Staphylococcus epidermidis

The following bacterial species were used: Bacillus subtilis (ATCC 10707), Enterobacter cloacae (human isolate), Escherichia coli (ATCC 0157:H7), Micrococcus flavus (ATCC 9341),Proteus mirabilis (human isolate), Pseudomonas aeruginosa (ATCC 27853), Salmonella enteritidis (ATCC 13076), S. epidermidis (ATCC 12228) S. typhimurium (ATCC 13311) Staphylococcus aureus (ATCC 25923). The antibacterial assays were carried out by the disc-diffusion [25 (link)] and microdilution method [26 ,27 (link),28 (link)] in order to determine the antibacterial activity of oils and their components against the human pathogenic bacteria. The bacterial suspensions were adjusted with sterile saline to a concentration of 1.0 × 10⁵ CFU/mL. The inocula were prepared daily and stored at +4 °C until use. Dilutions of the inocula were cultured on solid medium to verify the absence of contamination and to check the validity of the inoculum.

Sterilized titanium (Ti6Al4V) and steel (AIS1316-L) discs were colonized by 1 of 4 different bacterial strains (Figure 1). All strains were clinical isolates from patients with chronic PJI. The bacterial strains were identified to the species level by biotyping and/or standard microbiological procedures: Staphylococcus aureus (coagulase-positive, nuc-positive staphylococcus), Staphylococcus epidermidis (ID-32 STAPH; bioMèrièux, Marcy l'Etoile, France; profile: 166010210), Enterococcus faecalis (rapid ID 32 STREP; bioMèrièux; profile: 30721715171), and Propionibacterium acnes (rapid ID 32A; bioMèrièux; profile: 2503377604).
Confocal scanning laser microscopy (CSLM) was employed to confirm the 24-hour biofilm formation ability of each strain. 8 study groups were examined (Table 1). Bacteria were suspended in 25 mL of Mueller Hinton broth (BD, Franklin Lakes, NJ) and incubated at 35ºC until a spectrophotometric density of approximately 1 × 10⁸ colony forming units/mL (CFU/mL) had been reached in the exponential growth phase. A batch of 40 discs (one study group) was immersed in this bacterial suspension bath and incubated at 35ºC for 24 h on a gently stirring agitator (20 rpm).
To remove non-adherent bacteria, the discs were rinsed 6 times in sterile saline. First, the discs for each study group were placed in a sterile plastic tube (Sarstedt, Norway) containing 25 mL saline and gently vortex mixed (MS2 Minishaker; IKA Works Inc., Wilmington, NC) at 100 rpm for 10 seconds. The discs were then transferred to another tube, and the procedure was repeated twice. Each single disc was then transferred to a sterile glass test tube containing 5 mL saline and subjected to vortex mixing at 100 rpm. The single disc rinsing was also repeated 3 times.
Aliquots of 50 µL saline were incubated on agar (Merck, Darmstadt, Germany) with 5% ox blood at 35ºC for 3 days. For culture of P. acnes, FAA agar (Merck) was incubated in an anaerobic cabinet for 7 days. The bacteria cultured were enumerated by colony counting. The number of CFU after final rinsing was recorded as a quantitative baseline, facilitating evaluation of the different detachment methods.
Each experimental group (10 discs) was subjected to 1 of 4 methods for biofilm detachment and bacterial recovery. The experimental design is summarized in Table 1.

A 222 nm‐KrCl excimer lamp, emitting primarily at 222 nm, was used for laboratory testing for susceptibility. The lamp was operated with an optical bandpass filter to reduce the nonpeak emissions (Buonanno et al., 2021 (link)) and was used as a light source in laboratory experiments to assess the effectiveness of far‐UVC light to inactivate two of the most relevant Staphylococcus species identified as S. hominis and S. epidermidis. The three S. hominis isolates used for testing susceptibility to 222 nm‐far‐UVC were M1018, M1020, and M1022; and the two S. epidermidis isolates were A#2 and A#4; all of them isolated on February 24, 2022. Modified from Burlage (1998 ), chosen isolates were grown overnight in 50 mL Trypticase Soy Broth (TSB) in a shaker at 37°C/140 rpm. Then, 400 μL from this overnight culture were inoculated into 50 mL of fresh TSB, and cells were regrown to mid‐exponential phase for approximately 2.5–3 h until the absorption at 600 nm was verified to be between 0.3 and 0.4 using an Eppendorf Biophotometer D30. The cells were pelleted by centrifugation at 2103g for 8 min then resuspended to the same volume in phosphate‐buffered saline (PBS). Cells were centrifugated to a pellet again and resuspended again in 50 mL of PBS. A volume of 2 mL of this cell suspension was spread in a 4 cm diameter Petri dish swirling gently to cover the bottom of the plate. Dishes were exposed using the filtered excimer lamp at a distance of 20 cm. The irradiance at this distance was measured at 120 µW/cm² using a Hamamatsu C9536 UV power meter with an H9535‐222 sensor head (Hamamatsu Corporation). Radiant exposure doses of 2, 10, or 20 mJ/cm² were administered using respective exposure times of 17 s, 1 min 23 s, or 2 min 47 s. Plates were prepared in triplicate for each irradiation, and control plates remained unirradiated. Serial dilutions were prepared and 100 μL were spread onto TSA plates, incubated at 37°C in the dark, and colony forming units (CFUs) were counted after 48 h.