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

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 treatment duration and growth media on biofilm eradication in this study. The commonly used biofilm model 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 and concentration-dependent tobramycin, 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. This study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotics treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments.


Introduction
To improve diagnosis, treatment and prevention of infections, it is necessary to differentiate between acute infections with primarily planktonic microorganisms and biofilm infections with overweight of clusters of microbial cells (1)(2)(3)(4). Most microorganisms in a biofilm grow slowly with down-regulated virulence and are heterogeneously distributed. They are less susceptible to antibiotics compared with their planktonic counterpart and can often not be cleared by the immune system (5)(6)(7). Biofilm related infections can be device-related biofilm infections, such as prosthetic joint infections, or native tissue infections e.g. chronic osteomyelitis and cystic fibrosis. The most effective treatment of biofilm related infections is to remove the infected medical device and to debride the infected tissue in combination with antibiotic therapy (8). cation-adjusted Mueller Hinton Broth (CaMHB) media are often used in these studies. TSB is a complex nutrient-rich general-purpose medium, while CaMHB is recommended for MIC testing of nonfastidious organisms according to ISO standard 20776- 1 (2006) and is the standard medium in clinical laboratories in the US and European committee on antimicrobial susceptibility testing. High throughput methods for MBEC determination are most frequently used including 96-well microtiter plate combined with crystal violet staining, the Calgary biofilm device (CBD), or its commercial version the MBEC™ Assay (Innovotech, Canada) (9). As shown in Table 1-2, MIC values were similar for most of the studies. However, MBEC of vancomycin towards S. aureus varies from 1 to more than 8000 mg/L. Similarly, the MBEC of tobramycin towards P. aeruginosa varies from 2 to 2560 mg/L. This large discrepancy in MBEC values is surprising, especially in light of some studies using the same strain. We hypothesize that the different test parameters and lack of standardization contributed to the large disparity.
The purpose of this study was to demonstrate the influences of biofilm age, growth media, and antibiotics exposure time on S. aureus and P. aeruginosa biofilm removal using vancomycin and tobramycin, respectively. These two antibiotics were chosen because they are recommended for serious and life-threatening infections caused by Gram-positive bacteria and Gram-negative bacteria.
In addition, we investigated the possibility of biofilm eradication by local antibiotics treatment by testing OSTEOmycin, an allograft bone product loaded with either vancomycin or tobramycin (10,11).

Results
Four biofilm formation strains were chosen for this study. S. aureus strains SAU060112 (12) was isolated from prosthetic knee infection while S. aureus ATCC 49230 was originally from chronic osteomyelitis. Both infections are known to be associated with biofilms. P. aeruginosa strain PA14 is a well-known biofilm former (13) and P. aeruginosa ATCC 15442 is also known to form biofilms (14,15).
All four tested strains in this study were found susceptible to the tested antibiotics. The vancomycin MIC for both S. aureus strains was determined to be 1.25 mg L -1 , which is lower than breakpoint (2 mg L -1 ) for S. aureus. Likewise, the tobramycin MIC for both P. aeruginosa strains was 0.63 mg L -1 , which is lower than breakpoint (4 mg L -1 ) for P. aeruginosa according to Clinical breakpoints -bacteria (v 9.0) in European Committee on Antimicrobial Susceptibility Testing (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_9.0_Breakpoint_Tables.pdf)

Influence of biofilm age
Biofilm growth is dynamic and mature biofilms are thought to be more antibiotic tolerant. In this study biofilms grew for 24 h or 72 h first and then were subjected to antibiotics challenge for different duration. It was found that the number of colony forming units (CFUs) were higher for 72 h biofilms than for 24 h biofilms by up to 1-log difference (P<0.01, Figure S2

Media
Biofilm formation depends on many factors including nutrient availability. The main nutrients in both TSB and CaMHB media are amino acids. In addition, TSB contains glucose (2.5 g L -1 ) while CaMHB has starch (1.5 g L -1 ). The number of CFUs in the biofilms growing in these two media were different (P < 0.05, Figure S2 and S3). On average, slightly more CFUs were found in biofilms growing in CaMHB than TSB, except 72 h PA14 biofilms.
When challenged with antibiotics, biofilms were more difficult to kill in TSB than in CaMHB.

Antibiotics exposure time
Vancomycin is known to be a time-dependent antibiotic (16). Extending vancomycin exposure time from one to four days reduced survival ratio of SAU060112 biofilm in TSB (Figure 1a and 1c, Table 4) and CaMHB media (Figure 1b and 1d, Table 4). Prolonging treatment from four to seven days showed no further killing except 24 h biofilms in TSB (Table 4). Increased killing by prolonging vancomycin exposure was also found for S. aureus ATCC 49230 biofilms ( Figure 3a).
In contrast to vancomycin, tobramycin is known to exhibit concentration-dependent bactericidal activity (17). Removal efficacy of 24 h P. aeruginosa PA14 biofilm was not enhanced when duration was extended (Figure 2a and 2b, Table 5). However, increasing killing was indicated for 72 h PA14 biofilms (Figure 2c and 2d, Table 5) as well as for 72 h P. aeruginosa ATCC 15442 biofilms (Figure 3b).

Strains
The two S. aureus strains have the same vancomycin MIC value. Although the necessary concentration of vancomycin for biofilm eradication differed slightly, the same tendency is indicated for both strains that prolonged vancomycin treatment eradicated more biofilms. Similarly, the two P.

Biofilm age
Several biofilm models have been developed, each with many experimental parameters that can be adjusted. This flexibility inevitably makes it difficult to compare results obtained with varying conditions chosen in different studies. In the current study we confirmed previous findings that mature biofilm have reduced antibiotics susceptibility compared with young biofilms (18)(19)(20)(21).
However, no definition of young and mature biofilm has been universally adopted. In the case of P. aeruginosa, some considered 4 h biofilm as young and 24 h as mature (22), while others considered 7 young and 24 h as mature (23), whereas some considered 7 days old biofilm as mature (24). The inconsistency in the different biofilm studies underlines the need for a form of consensus definition and a simple way to measure maturity. The textbook version of biofilm formation involves bacterial initial attachment to a solid surface, the formation of microcolonies on the surface, and finally differentiation of microcolonies into exopolysaccharide-encased, mature biofilms. However, studies often assume the maturity of the biofilm without looking into the structure of the biofilms or even CFUs of biofilm. In the case of MIC testing, a crucial parameter is inoculum size which is set to be 5 x

Growth media
Biofilm eradication was found different with the two media ( Figure 1 and Figure 2). Different composition of media is reported to change the activity of antibiotics (27)(28)(29). The Ca 2+ and Mg 2+ ions in CaMHB media are required for a correct antimicrobial susceptibility testing because those ions reflect the divalent cation concentration in human blood (30-33). However, in vivo conditions are far more complex, and the biofilms formed in vivo often incorporate human factors such as blood components. Hence, the antibiotics concentration needed for biofilm eradication will mostly likely be different from in vitro results. For comparison between different studies, a simple and widely available culture media is suitable, but for estimation of in vivo biofilm killing host factors in form of, for example, serum, plasma, or blood should be included in testing medium.

Antibiotics exposure time
Vancomycin displayed a time-dependent eradication of S. aureus biofilms ( Table 2) (42). Therefore, a killing curve is much more informative than a definitive MBEC value determined at a fixed time point.

OSTEOmycin
Since the antibiotic concentration needed for biofilm eradication is far above the parenterally administrated levels, local delivery of antibiotics may achieve concentrations high enough for biofilm killing. In this study, OSTEOmycin showed a strong biofilm eradication efficacy and completely removed biofilm in all tested conditions except three 72 h S. aureus biofilms. OSTEOmycin is a product developed based on Winkler et al. 2000 (43). According to the study, 1 g human cancellous bone impregnated with vancomycin released around 20000 mg L -1 vancomycin in 3 mL of 5% human albumin solution after one day and decreased to around 100 mg L -1 after seven days. When impregnated with tobramycin, it released more than 10000 mg L -1 tobramycin after one day and decreased to around 30 mg L -1 after seven days (43). These concentrations are much higher than the MBEC values found in Figure 1 and 2, which likely explains the high efficacy. Osteomycin was also shown to be a promising product for local treatment of osteomyelitis in the clinic although recurrence may still occur in complex cases within an unknown period of time (11).

Conclusion
This study showed biofilm removal efficacy was influenced by media, biofilm age and antibiotics treatment duration. It is therefore necessary to take these parameters into consideration when designing experiments. We recommend choosing the conditions most similar to the in vivo situation and explaining the rationale when reporting. This study also showed that in vitro biofilms were possible to be eradicated when treated with long-term high concentrations of antibiotics. This finding needs to be confirmed by in vivo studies.

Material And Method
Bacterial strains, growth media and antibiotics S. aureus strains SAU060112 (12) and ATCC 49230 were tested with vancomycin (Sigma-Aldrich). P. aeruginosa strains PA14 and ATCC 15442 were tested with tobramycin (Sigma-Aldrich). Both TSB (Sigma-Aldrich) and CaMHB (Sigma-Aldrich) media were employed in susceptibility testing.

Minimum inhibitory concentration (MIC) determined by the broth microdilution method
The broth microdilution method was used to determine the MIC of each strain according to the procedures described in Wiegand et al. (44). Briefly, each strain was inoculated on TSB agar plate for 24 h. Then five well-isolated colonies were selected and inoculated in a 50 mL tube with 20 mL CaMHB until the OD600 value of the culture reached around 0.6. The culture was diluted to approximately 1 × 10 6 colony-forming unit (CFU) mL -1 . Then, 100 µl of the diluted culture was added into each well of a 96-well-plate containing 100 µl of antibiotics at different concentrations. The plate was covered and inoculated at 37℃ with shaking at 150 rpm for 24 h. After that, OD595 of each well was measured by Infinite F200 Pro (Tecan Group Ltd., Switzerland) to determine MIC.

Biofilm antibiotics susceptibility testing by Calgary Biofilm Device (CBD)
CBD (45) was used to grow biofilms. An illustration of the experimental procedure is given in Figure   S1. Briefly, biofilms were formed by immersing the pegs of a microtiter lid (Nunc TM 445497) into the biofilm growth microtiter plate ( Figure S1), 150 µl of the diluted culture containing 10 4 CFU was added into the wells of 96 well microtiter plate (Thermo Fisher Scientific) and then covered with peg lid The biofilms were allowed to grow in TSB or CaMHB media at 37℃ with shaking at 150 rpm for intended time (Table 3). After incubation, the lid with biofilms was transferred to a rinse plate containing 200 µl saline in each well and incubated for 1 minute. The rinsed lid was then transferred to a challenge plate containing 200 µl antibiotics solution in each well. The antibiotics were prepared in the media used for growing biofilms. After challenged in the antibiotics solution, the lid containing biofilms was rinsed twice with fresh saline each time and then transferred to a recovery plate containing 200 µl sterile media followed by sonication at 40KHz for five minutes.
The recovery plate was inoculated for another 24 h at 37℃ with shaking at 150 rpm and OD595 measured by Infinite F200 Pro to determine the biofilm removal efficacy. All tests were repeated at least on two occasions with minimum 10 replicates each time. Percentage of the surviving replicates was calculated and presented as biofilm survival ratio.

Biofilm eradication by OSTEOmycin TM
OSTEOmycin TM samples were obtained from European Cell and Tissue Bank (ECTB). Two clinical strains S. aureus ATCC 49230 and P. aeruginosa PA14 were chosen for this test. Seventy-two h (3days) or 168 h (7-days) biofilms were challenged with OSTEOmycin TM for 1, 2, 4 or 7 days following the method described above. S. aureus ATCC 49230 biofilms were challenged with 186 g L -1 OSTEOmycin V TM in CaMHB, while P. aeruginosa PA14 biofilms were subjected to 220 g L -1 OSTEOmycin T TM .

Statistics analysis
ANOVA was used to calculate the difference between biofilm formation on CBD pegs. Binary logistic regression model was used to compare biofilm removal efficacy under different conditions.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information file.

Competing interests
The authors declare that they have no competing interests.

Funding
This study is partly supported by Det Obelske Familiefond. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' contributions
The study was conceived and designed by YX, HW and TRT. XC performed laboratory experiments. XC and YX analyzed the data. YX, XC and TRT wrote the manuscript. All authors have read and approved the manuscript.

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