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

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background: </jats:bold>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<jats:sup>TM</jats:sup>, a clinically used antibiotic containing allograft bone product, was tested for antibiofilm efficacy. <jats:bold>Results: </jats:bold>The commonly used Calgary biofilm device was used to grow 24 h and 72 h biofilms of <jats:italic>Staphylococcus aureus</jats:italic> and <jats:italic>Pseudomonas aeruginosa</jats:italic>, which were treated with time-dependent vancomycin (up to 3000 mg/L) and concentration-dependent tobramycin (up to 80 mg/L), 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 <jats:italic>S. aureus</jats:italic> biofilms. Tobramycin treatment of 24 h <jats:italic>P. aeruginosa</jats:italic> biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h <jats:italic>P. aeruginosa</jats:italic> biofilms. Treatment with tobramycin containing OSTEOmycin T<jats:sup>TM</jats:sup> removed 72 h and 168 h <jats:italic>P. aeruginosa</jats:italic> biofilms after one day treatment, while few 72h <jats:italic>S. aureus</jats:italic> biofilms survived after two days treatment with vancomycin containing OSTEOmycin V<jats:sup>TM</jats:sup>. <jats:bold>Conclusions:</jats:bold> 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 <jats:italic>in vitro</jats:italic> 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.</jats:p>

Halicin Is Effective Against Staphylococcus aureus Biofilms In Vitro

“…Like halicin, tobramycin was as effective against S. aureus in less mature 3-day biofilms as those in planktonic cultures, but higher concentrations were needed for more mature 7-day biofilms (Table 1). The tobramycin results are consistent with previous studies showing that its effectiveness against biofilms depends on biofilm maturity [2,9,10,12,13,[20][21][22][23]36]. Despite its activity against biofilms, tobramycin resistance is common in methicillinresistant S. aureus and other staphylococci isolates in patients with prosthetic joint infection [5], whereas halicin is effective against drug-resistant bacteria, including methicillin-resistant S. aureus, and did not induce resistance in vitro [48].…”

“…We also did not use imaging-based techniques to confirm biofilm formation in our cultures. However, biofilm formation and the expected time-based increase in biofilm maturity [9, 13, 27, 39, 57] were functionally confirmed by the large increases in the MBEC 75 of cefazolin, vancomycin, rifampicin for both 3-day and 7-day biofilms compared with their MIC and the increased MBEC 75 of tobramycin for 7-day biofilms (Table 1). Another limitation is that our study focused exclusively on a single bacteria type.…”

“…Results of studies assessing the effects of antibiotics on biofilms are highly dependent on the experimental conditions that lead to differing levels of biofilm maturity [2, 9, 10, 12, 13, 20-23, 27, 28, 36, 39, 57, 59]. However, biofilm maturity is difficult to measure directly but can be assessed functionally by comparing MICs and MBECs for antibiotics, such as tobramycin, cefazolin, vancomycin, and rifampicin, which are known to have different effects on planktonic cultures, less mature biofilms, and more mature biofilms.…”

A methodological review on the characterization of microalgal biofilm and its extracellular polymeric substances

“…To date, there is no universally adopted definition of young and mature biofilm. Some researchers considered 4 h biofilm as young and 24 h as mature while the others considered 24 h as young and 12 days as mature in the case of Pseudomonas aeruginosa (Chen et al, 2020). Biofilm age influences micro‐community structure and its corresponding metabolic activities.…”

In vitro investigations of Staphylococcus aureus biofilms in physiological fluids suggest that current antibiotic delivery systems may be limited

“…Importantly, in vivo results demonstrated a dynamic antibiotic concentration profile, whereas in vitro experiments maintained the designated antibiotic concentration for the duration of the experiment. In vitro biofilm characteristics, such as growth age and exposure time (Chen et al, 2020) as well as timeline of antibiotic therapy (Post et al, 2017), are important to consider when evaluating in vitro results (Chen et al, 2020). In vivo, Castaneda et al (2016) demonstrated that a 5 d course of antibiotic exposure lower the MBEC compared to a 24 h exposure, suggesting that a 24 h model may overestimate minimum concentrations needed to eradicate biofilms in vivo.…”