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 inTable 1 .
Confocal scanning laser microscopy (CSLM) was employed to confirm the 24-hour biofilm formation ability of each strain. 8 study groups were examined (
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
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