posaconazole (PSC) is a triazole group anti-fungal agent with the widest spectrum. Although there is no commercially available ocular dosage form, its diluted oral suspension preparation (Noxafil®) is used as off-label in topical treatment of severe keratitis and sclerokeratitis in the clinic. However, ocular bioavailability of PSC suspension form is extremely low due to its highly lipophilic character. Thus, there is a clinical need to improve its ocular bioavailability and to develop novel delivery system for the treatment of ocular fungal infections. Herein, we studied ex vivo permeation, penetration, anti-fungal activity, and Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) toxicity tests in order to assess ocular targeting of PSC micelles, which were optimized in our previous study. The results indicated that micellar carrier system increased the permeability of PSC to eye tissues. Micelles showed higher affinity to ocular tissues than that of commercial oral suspension of PSC (Noxafil®). In vitro anti-fungal activity data also confirmed the efficacy of PSC loaded micellar formulations against Candida. albicans strains. The relative safety of the optimized micelles on the ocular tissue was shown with the HET-CAM toxicity test. In conclusion, micellar systems could be a promising strategy for the effective and safe delivery of PSC in the treatment of ocular fungal infections.

Oregano essential oil inhibits Candida spp. biofilms.

Candida spp. can form biofilms on mucosal surfaces and epithelial cells as well as on devices implanted in the body such as catheters and dentures, which are thought to underlie the most recalcitrant infections. It was aimed to show antifungal and antibiofilm activities of oregano oil (Origanum onites). The antifungal activities of some essential oils were investigated against C. spp. and among them, oregano oil was found to be the most effective oil and further biofilm studies were conducted with it. Oregano oil inhibited biofilm adhesion and formation of C. spp. and mature biofilms and also displayed the ability to reduce biofilm formation when they were allowed to form on surfaces previously coated with oil (up to 50% inhibition rates). In addition, oregano oil was found to be effective against dual biofilms of Candida albicans + Staphyloccocus aureus at different concentrations. This study suggests that O. onites essential oil has useful antibiofilm effects against C. spp. The inhibitory effects of O. onites essential oil, against C. spp., were demonstrated for the first time. It also had antifungal effect on biofilm formation and established biofilm even at MIC level.

Biofilm modelling on the contact lenses and comparison of the in vitro activities of multipurpose lens solutions and antibiotics.

During the contact lens (CL) usage, microbial adhesion and biofilm formation are crucial threats for eye health due to the development of mature biofilms on CL surfaces associated with serious eye infections such as keratitis. For CL related eye infections, multi drug resistant Pseudomonas aeruginosa or Staphylococcus aureus (especially MRSA) and Candida albicans are the most common infectious bacteria and yeast, respectively. In this study, CL biofilm models were created by comparing them to reveal the differences on specific conditions. Then the anti-biofilm activities of some commercially available multipurpose CL solutions (MPSs) and antibiotic eye drops against mature biofilms of S. aureus, P. aeruginosa, and C. albicans standard and clinical strains were determined by the time killing curve (TKC) method at 6, 24 and 48 h. According to the biofilm formation models, the optimal biofilms occurred in a mixture of bovine serum albumin (20% v/v) and lysozyme (2 g/L) diluted in PBS at 37 °C for 24 h, without shaking. When we compared the CL types under the same conditions, the strongest biofilms according to their cell density, were formed on Pure Vision ≥ Softens 38 > Acuve 2 âˆ¼ Softens Toric CLs. When we compared the used CLs with the new ones, a significant increase at the density of biofilms on the used CLs was observed. The most active MPS against P. aeruginosa and S. aureus biofilms at 24 h was Opti-Free followed by Bio-True and Renu according to the TKC analyses. In addition, the most active MPS against C. albicans was Renu followed by Opti-Free and Bio-True at 48 h. None of the MPSs showed 3 Log bactericidal/fungicidal activity, except for Opti-Free against S. aureus and P. aeruginosa biofilms during 6 h contact time. Moreover, all studied antibiotic eye drops were active against S. aureus and P. aeruginosa biofilms on CLs at 6 h and 24 h either directly or as 1/10 concentration, respectively. According to the results of the study, anti-biofilm activities of MPSs have changed depending on the chemical ingredients and contact times of MPSs, the type of infectious agent, and especially the CL type and usage time.

Antibiofilm activities of ceragenins and antimicrobial peptides against fungal-bacterial mono and multispecies biofilms.

Multispecies biofilms, in which both fungus and bacteria species can be present, play a significant role in persistent infections, and new therapeutic options are needed against them. In this study, the activities of ceragenins and antimicrobial peptides (AMPs) (magainin, cecropin A, LL-37) were investigated against multispecies biofilms formed by Candida albicans and four clinically important Gram-negative bacteria, including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae. Our results show that CSA-13 and CSA-90 were the most effective agents against both mono and multispecies biofilms (P < 0.05). CSA-131 and CSA-192 showed the least antimicrobial activity against mono and fungal-bacterial multispecies biofilms. Inhibition of multispecies biofilms with CSA-13 and CSA-90 was also confirmed through fluorescence microscopy images. When AMPs evaluated alone, they proved ineffective against both C. albicans and Gram-negative bacteria at the concentrations tested. In these studies, ceragenins were much more effective than AMPs against multi or monospecies biofilms, especially those containing C. albicans.

Effects of ceragenins and conventional antimicrobials on Candida albicans and Staphylococcus aureus mono and multispecies biofilms.

It is known that synergy between Candida albicans and Staphylococcus aureus results in enhanced biofilm formation and increased resistance to antimicrobials. Ceragenins (CSAs) are derivatives of cholic acid designed to mimic the antimicrobial activities of endogenous antimicrobial peptides. In this study, various CSAs were tested on C. albicans and methicillin-susceptible S. aureus or methicillin-resistant S. aureus mono or multispecies biofilms at 2 different concentrations (16 and 64 µg/mL) and compared with conventional antimicrobials. CSA-8 was active agent both with mono and multispecies biofilms (P < 0.05). Among antifungals, amphotericin B and, among antibacterials, ciprofloxacin and gentamicin were active agents against all studied microorganisms. This study suggests that CSAs, especially CSA-8, have useful antibiofilm effects against monomicrobial or fungal-bacterial multispecies biofilms.

In vitro activities of antimicrobial peptides and ceragenins against Legionella pneumophila.

Legionella pneumophila is a waterborne intracellular pathogenic bacterium, the most frequent cause of human legionellosis and a relatively common cause of community-acquired and nosocomial pneumonia. Some legionellosis outbreaks are related to the presence of biofilms, which provide a reservoir for L. pneumophila strains. We investigated the in vitro activities of antibiotics; erythromycin and doxycycline, antimicrobial peptides AMPs; melittin, LL-37 and CAMA (cecropin A (1-7)-Melittin A (2-9) and ceragenins; CSA-8, CSA-13, CSA-44, CSA-131 and CSA-138 against L. pneumophila. Isolation of Legionella strains was conducted according to ISO 1998. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum biofilm eradication concentrations (MBECs) were determined using microbroth dilution techniques. MIC ranges for melittin, LL-37, and CAMA were 0.25-1, 1-4, and 2-8 µg ml-1, respectively. MIC ranges for CSA-8, 13, 44, 131, and 138 were 0.5-2, 0.5-1, 1-4, 0.5-2, and 1-2 µg ml-1, respectively, and MBEC values for the ceragenins were 10-160 µg ml-1. These results demonstrate that AMPs and ceragenins display broad-spectrum, in vitro activity against L. pneumophila. In particular, CSA-8, CSA-13 and melittin gave the lowest MICs and MBCs. We also observed that ceragenins are active against established L. pneumophila biofilms.

Antifungal susceptibilities, in vitro production of virulence factors and activities of ceragenins against Candida spp. isolated from vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC) is the second most common cause of vaginitis after bacterial vaginosis, affecting millions of women worldwide every year. Candida albicans is the most frequent agent of VVC followed by other species of Candida such as C. glabrata and C. parapsilosis. Out of a total of 100 clinical isolates of Candida spp. obtained from patients diagnosed with VVC, 84 were identified as C. albicans, while the remaining isolates were identified as non--albicans Candida strains. Phospholipases and proteinases were produced by a majority of the C. albicans strains and esterases and hemolysins a minority of these strains. Among the non-C. albicans strains, only a few of the strains produced these proteins. Nearly all of the isolates formed biofilms. Our results showed that the butoconazole, clotrimazole, and fluconazole were active against C. albicans and less so against the non-albicans Candida strains. The MIC90 of amphotericin B and nystatins were 2 and 4 µg/ml, respectively, against either C. albicans or non-albicans Candida spp. Representative ceragenins (CSA-13, CSA-131, and CSA-138), developed as mimics of endogenous antimicrobial peptides, were active against fluconazole-resistant strains, both alone and in combination with fluconazole. These results suggest the potential use of ceragenins in treating VVC, including infections caused by fluconazole-resistant isolates.

Antibacterial and Antibiofilm Activities of Ceragenins against Pseudomonas aeruginosa Clinical Isolates.

OBJECTIVES: Pseudomonas aeruginosa can cause life-threatening infections that are difficult to treat due to its high resistance to antibiotics and its ability to form antibiotic tolerant biofilms. Ceragenins, designed to mimic the activities of antimicrobial peptides, represent a promising new group of antibacterial agents that display potent anti-P. aeruginosa activity. The aim of this study was to evaluate the antibacterial and antibiofilm activities of ceragenins in comparison to colistin and ciprofloxacin against P. aeruginosa strains. MATERIALS AND METHODS: Biofilm formation and determination of minimum inhibitory concentration (MIC) values of ceragenins (CSA-13, CSA-44, CSA-131, and CSA-138), ciprofloxacin, and colistin were evaluated against 25 P. aeruginosa isolates. Four good biofilm-producing strains were chosen for biofilm studies, and sessile MICs and inhibition of molecule adhesion and biofilm formation were evaluated. RESULTS: The MIC50 (µg/mL) values of CSA-13, CSA-44, CSA-131, CSA-138, ciprofloxacin, and colistin were 8, 8, 8, 16, 1, and 2, respectively. The sessile MICs for molecules were greater than planktonic MICs. CSA-13, CSA-44, and CSA-131 were more efficient after 4 h incubation while CSA-138, ciprofloxacin and colistin were more efficient after 1 h incubation. The most efficient agent for inhibition of adhesion was colistin (up to 45%). CSA-131, CSA-138, and colistin were the most efficient agents for inhibition of biofilm formation (up to 90%). CONCLUSION: Our study highlights the potential of CSA-131 and CSA-138 as potential alternative agents to conventional antibiotics for the eradication of biofilms of P. aeruginosa.

In vitro activities of antifungals alone and in combination with tigecycline against Candida albicans biofilms.

BACKGROUND: Candida may form biofilms, which are thought to underlie the most recalcitrant infections. METHODS: In this study, activities of antifungal agents alone and in combination with tigecycline against planktonic cells and mature and developing biofilms of Candida albicans isolates were evaluated. RESULTS: Amphotericin B and echinocandins were found to be the most effective agents against mature biofilms, whereas the least effective agent was fluconazole. Furthermore, the most effective anti-fungal monotherapies against biofilm formation were amphotericin B and anidulafungin, and the least effective monotherapy was itraconazole. The combination of tigecycline and amphotericin B yielded synergistic effects, whereas combinations containing itraconazole yielded antagonist effects against planktonic cells. The combination of tigecycline and caspofungin exhibited maximum efficacy against mature biofilms, whereas combinations containing itraconazole exhibited minimal effects. Combinations of tigecycline with amphotericin B or anidulafungin were highly effective against C. albicans biofilm formation. DISCUSSION: In summary, tigecycline was highly active against C. albicans particularly when combined with amphotericin B and echinocandins.

Investigation of the in vitro antifungal and antibiofilm activities of ceragenins CSA-8, CSA-13, CSA-44, CSA-131, and CSA-138 against Candida species.

Cationic steroid antimicrobials (CSA-ceragenin) are a new class of antimicrobial agent. In vitro activities of CSA-8, CSA-13, CSA-44, CSA-131, and CSA-138 and amphotericin B (AMP-B) were assessed against 50 nonrepeat Candida spp. isolates MICs, MFCs and combination studies were determined. Antibiofilm activities of CSAs, AMP-B, 2 azoles, and 2 echinocandins against Candida albicans were performed. Also, effects of coating the wells of plate with selected CSAs and antifungals were measured. The MIC50 (µg/mL) values of CSA-8, CSA-13, CSA-44, CSA-131, CSA-138, and AMP-B were 16, 1, 2, 1, 1, and 1, respectively. The MFCs were equal to or 2-fold greater than those of the MICs. Synergistic interactions were mostly seen with CSA-13+ AMP-B combination, whereas the least synergistic interactions were observed with the CSA-131+ AMP-B combination. CSAs inhibited the attachment of Candida biofilms. The studied CSAs and antifungals inhibited C. albicans biofilm formation. In conclusion, CSA-13, CSA-131, and CSA-138 appear to be good candidates (alone or in combination) in the treatment of Candida infections as well as biofilm-related ones.

Antimicrobial activities of widely consumed herbal teas, alone or in combination with antibiotics: an in vitro study.

BACKGROUND: Because of increasing antibiotic resistance, herbal teas are the most popular natural alternatives for the treatment of infectious diseases, and are currently gaining more importance. We examined the antimicrobial activities of 31 herbal teas both alone and in combination with antibiotics or antifungals against some standard and clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, methicillin susceptible/resistant Staphylococcus aureus and Candida albicans. METHODS: The antimicrobial activities of the teas were determined by using the disk diffusion and microbroth dilution methods, and the combination studies were examined by using the microbroth checkerboard and the time killing curve methods. RESULTS: Rosehip, rosehip bag, pomegranate blossom, thyme, wormwood, mint, echinacea bag, cinnamon, black, and green teas were active against most of the studied microorganisms. In the combination studies, we characterized all the expected effects (synergistic, additive, and antagonistic) between the teas and the antimicrobials. While synergy was observed more frequently between ampicillin, ampicillin-sulbactam, or nystatine, and the various tea combinations, most of the effects between the ciprofloxacin, erythromycin, cefuroxime, or amikacin and various tea combinations, particularly rosehip, rosehip bag, and pomegranate blossom teas, were antagonistic. The results of the time kill curve analyses showed that none of the herbal teas were bactericidal in their usage concentrations; however, in combination with antibiotics they showed some bactericidal effect. DISCUSSION: Some herbal teas, particularly rosehip and pomegranate blossom should be avoided because of their antagonistic interactions with some antibiotics during the course of antibiotic treatment or they should be consumed alone for their antimicrobial activities.

Community Composition Determines Activity of Antibiotics against Multispecies Biofilms.

In young cystic fibrosis (CF) patients, Staphylococcus aureus is typically the most prevalent organism, while in adults, Pseudomonas aeruginosa is the major pathogen. More recently, it was observed that also Streptococcus anginosus plays an important role in exacerbations of respiratory symptoms. These species are often coisolated from CF lungs, yet little is known about whether antibiotic killing of one species is influenced by the presence of others. In the present study, we compared the activities of various antibiotics against S. anginosus, S. aureus, and P. aeruginosa when grown in monospecies biofilms with the activity observed in a multispecies biofilm. Our results show that differences in antibiotic activity against species grown in mono- and multispecies biofilms are species and antibiotic dependent. Fewer S. anginosus cells are killed by antibiotics that interfere with cell wall synthesis (amoxicillin plus sulbactam, cefepime, imipenem, meropenem, and vancomycin) in the presence of S. aureus and P. aeruginosa, while for ciprofloxacin, levofloxacin, and tobramycin, no difference was observed. In addition, we observed that the cell-free supernatant of S. aureus, but not that of P. aeruginosa biofilms, also caused this decrease in killing. Overall, S. aureus was more affected by antibiotic treatment in a multispecies biofilm, while for P. aeruginosa, no differences were observed between growth in mono- or multispecies biofilms. The results of the present study suggest that it is important to take the community composition into account when evaluating the effect of antimicrobial treatments against certain species in mixed biofilms.