Minimum Biofilm Eradication Concentration Values Research Articles

The antimicrobial and antibiofilm effects of gentamicin, imipenem, and fucoidan combinations against dual-species biofilms of Staphylococcus aureus and Acinetobacter baumannii isolated from diabetic foot ulcers

IntroductionDiabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia due to impaired insulin production or utilization, leading to severe health complications. Diabetic foot ulcers (DFUs) represent a major complication, often exacerbated by polymicrobial infections involving Staphylococcus aureus and Acinetobacter baumannii. These pathogens, notorious for their resistance to antibiotics, complicate treatment efforts, especially due to biofilm formation, which enhances bacterial survival and resistance. This study explores the synergistic effects of combining gentamicin, imipenem, and fucoidan, a sulfated polysaccharide with antimicrobial properties, against both planktonic and biofilm forms of S. aureus and A. baumannii.MethodsIsolates of S. aureus and A. baumannii were collected from DFUs and genetically confirmed. Methicillin resistance in S. aureus was identified through disk diffusion and PCR. Biofilm formation, including dual-species biofilms, was analyzed using the microtiter plate method. The antimicrobial efficacy of gentamicin, imipenem, and fucoidan was assessed by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC). Synergistic interactions were evaluated using the fractional inhibitory concentration index (FICi) and fractional bactericidal concentration index (FBCi). The expression of biofilm-associated genes (icaA in S. aureus and bap in A. baumannii) was analyzed, and the cytotoxicity of fucoidan was assessed.ResultsThe study revealed that 77.4% of S. aureus and all A. baumannii isolates showed multidrug resistance. Among 837 tested conditions for dual-species biofilm formation, 72 resulted in strong biofilm formation and 67 in moderate biofilm formation. The geometric mean MIC values for gentamicin were 12.2 µg/mL for S. aureus, 22.62 µg/mL for A. baumannii, and 5.87 µg/mL for their co-culture; for imipenem, they were 19.84, 9.18, and 3.70 µg/mL, respectively, and for fucoidan, 48.50, 31.20, and 19.65 µg/mL, respectively. The MBC values for gentamicin were 119.42, 128, and 11.75 µg/mL; for imipenem, they were 48.50, 14.92, and 8 µg/mL; and for fucoidan, they were 88.37, 62.62, and 42.48 µg/mL. The MBIC values were 55.71, 119.42, and 18.66 µg/mL for gentamicin; 68.59, 48.50, and 25.39 µg/mL for imipenem; and 153.89, 101.49, and 53.53 µg/mL for fucoidan. The MBEC values were 315.17, 362.03, and 59.25 µg/mL for gentamicin; 207.93, 157.58, and 74.65 µg/mL for imipenem; and 353.55, 189.46, and 99.19 µg/mL for fucoidan. When cultured in planktonic form, the geometric mean FICi and FBCi values indicated additive effects, while co-culture showed FICi values of ≤ 0.5, suggesting a synergistic interaction. Treatment with gentamicin and fucoidan led to significant downregulation of the icaA and bap genes in both single-species and dual-species biofilms of S. aureus and A. baumannii. The reductions in gene expression were more pronounced in dual-species biofilms compared to single-species biofilms. Additionally, treatment with imipenem and fucoidan also resulted in significant downregulation of these genes in both biofilm types. Cytotoxicity assessments indicated that higher concentrations of fucoidan were toxic, yet no harmful effects were noted at the optimal synergistic concentrations used with antibiotics.ConclusionIn our investigation, we found that combining gentamicin, imipenem, and fucoidan had a synergistic effect on dual-species biofilms of S. aureus and A. baumannii, suggesting potential benefits for treating such infections effectively. This underscores the importance of understanding microbial interactions, antibiotic susceptibility, and biofilm formation in DFUs.

Investigating the antimicrobial and antibiofilm properties of marine halophilic Bacillus species against ESKAPE pathogens.

Antimicrobial resistance (AMR), known as the "silent pandemic," is exacerbated by pathogenic bacteria's ability to form biofilms. Marine compounds hold promise for novel antibacterial drug discovery. Two isolates from preliminary saltwater environment screening demonstrated antimicrobial activity and were subsequently identified as Bacillus subtilis MTUA2 and Bacillus velezensis MTUC2. Minimum inhibitory concentrations (MICs), minimum biofilm inhibition concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) required to prevent and/or disrupt bacterial growth and biofilm formation were established for MRSA, Staphylococcus aureus, Acinetobacter baumannii and Escherichia coli. The metabolic activity within biofilms was determined by the 2,3,5-triphenyltetrazolium chloride assay. Both Bacillus species exhibited unique antimicrobial effects, reducing MRSA and S. aureus planktonic cell growth by 50% and sessile cell growth for S. aureus and E. coli by 50% and 90%, respectively. No effect was observed against A. baumannii. Significant MBIC and MBEC values were achieved, with 99% inhibition and 90% reduction in MRSA and S. aureus biofilms. Additionally, 90% and 50% inhibition was observed in E. coli and A. baumannii biofilms, respectively, with a 50% reduction in E. coli biofilm. These findings suggest that the mode of action employed by B. subtilis MTUA2 and B. velezensis MTUC2 metabolites should be further characterized and could be beneficial if used independently or in combination with other treatments.

Effective biofilm eradication in MRSA isolates with aminoglycoside-modifying enzyme genes using high-concentration and prolonged gentamicin treatment.

Our analysis of 101 MRSA clinical isolates has provided valuable insights that could enhance treatment selection for biofilm infections in orthopedics. We found that high concentrations of gentamicin were effective against MRSA biofilms, and even prolonged exposure to concentrations lower than the minimum biofilm eradication concentration (MBEC) value could eliminate biofilms. The presence of the aac(6')-aph(2″) gene, an aminoglycoside resistance gene, was found to correlate with the minimum inhibitory concentration (MIC) and MBEC values of gentamicin, providing a potential predictive tool for treatment susceptibility. These results suggest that extended high concentrations of local gentamicin treatment could effectively eliminate MRSA biofilms in orthopedic infections. Furthermore, testing for gentamicin MIC or the possession of the aac(6')-aph(2″) gene could help select treatment, including topical gentamicin administration and surgical debridement.

Cannabidiol Demonstrates Remarkable Efficacy in Treating Multidrug-Resistant Enterococcus Faecalis Infections In Vitro and In Vivo

The growing prevalence of multidrug-resistant Enterococcus faecalis infection has become a global concern. There is a demand for alternative antibacterial agents, such as herbal alternatives, such as Cannabidiol, that are cost-effective, non-toxic and efficient. This study investigates the Minimum Inhibitory Concentration (MIC) for planktonic cells and the Minimum Biofilm Eradication Concentration (MBEC) for biofilm formation in multidrug- resistance isolates of E. faecalis isolates, particularly focusing cannabidiol activity. E. faecalis isolates with strong biofilm presence, vancomycin, levofloxacin, and daptomycin displayed high MIC and MBEC values. Cannabidiol exhibited a significantly lower MIC (1 μg/mL) for planktonic cells and a low MBEC (2 μg/mL). Moreover, at low concentrations (2 µg/mL), cannabidiol demonstrated notable reductions in biofilm biovolume, bacterial cell viability and colony-forming unit compared to vancomycin, levofloxacin, and daptomycin. The mice treated with cannabidiol (100 mg/kg) exhibited a significant reduction in E. faecalis bacterial load in internal organs and increased the survival. In conclusion, the findings underscore the promising antibiofilm properties of cannabidiol against E. faecalis, indicating its potential as a novel therapeutic agent.

Increased antibiotic resistance of Pseudomonas aeruginosa isolates from chronic rhinosinusitis patients grown in anaerobic conditions.

In chronic rhinosinusitis (CRS), the congestion and blockage of the nose can cause anaerobic conditions within the sinus cavities which may promote the expression of virulence and antibiotic resistance genes in invading pathogens. Pseudomonas aeruginosa is a facultative anaerobic bacteria and causes severe recalcitrant CRS. In this study, we aimed to evaluate the antimicrobial resistance of P. aeruginosa isolates of CRS patients in planktonic and biofilm form grown in aerobic and anaerobic conditions. P. aeruginosa clinical isolates of CRS patients (n = 25) were grown in planktonic and biofilm form in aerobic and anaerobic conditions. Minimum inhibitory concentrations (MIC) of planktonic forms and minimum biofilm eradication concentrations (MBEC) were determined. Additionally, metabolic activity by fluorescein diacetate assay, biofilm biomass by crystal violet assay and eDNA concentration were assessed in both conditions. P. aeruginosa planktonic cells grown in anaerobic condition exhibited increased gentamicin resistance (p < .01), whereas P. aeruginosa biofilms grown in anaerobic condition displayed significantly increased MBEC values for gentamicin (p < .0001) and levofloxacin (p < .001). The metabolic activity of anaerobic biofilms was significantly higher compared with aerobic biofilms (p < .0001). However, the biofilm biomass of isolates grown in aerobic conditions was higher than anaerobic conditions (p < .5). P. aeruginosa isolates from CRS patients grown in anaerobic conditions showed significantly increased resistance to antibiotics with an increased metabolic activity but decreased biofilm biomass. NA.

A hope for ineffective antibiotics to return to treatment: investigating the anti-biofilm potential of melittin alone and in combination with penicillin and oxacillin against multidrug resistant-MRSA and -VRSA.

The emergence and rapid spread of multi-drug resistant (MDR) bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), have posed a significant challenge to the medical community due to their ability to form biofilm and develop resistance to common antibiotics. Traditional antibiotics that were once effective in treating bacterial infections are now becoming increasingly ineffective, leading to severe consequences for patient outcomes. This concerning situation has called for urgent research to explore alternative treatment strategies. Recent studies have shown that antimicrobial peptides (AMPs) hold promise as effective agents against biofilm-associated drug-resistant infections as well as to enhance the efficacy of conventional antibiotics. Accordingly, we aimed to investigate the antimicrobial and antibiofilm effects of melittin AMP, both alone and in combination with penicillin and oxacillin, against biofilm-forming MDR-MRSA and -VRSA. In this study, we investigated the kinetics of biofilm formation and assessed various parameters related to the antimicrobial and antibiofilm efficacy of melittin and antibiotics, both alone and in combination, against MDR-MRSA and -VRSA. The antimicrobial parameters included the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Fractional Inhibitory Concentration Index (FICi), Fractional Bactericidal Concentration Index (FBCi), and the antibiofilm activity of melittin and antibiotics indicated by the Minimum Biofilm Inhibitory Concentration (MBIC), Minimal Biofilm Eradication Concentration (MBEC), Fractional Biofilm Inhibitory Concentration Index (FBICi), and Fractional Biofilm Eradication Concentration Index (FBECi). The MIC results showed that all S. aureus isolates were resistant to penicillin (≥0.25 μg/mL), and 66% of isolates were resistant to oxacillin. The geometric means of the MIC values for penicillin, oxacillin, and melittin were 19.02, 16, and 1.62 μg/ml, respectively, and the geometric means of the MBC values for penicillin, oxacillin, and melittin were 107.63, 49.35, and 5.45 μg/ml, respectively. The study revealed that the combination indexes of melittin-penicillin and melittin-oxacillin, as determined by FIC values against all isolates, were 0.37 and 0.03, respectively. Additionally, melittin-penicillin and melittin-oxacillin exhibited combination indexes based on FBC values against all isolates at 1.145 and 0.711, respectively. Besides, melittin inhibited the biofilm formation of all S. aureus isolates, with MBIC values ranging from 10 to 1.25 μg/mL, and MBEC values ranging from 40 to 10 μg/mL. Generally, the combination indexes of melittin-penicillin and melittin-oxacillin, determined using FBIC values against all isolates, were 0.23 and 0.177, respectively. Moreover, melittin-penicillin and melittin-oxacillin typically had combination indexes based on FBEC values against all isolates at 5 and 2.97, respectively. In conclusion, our study provides evidence that melittin is effective against both planktonik and biofilm forms of MRSA and VRSA and exhibits significant synergistic effects when combined with antibiotics. These results suggest that melittin and antibiotics could be a potential candidate for further investigation for in vivo infections caused by MDR S. aureus. Furthermore, melittin has the potential to restore the efficacy of penicillin and oxacillin antibiotics in the treatment of MDR infections. Applying AMPs, like melittin, to revive beta-lactam antibiotics against MRSA and VRSA is an innovative approach against antibiotic-resistant bacteria. Further research is needed to optimize dosage and understand melittin mechanism and interactions with beta-lactam antibiotics for successful clinical applications.

Poly-Gamma-Glutamic Acid Nanopolymer Effect against Bacterial Biofilms: In Vitro and In Vivo Study.

In this study, a biodegradable poly-gamma-glutamic-acid nanopolymer (Ɣ-PGA NP) was investigated for its activity against clinical strains of Gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli), and reference strains of S. aureus ATCC 6538, S. pyogenes ATCC 19615 (Gram-positive), and Gram-negative E. coli ATCC 25922, and K. pneumoniae ATCC 13884 bacterial biofilms. The minimum inhibitory concentration (MIC) effect of Ɣ-PGA NP showed inhibitory effects of 0.2, 0.4, 1.6, and 3.2 μg/mL for S. pyogenes, S. aureus, E. coli, and K. pneumoniae, respectively. Also, MIC values were 1.6, 0.8, 0.2, and 0.2 μg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Afterwards, MBEC (minimum biofilm eradication concentration) and MBIC (minimum biofilm inhibitory concentration) were investigated to detect Ɣ-PGA NPs efficiency against the biofilms. MBEC and MBIC increased with increasing Ɣ-PGA NPs concentration or time of exposure. Interestingly, MBIC values were at lower concentrations of Ɣ-PGA NPs than those of MBEC. Moreover, MBEC values showed that K. pneumoniae was more resistant to Ɣ-PGA NPs than E. coli, S. aureus, and S. pyogenes, and the same pattern was observed in the reference strains. The most effective results for MBEC were after 48 h, which were 1.6, 0.8, 0.4, and 0.2 µg/mL for K. pneumoniae, E. coli, S. aureus, and S. pyogenes, respectively. Moreover, MBIC results were the most impactful after 24 h but some were the same after 48 h. MBIC values after 48 h were 0.2, 0.2, 0.2, and 0.1 μg/mL for K. pneumoniae, E. coli, S. aureus, and S. pyogenes, respectively. The most effective results for MBEC were after 24 h, which were 1.6, 0.8, 0.4, and 0.4 µg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Also, MBIC results were the most impactful after an exposure time of 12 h. MBIC values after exposure time of 12 h were 0.4, 0.4, 0.2, and 0.2 μg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Besides that, results were confirmed using confocal laser scanning microscopy (CLSM), which showed a decrease in the number of living cells to 80% and 60% for MBEC and MBIC, respectively, for all the clinical bacterial strains. Moreover, living bacterial cells decreased to 70% at MBEC while decreasing up to 50% at MBIC with all bacterial refence strains. These data justify the CFU quantification. After that, ImageJ software was used to count the attached cells after incubating with the NPs, which proved the variation in live cell count between the manual counting and image analysis methods. Also, a scanning electron microscope (SEM) was used to detect the biofilm architecture after incubation with the Ɣ-PGA NP. In in vivo wound healing experiments, treated wounds of mice showed faster healing (p < 0.00001) than both the untreated mice and those that were only wounded, as the bacterial count was eradicated. Briefly, the infected mice were treated faster (p < 0.0001) when infected with S. pyogenes > S. aureus > E. coli > K. pneumoniae. The same pattern was observed for mice infected with the reference strains. Wound lengths after 2 h showed slightly healing (p < 0.001) for the clinical strains, while treatment became more obvious after 72 h > 48 h > 24 h (p < 0.0001) as wounds began to heal after 24 h up to 72 h. For reference strains, wound lengths after 2 h started to heal up to 72 h.

Nanoemulsion of Lavandula angustifolia Essential Oil/Gold Nanoparticles: Antibacterial Effect against Multidrug-Resistant Wound-Causing Bacteria.

Hospitalized patients are severely impacted by delayed wound healing. Recently, there has been a growing focus on enhancing wound healing using suitable dressings. Lavandula angustifolia essential oil (LEO) showed potential antibacterial, anti-inflammatory, and wound healing properties. However, the prepared gold nanoparticles possessed multifunctional properties. Consequently, the present investigation aimed to synthesize a novel nanosystem consisting of nano-Lavandula angustifolia essential oil and gold nanoparticles prepared through ultrasonic nanoemulsifying techniques in order to promote wound healing and combat bacterial infection. LEO showed potent antibacterial activity against Klebsiella pneumoniae, MRSA and Staphylococcus aureus with minimum inhibitory concentration (MIC) values of 32, 16 and 16 µg/mL, respectively, while exhibiting low activity against Proteus mirabilis. Interestingly, the newly formulated nano-gold/nano-Lavandula angustifolia penetrated the preformed P. mirabilis biofilm with a full eradication of the microbial cells, with MIC and MBEC (minimal biofilm eradication concentration) values reaching 8 and 16 µg/mL, respectively. The cytotoxic effect of the novel nanoformula was also assessed against WI-38 fibroblasts vero (normal) cells (IC50 = 0.089 mg/mL) while nano-gold and nano-Lavandula angustifolia showed higher results (IC50 = 0.529, and 0.209 mg/mL, respectively). Nano-gold/nano-Lavandula angustifolia formula possessed a powerful wound healing efficacy with a 96.78% wound closure. These findings revealed that nano-gold/nano-Lavandula angustifolia nanoemulsion can inhibit bacterial growth and accelerate the wound healing rate.

Biofilm Formation and Phospholipase and Proteinase Production in Cryptococcus neoformans Clinical Isolates and Susceptibility towards Some Bioactive Natural Products.

Cryptococcosis is one of the most common fungal infections in immunocompromised patients, which is caused by Cryptococcus neoformans. However, relatively little is known about the virulence factors of C. neoformans and the incidence of antifungal drug resistance in C. neoformans is rapidly increasing. This study was undertaken to investigate the virulence factors in C. neoformans, thymol, curcumin, piperine, gallic acid, eugenol, and plumbagin for their potential antimicrobial activity against C. neoformans. The production of phospholipase and proteinase was detected using standard methods. Biofilm formation was determined using the microtiter plate method. The broth microdilution method was used to determine the antifungal activity. The antibiofilm activity was assessed using the safranin staining method. All isolates of C. neoformans produced biofilms with optical density values ranging from 0.16 to 0.89. A majority of C. neoformans isolates that were tested exhibited strong phospholipase (7/8) and proteinase (5/8) production. Plumbagin (with minimum inhibitory concentration values ranging from 4 to 16 μg/mL) showed the highest antifungal activity followed by thymol (with minimum biofilm inhibitory concentration values ranging from 8 to 64 μg/mL). In addition, plumbagin showed the highest antibiofilm activity with minimum biofilm inhibitory concentration and minimum biofilm eradication concentration values ranging from 4 to 16 μg/mL and 32 to 256 μg/mL, respectively. Plumbagin, compared to other natural products studied, was the most efficient in terms of antifungal and antibiofilm activities. Hence, plumbagin could be used in combination with antifungals for the development of new anticryptococcal drugs.

Antimicrobial, Antibiofilm, and Antioxidant Potentials of Four Halophytic Plants, Euphorbia chamaesyce, Bassia arabica, Fagonia mollis, and Haloxylon salicornicum, Growing in Qassim Region of Saudi Arabia: Phytochemical Profile and In Vitro and In Silico Bioactivity Investigations.

The current study aimed to investigate the phytochemical contents and antioxidant, antimicrobial, and antibiofilm activities of four halophytic plants, namely, Euphorbia chamaesyce, Bassia arabica, Fagonia mollis, and Haloxylon salicornicum, native to central Saudi Arabia. The alcoholic extract of E. chamaesyce was found to be the most potent in various bioactivities-based evaluations and rich in polyphenols and flavonoid secondary metabolites, with 68.0 mg/g and 39.23 mg/g gallic acid and quercetin equivalents, respectively. Among all plants' extracts, the alcoholic extract of E. chamaesyce had the highest DPPH scavenging and metal chelating antioxidant activities at 74.15 Trolox equivalents and 16.28 EDTA equivalents, respectively. The highest antimicrobial activity of E. chamaesyce extract was found to be against Shigella flexneri, with a mean zone of inhibition diameter of 18.1 ± 0.2 mm, whereas the minimum inhibitory concentration, minimum biocidal concentration, minimum biofilm inhibitory concentration, and minimum biofilm eradication concentration values were 12.5, 25, 25, and 50 mg/mL, respectively. The LC-ESI-MS/MS analysis of the E. chamaesyce extract showed the presence of six flavonoids and ten phenolic constituents. The in silico binding of the E. chamaesyce extract's constituents to Staphylococcus aureus tyrosyl-tRNA synthetase enzyme displayed -6.2 to -10.1 kcal/mol binding energy values, suggesting that these constituents can contribute to the antimicrobial properties of the plant extract, making it an essential medicinal ingredient. In conclusion, these results warrant further investigation to standardize the antimicrobial profiles of these plant extracts.

Antibiofilm Activity of Essential Fatty Acids Against Candida albicans from Vulvovaginal Candidiasis and Bloodstream Infections.

PurposeThe biofilm formation of Candida albicans is an important virulence factor as it can increase tolerance to conventional antifungal drugs and the host immune system. The study aimed to assess the effect of essential fatty acids (EFAs) against biofilm formation and mature biofilms of C. albicans strains, which were isolated from vulvovaginal candidiasis and candidemia.MethodsThe biofilm formation ability of C. albicans and antifungal activities of fluconazole were determined. Additionally, the effects of six EFAs [α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LOA), γ-linolenic acid (GLA), and arachidonic acid (AA)] against C. albicans under planktonic and biofilm conditions were evaluated.Results94.1% of C. albicans exhibited biofilm formation capacity, and 98.5% of C. albicans were susceptible to fluconazole. The biofilms of C. albicans were highly resistant to fluconazole with minimum biofilm eradication concentration values ≥ 64 µg/mL. The EFAs attenuated biofilm formation in a dose-dependent manner, and GLA displayed a remarkable inhibitory activity against biofilm formation of C. albicans. In addition, EPA, DHA, and GLA at 0.1 mM could inhibit the biofilm formation of C. albicans without affecting the planktonic growth rate. Notably, EPA and AA at 1 mM had both inhibitory and eradication activities on C. albicans biofilms.ConclusionThis is the first study to directly compare different EFAs for their capacity to affect C. albicans biofilm formation as well as biofilm eradication. These results suggest EPA and AA could serve as potential new antifungal agents for the treatment of clinical infections caused by C. albicans biofilms.

Antibiofilm Synergistic Activity of Streptomycin in Combination with Thymol-Loaded Poly (Lactic-co-glycolic Acid) Nanoparticles against Klebsiella pneumoniae Isolates.

Background Thymol is an important component of essential oils found in the oil of thyme, is extracted mainly from Thymus vulgaris, and was shown to act synergistically with streptomycin against Klebsiella pneumoniae biofilms. Additionally, thymol could be encapsulated into poly (lactic-co-glycolic acid) (PLGA) nanoparticles to overcome issues related to its low water solubility and high volatility. The present study aimed to investigate the antibiofilm activity of thymol-loaded PLGA nanoparticles (Thy-NPs) alone and in combination with streptomycin against biofilms of K. pneumoniae isolates. Methods The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The antibiofilm activities were determined by the safranin dye assay. The synergistic effect of Thy-NPs with streptomycin was assessed by the checkerboard method. The kinetic study of the biofilm biomass and time-kill assay were further performed. Results Thy-NPs exhibited the highest antibacterial activity against K. pneumoniae isolates, with MIC values ranging from 1 to 8 µg/mL. Additionally, Thy-NPs showed the highest antibiofilm activity against K. pneumoniae isolates with minimal biofilm inhibitory concentration (MBIC) and minimal biofilm eradication concentration (MBEC) values ranging from 16 to 64 µg/mL and from 32 to 128 µg/Ml, respectively. The combination treatment combining Thy-NPs with streptomycin showed a synergistic effect against the inhibition of biofilm formation and eradication of biofilms of K. pneumoniae isolates with fractional inhibitory concentration index values ranging from 0.13 to 0.28. In addition, the MBIC and MBEC values of streptomycin against K. pneumoniae isolates were dramatically reduced (up to 128-fold) in combination with Thy-NPs, suggesting that Thy-NPs would enhance the antibiofilm activity of streptomycin. The biomass and time-kill kinetics analysis confirmed the observed synergistic interactions and showed the bactericidal activity of streptomycin in combination with Thy-NPs. Conclusions Our results indicate that the synergistic bactericidal effect between streptomycin and Thy-NPs could be a promising approach in the control of biofilm-associated infections caused by K. pneumoniae.

Improvement of antimicrobial susceptibility testing in biofilm-growingcoagulase-negative Staphylococcus hominis

Coagulase-negative Staphylococcus hominis causes bloodstream infections and often can form biofilms on medical devices. This study aimed to improve the current methodology for antimicrobial susceptibility testing (AST) in biofilm-growing S. hominis isolates. Biofilm production of S. hominis was assessed using the crystal violet staining method in trypticase soy broth supplemented with 1% glucose (TSBglu1%), Mueller-Hinton broth (MHB), or MHBglu1% using flat-bottom plates or the Calgary device. Susceptibility to antibiotics was assessed using the broth microdilution method (MHB and TSBglu1%) in planktonic cells (round-bottom plates) and biofilm cells (flat-bottom plates and the Calgary device). Biofilm determination using TSBglu1% yielded better performance over MHB, and flat-bottom plates without agitation were preferred over the Calgary device. Higher fold dilution values between the minimum biofilm eradication concentration (MBEC) and the minimum inhibitory concentration (MIC) were obtained in MHB for almost all antibiotics, except for linezolid. TSBglu1% and flat-bottom polystyrene plates were preferred over MHB and the Calgary device for biofilm determination. AST in biofilm-growing S. hominis showed better performance using TSBglu1% compared to MHB. Therefore, when comparing MBEC and MIC values, AST in planktonic cells could also be performed using TSBglu1% instead of MHB.

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices.

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

Antibiofilm and anticancer activities of unripe and ripe Azadirachta indica (neem) seed extracts

BackgroundAntibiotic resistances of pathogens and breast cancer warrant the search for new alternative strategies. Phytoextracts can eradicate microbe-borne diseases as well as cancer with lower side effects compared to conventional antibiotics.AimUnripe and ripe Azadirachta indica (neem) seed extracts were explored as potential antibiofilm and anticancer agents in combating multidrug-resistant infectious bacteria as well as anticancer agents against the MDR breast cancer cell lines.Methods Shed-dried neem seeds (both unripe and ripe) were pulverized and extracted using methanol. The chemical components were identified with FTIR and gas chromatography - mass spectrometry. Antibiofilm activity of neem seed extracts were assessed in terms of minimum biofilm inhibitory concentration (MBIC), minimum biofilm eradication concentration (MBEC), and fluorescence microscopic studies on Staphylococcus aureus and Vibrio cholerae. Bacterial cells were studied by fluorescence microscopy using acridine orange/ethidium bromide as the staining agents. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were evaluated to observe the antibacterial activities. Cytotoxicity of the extracts against human blood lymphocytes and the anticancer activity against drug-resistant breast cancer cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence-activated cell sorting (FACS) studies.Results 4-Ethyl-2-hydroxy-2-cyclopentene-1-one, phthalic acid, and 2-hexyl-tetrahydro thiophane were the major compounds in unripe neem seed, whereas 3,5-dihydroxy-6-methyl-2,3-dihydro-4-H-pyran-4-one and 4-ethylbenzamide were predominant in ripe neem seed. Triazine derivatives were also common for both the extracts. MBIC values of unripe and ripe neem seed extracts for S. aureus are 75 and 100 µg/mL, respectively, and for V. cholerae, they are 100 and 300 µg/mL, respectively. MBEC values of unripe and ripe seed extracts are 500 and 300 µg/mL, respectively for S. aureus and for V. cholerae the values are 700 and 500 µg/mL, respectively. Fluorescence microscopic studies at 16 and 24 h, after bacterial culture, demonstrate enhanced antibiofilm activity for the ripe seed extract than that of the unripe seeds for both the bacteria. MTT assay reveals lower cytotoxicity of both the extracts towards normal blood lymphocytes, and anticancer activity against breast cancer cell line (MDA-MB-231) with superior activity of ripe seed extract. FACS studies further supported higher anticancer activity for ripe seed extract.ConclusionsMethanolic extract of neem seeds could substantially inhibit and eradicate biofilm along with their potent antibacterial and anticancer activities. Both the extracts showed higher antibiofilm and antibacterial activity against S. aureus (gram-positive) than V. cholerae (gram-negative). Moreover, ripe seed extract showed higher antibiofilm and anticancer activity than unripe extracts.Graphical

Prevention, inhibition, and degradation effects of melittin alone and in combination with vancomycin and rifampin against strong biofilm producer strains of methicillin-resistant Staphylococcus epidermidis

Methicillin-resistant Staphylococcus epidermidis (MRSE) bacteria are being recognized as true pathogens as they are able to resist methicillin and commonly form biofilms. Recent studies have shown that antimicrobial peptides (AMPs) are promising agents against biofilm-associated bacterial infections. In this study, we aimed to explore the antibiofilm activity of melittin, either alone or in combination with vancomycin and rifampin, against biofilm-producing MRSE strains. Minimum biofilm preventive concentration (MBPC), minimum biofilm inhibition concentration (MBIC), and minimum biofilm eradication concentration (MBEC), as well as fractional biofilm preventive-, inhibitory-, and eradication concentrations (FBPCi, FBICi, and FBECi), were determined for the antimicrobial agents tested. Cytotoxicity and hemolytic activity of melittin at its synergistic concentration were examined on human embryonic kidney cells (HEK-293) and Red Blood Cells (RBCs), respectively. The effect of melittin on the downregulation of biofilm-associated genes was explored using Real-Time PCR. MBPC, MBIC, and MBEC values for melittin were in the range of 0.625–20, 0.625–20, and 10–40 μg/μL, respectively. Melittin showed high synergy (FBPCi, FBICi and FBECi < 0.5). The synergism resulted in a 64–512-fold, 2–16 and 2–8-fold reduction in melittin, rifampicin and vancomycin concentrations, respectively. The synergistic melittin concentration found to be effective did not manifest either cytotoxicity on HEK-293 or hemolytic activity on RBCs. Results showed that melittin downregulated the expression of biofilm-associated icaA, aap, and psm genes in all isolates tested, ranging from 0.04-folds to 2.11-folds for icaA and from 0.05 to 3.76-folds for aap and psm. The preventive and therapeutic indexes of melittin were improved 8-fold when combined with vancomycin and rifampin. Based on these findings, the combination of melittin with conventional antibiotics could be proposed for treating or preventing biofilm-associated MRSE infections.

Rifabutin versus rifampicin bactericidal and antibiofilm activities against clinical strains of Staphylococcus spp. isolated from bone and joint infections

Staphylococci account for approximately 60% of periprosthetic joint infections (PJIs). Rifampicin (RMP) combination therapy is generally considered to be the treatment of choice for staphylococcal PJIs but carries an important risk of adverse events and drug-drug interactions. Rifabutin (RFB) shares many of the properties of rifampicin but causes fewer adverse events. To compare the minimal inhibitory concentration (MIC), the minimum bactericidal concentrations (MBC), and the minimum biofilm eradication concentrations (MBEC) of rifabutin and rifampicin for staphylococcal clinical strains isolated from PJIs. 132 clinical strains of rifampicin-susceptible staphylococci [51 Staphylococcus aureus (SA), 48 Staphylococcus epidermidis (SE) and 33 other coagulase-negative staphylococci (CoNS)] were studied. The MBC and the MBEC were determined using the MBEC® Assay for rifabutin and rifampicin and were compared. When compared with the rifampicin MIC median value, the rifabutin MIC median value was significantly higher for SA (P < 0.05), but there was no statistically significant difference for SE (P = 0.25) and CoNS (P = 0.29). The rifabutin MBC median value was significantly higher than that of rifampicin for SA (P = 0.003) and was lower for SE (P = 0.003) and CoNS (P = 0.03). Rifabutin MBEC median value was statistically lower than that of rifampicin for all strains tested. Using the determination of MBEC values, our study suggests that rifabutin is more effective than rifampicin against clinical strains of Staphylococcus spp. obtained from PJIs. Using MBECs instead of MICs seems to be of interest when considering biofilms. In vivo higher efficacy of rifabutin when compared with rifampicin needs to be confirmed.

Synergistic Antibiofilm Efficacy of Thymol and Piperine in Combination with Three Aminoglycoside Antibiotics against Klebsiella pneumoniae Biofilms.

Background Thymol and piperine are two naturally occurring bioactive compounds with several pharmacological activities. In this study, their antibiofilm potential either alone or in combination with three aminoglycoside antibiotics was evaluated against a biofilm of Klebsiella pneumoniae. Methods Determination of antimicrobial susceptibility was performed using the broth microdilution method. Biofilm formation was evaluated by the microtiter plate method. Antibiofilm activity was determined using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay. The combination studies were performed by the checkerboard microdilution method. Results The minimum biofilm inhibitory concentration (MBIC) of streptomycin was reduced by 16- to 64-fold when used in combination with thymol, while the MBIC of kanamycin was reduced by 4-fold when combined with piperine. The minimum biofilm eradication concentration (MBEC) values of streptomycin, amikacin, and kanamycin were, respectively, 16- to 128-fold, 4- to 128-fold, and 8- to 256-fold higher than the planktonic minimum inhibitory concentration (MIC). Thymol combined with streptomycin or kanamycin showed synergic effects against the preformed biofilm with 16- to 64-fold reduction in the minimum biofilm eradication concentration values of each antibiotic in combination. Piperine acted also synergically with kanamycin with an 8- to 16-fold reduction in the minimum biofilm eradication concentration values of kanamycin in combination. Conclusion The association of thymol with antibiotics showed a strong synergistic effect both in the inhibition of biofilm formation and the destruction of the preformed biofilm of K. pneumoniae. This study suggests that a combination of thymol with streptomycin, amikacin, or kanamycin could be a promising alternative therapy to overcome the problem of K. pneumoniae biofilm-associated infections.

Anti-biofilm activity of murepavadin against cystic fibrosis Pseudomonas aeruginosa isolates.

To determine the activity of murepavadin in comparison with tobramycin, colistin and aztreonam, against cystic fibrosis (CF) Pseudomonas aeruginosa isolates growing in biofilms. The biofilm-epidemiological cut-off (ECOFF) values that include intrinsic resistance mechanisms present in biofilms were estimated. Fifty-three CF P. aeruginosa isolates from respiratory samples were tested using the Calgary (closed system) device, while 4 [2 clinical (one smooth, one mucoid) and 2 reference strains] were tested using the BioFlux, a microfluidic open model of biofilm testing. Biofilm was stained with SYTO9® and propidium iodide. The minimal biofilm inhibitory concentration (MBIC) and the minimal biofilm eradication concentration (MBEC) were determined. The MBIC-ECOFF and the MBEC-ECOFF were calculated. Colistin, tobramycin and murepavadin presented similar MBIC50/MBIC90 values (4/32, 8/64 and 2/32, respectively). Murepavadin exhibited the lowest MBEC90 (64 mg/L). Aztreonam MBIC and MBEC values were higher than those of the other antibiotics tested. Tobramycin and murepavadin had the lowest MBEC-ECOFF (64 and 128 mg/L, respectively), while those of aztreonam and colistin exceeded 512 mg/L. Using the BioFlux, for the PAO1, PAO mutS and the smooth clinical strain, a significant difference (P < 0.0125) was observed when comparing the fluorescence of treated and untreated biofilms. For the mucoid strain, only the biofilm treated with aztreonam (MBIC and MBEC) and tobramycin (MBEC) showed differences with respect to the untreated biofilm. Murepavadin demonstrated good activity against P. aeruginosa biofilms both in open and closed systems. The MBIC-ECOFF and the MBEC-ECOFF are proposed as new parameters to estimate the activity of antibiotics on biofilms.