Quantifying Staphylococcus aureus biofilm formation on stainless steel and titanium alloy used in 1 orthopedic fracture fixation 2

18 In order to assess and compare the antibacterial property of implants surfaces, a standard method 19 is needed to quantify bacterial load. This study evaluated the effectiveness of three quantifying methods, 20 namely, (I) crystal violet staining analysis, (II) ultrasound detachment with viable cell counts, and (III) 21 confocal laser scanning microscopy for characterizing S. aureus Seattle 1945 (ATCC 25923) biofilm on 22 metallic coupons. The accuracy of the results, time for completion, and ease of use of methods were 23 compared. The crystal violet method is relatively faster and more straightforward for analyzing biofilm 24 formation. However, the accuracy of the confocal laser scanning microscopy method is found to be 25 considerably higher than that of the other methods. Confocal laser scanning microscopy method is considered to be more time-consuming for data collection and analysis and costlier. The ultrasound 27 detachment followed by viable cell count of recovered cells is recommended for biofilm quantification 28 analysis on orthopedic materials when there is a large number of samples (more than ten samples). This 29 info could provide guidelines that would facilitate the selection of suitable method for quantifying biofilm 30 formation on orthopedic implants based on investigators’ consideration on method accessibility, assay cost, 31 assay time, and complexity of method. 32 33

considerably higher than that of the other methods. Confocal laser scanning microscopy method is 26 considered to be more time-consuming for data collection and analysis and costlier. The ultrasound 27 detachment followed by viable cell count of recovered cells is recommended for biofilm quantification 28 analysis on orthopedic materials when there is a large number of samples (more than ten samples). This 29 info could provide guidelines that would facilitate the selection of suitable method for quantifying biofilm 30 formation on orthopedic implants based on investigators' consideration on method accessibility, assay cost, 31 assay time, and complexity of method. 32

INTRODUCTION 36
Biofilms consist of a group of microorganisms attached to a surface within an extracellular 37 polymeric substance matrix. When the film structure is established, the microorganisms inside it are more 38 resistant to various antimicrobial agents 1 . Post-surgery biofilm formation on orthopedic implants is a major 39 problem in the treatment of the patients 2 A biofilm delays the healing process and increases the probability 40 that revision surgeries may be needed to remove the contaminated implant 2 . The attachment of the biofilm 41 has been shown to be a complex process that is influenced by different environmental factors and surface 42 characteristics 3 . Surface roughness, and associated changes in effective surface area, is one of the known 43 factors that affect microorganism's colonization. It appears that colonization increases with escalations in 44 surface roughness 4 . In addition to roughness, surface hydrophobicity can also play a role in the initial 45 attachment. Some studies showed that microorganisms attach faster to hydrophobic, nonpolar surfaces such 46 as Teflon than hydrophilic surfaces like glass or metals 3,4 . 47 New implant materials or surface modification methods have been developed to reduce biofilm 48 formation 5-7 . Prior to in vivo application of these materials or surface modifications, the anti-biofilm 49 properties need to be evaluated in vitro. Currently, there is a range of methods that have been used to 50 quantify biofilms on solid surfaces in vitro and these can be classified into three groups: (I) microscopic faster than laser confocal microscopy 17,18 . The method is based on recovering the live bacteria from the 78 surface of biofilm by detaching them through a sonication technique followed by a plate counting method 8 . 79 The low-moderate sonication (20-40 kHz) of the bacterial suspension in the water bath for a short duration 80 (2-10 min), has been shown to have the optimum effect on bacterial detachment. Longer exposure time or 81 higher ultrasound power leads to disruption of bacterial cell wall 19,20 . 82 In this study, three different quantification methods were compared for the characterization of Stainless steel and titanium are the most common alloys used for fracture fixation implants 9 . In this 93 study, biofilms were grown on titanium-6Al-4Vand 316 stainless steel cylindrical disks (Lisnabrin 94 Engineering, Ireland) and will be referred to in this manuscript as Ti and SS coupons respectively. Each 95 coupon (1 cm diameter, 1 mm thickness) were laser cut from 1 mm thick sheets of either grade 5 Ti-6Al-96  To be able to quantify the amount of biofilm formed on just the upper side of the SS and Ti coupons, 120 the underside of individual coupons was coated a mixture of vaseline, lanolin and paraffin wax in equal 121 rations (VaLaP). This is a biologically inert material that has been shown to prevent bacterial attachment 26 . 122 Covering one side of the surface is important when quantifying biofilm using the CV and ultrasound with 123 VCC methods, while it is not necessary when using the CLSM method as only the upper surface is imaged. 124 125

Cultivation and quantification of biofilm 126
Cultivation of biofilm growth were conducted using the following protocol. Briefly, 24-well flat-127 bottom non-tissue-culture treated microtiter plates were used to minimize binding of bacteria onto the wells and to facilitate the cultivation of biofilm on metal coupon surfaces. Coupons were dipped gently into 129 melted VaLaP until the bottom side was covered. Each coupon was then placed into a separate well of the 130 24-well plate. 600 µL of TSB+1% glucose was added to each well to cover the entire surface of the coupon. 131 The concentration of the overnight S. aureus was determined spectrophotomically and 10 3 bacterial cells 132 were added to each well containing coupon and the plate was incubated at 37 °C for 24 h. Washing of metal 133 coupons after growth of biofilms was carried out by dipping the coupons in wells containing 500 µL of 134 deionized (D.I.) water. This process was repeated three times to wash away planktonic bacteria. The 135 biofilms on metal coupons were then observed using an epi-fluorescence microscope or a laser confocal 136 microscope. For quantification of biofilm, the CV staining method or viable cell count experiment was 137 used. A positive control, i.e., a well containing TSB and bacterial inoculum, and a negative control, i.e., a 138 well containing TSB without bacteria were included for each individual experiment 14,16 . 139 140

Quantification of biofilm via CV staining 141
For quantification of biofilm growth on the coupons using CV method, they were processed as 142 follows: After the last wash to remove unbound planktonic cells, the coupons were placed into empty wells 143 and allowed to air-dry at 37 °C for 30 min. Metal coupons were placed into wells containing 600 µL of

Quantification of biofilm using viable cell count 181
A viable count test was performed to quantify biofilm formation on the coupons since this method 182 yields more consistent and sensitive results than the CV method. We followed the Bjerkan method with 183 modification; briefly, after cultivation of biofilm on the coupon surface, each disk was placed into a 15 mL 184 sterile conical tube containing 5 mL of PBS and gently vortexed for 15 seconds to rinse off planktonic cells. 185 After addition of 5 mL of PBS, each tube was then subjected to sonication for 5 min at full wave (40 KHz) 186 in a DK Sonic water bath ultra-sonic cleaner. The power was enough to detach the bacterial cells from the 187 surface, but not to damage the cells. This wash 1 solution (W1) was serially diluted 10 -1 , 10 -2 , 10 -3 , and 10 - Images were taken using CLSM with a 63x objective and oil immersion. For each coupon, three image 247 stacks were taken in three discrete randomly picked locations. Figure 3 (a, b) shows a 3D structure of 248 biofilm on the metal coupon surface, and Figure 3 (c, d) shows the wide filed images of biofilm on the 249 metal coupon surface. To analyze the average thickness and biomass of biofilm formed on coupon surfaces, 250 multiple laser confocal image stacks were taken and analyzed using COMSTAT program. From the 251 collected data (Figure 2 b, c), Ti and SS material surfaces showed different amounts of biofilm formed on 252 them. However, no significant difference was observed between the material surfaces. To validate the consistency and reproducibility of the methods, biofilm was grown on the glass 272 coverslips and analyzed using the same three quantification methods used to evaluate biofilm on metal 273 coupons. The results showed that the biofilm formed successfully on the glass coverslips (Figure 4).