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Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro.

ABSTRACT: BACKGROUND:Biofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported minimum biofilm eradication concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic concentration and treatment duration, and growth media on biofilm eradication. Additionally, OSTEOmycin™, a clinically used antibiotic containing allograft bone product, was tested for antibiofilm efficacy. RESULTS:The commonly used Calgary biofilm device was used to grow 24?h and 72?h biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, which were treated with time-dependent vancomycin (up to 3000?mg?L-?1) and concentration-dependent tobramycin (up to 80?mg?L-?1), respectively. Two common bacteriological growth media, tryptic soy broth (TSB) and cation-adjusted Mueller Hinton broth (CaMHB), were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24?h) were easier to eradicate than old biofilms (72?h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24?h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72?h P. aeruginosa biofilms. Treatment with tobramycin containing OSTEOmycin T™ removed 72?h and 168?h P. aeruginosa biofilms after 1 day treatment, while few 72?h?S. aureus biofilms survived after 2 days treatment with vancomycin containing OSTEOmycin V™. CONCLUSIONS:This study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotic concentration and treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments. The results of OSTEOmycin™ products indicated that simple in vitro biofilm test could be used for initial screening of antibiofilm products. For clinical application, a more clinically relevant biofilm model for the specific biofilm infection in question should be developed to guide the amount of antibiotics used for local antibiofilm treatment.

SUBMITTER: Chen X

PROVIDER: S-EPMC7444035 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro.

Chen Xiaofeng X Thomsen Trine Rolighed TR Winkler Heinz H Xu Yijuan Y

BMC microbiology 20200824 1

<h4>Background</h4>Biofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported minimum biofilm eradication concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental paramete ...[more]

PMID: 32831025

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Project description:ObjectivesStaphylococcus aureus and Pseudomonas aeruginosa were the most common bacteria in nosocomial infections. Different bacteriocins are currently being studied as antibiotics or in conjunction with antibiotics as potential strategies to treat resistant infectious agents. The study aimed to determine nisin's effect on the biofilm production, antimicrobial susceptibility, and biofilm formation of S. aureus and P. aeruginosa.Materials and methodsThe experimental research tested two antibiotic-resistant isolates of S. aureus and P. aeruginosa strains. The experimental study tested two antibiotic-resistant isolates of S. aureus and P. aeruginosa strains. The MIC of bacteriocin nisin was determined using the micro broth dilution method, and crystal violet was used to assess the effect of bacteriocin on the biofilm. In addition, L929 cell culture was used to determine the effectiveness of bacteriocin on the isolate under similar cell conditions. Moreover, the MTT assay was used to and evaluate bacteriocin toxicity. In this study, the software Prism version 9 and Graph pad software were utilized.ResultsThe results of this study reveal that the nisin has different activities at different doses and is considered dose-dependent. At various times and doses, nisin inhibits biofilm formation in S. aureus, and P. aeruginosa isolates. Nisin also showed a decreasing survival of the isolates. Antibiotic-resistant bacteria can be made more vulnerable by nisin. Furthermore, nisin treatment affected the production of virulence factors such as hemolysins in S. aureus and had little or a negative effect on P. aeruginosa virulence factors. This medication stops S. aureus and P. aeruginosa from growing and causes bacterial cell damage.ConclusionsAntibacterial properties of nicin against S. aureus and P. aeruginosa were successfully studied. This bacteriocin stops S. aureus and P. aeruginosa from growing and causes bacterial cell damage or death. Damage to the membrane among the fundamental causes is reduced membrane potential and enzyme inactivation.

Project description:Pseudomonas aeruginosa and Staphylococcus aureus often cause chronic, recalcitrant infections in large part due to their ability to form biofilms. The biofilm mode of growth enables these organisms to withstand antibacterial insults that would effectively eliminate their planktonic counterparts. We found that P. aeruginosa supernatant increased the sensitivity of S. aureus biofilms to multiple antimicrobial compounds, including fluoroquinolones and membrane-targeting antibacterial agents, including the antiseptic chloroxylenol. Treatment of S. aureus with the antiseptic chloroxylenol alone did not decrease biofilm cell viability; however, the combination of chloroxylenol and P. aeruginosa supernatant led to a 4-log reduction in S. aureus biofilm viability compared to exposure to chloroxylenol alone. We found that the P. aeruginosa-produced small molecule 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) is responsible for the observed heightened sensitivity of S. aureus to chloroxylenol. Similarly, HQNO increased the susceptibility of S. aureus biofilms to other compounds, including both traditional and nontraditional antibiotics, which permeabilize bacterial membranes. Genetic and phenotypic studies support a model whereby HQNO causes an increase in S. aureus membrane fluidity, thereby improving the efficacy of membrane-targeting antiseptics and antibiotics. Importantly, our data show that P. aeruginosa exoproducts can enhance the ability of various antimicrobial agents to kill biofilm populations of S. aureus that are typically difficult to eradicate. Finally, our discovery that altering membrane fluidity shifts antimicrobial sensitivity profiles of bacterial biofilms may guide new approaches to target persistent infections, such as those commonly found in respiratory tract infections and in chronic wounds.IMPORTANCE The thick mucus in the airways of cystic fibrosis (CF) patients predisposes them to frequent, polymicrobial respiratory infections. Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from the airways of individuals with CF, as well as from diabetic foot ulcers and other wounds. Both organisms form biofilms, which are notoriously difficult to eradicate and promote chronic infection. In this study, we have shown that P. aeruginosa-secreted factors can increase the efficacy of compounds that alone have little or no bactericidal activity against S. aureus biofilms. In particular, we discovered that P. aeruginosa exoproducts can potentiate the antistaphylococcal activity of phenol-based antiseptics and other membrane-active drugs. Our findings illustrate that polymicrobial interactions can dramatically increase antibacterial efficacy in vitro and suggest that altering membrane physiology promotes the ability of certain drugs to kill bacterial biofilms-knowledge that may provide a path for the discovery of new biofilm-targeting antimicrobial strategies.

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Dataset Information

Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro.

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Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro.

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