Microbiological samples were collected from all patients for:

1. Isolation of C. acnes
2. Antibiotic susceptibility testing for C. acnes isolates against the most commonly used antibiotics in the treatment of acne(Azithromycin and Doxycycline) in the presence and absence of Salicylic acid, Isotretinoin, and N-acetylcysteine
3. Testing biofilm forming ability of C. acnes isolates in the presence and absence of Salicylic acid, Isotretinoin, and N-acetylcysteine

Biofilm forming ability of P. acnes isolates was adapted from Coenye et al., 2007 and Abbott et al., 2022

G. The results were obtained according to (Stepanovic et al., 2007); the average OD values were calculated for all tested isolates & negative controls, and the cut-off value (ODc) was detected. It is defined as a 3 standard deviation (SD) above the mean OD of the negative control. The final OD value of a tested strain was expressed as the average OD value of the strain reduced by ODc value; ODc value was calculated for each plate separately. For easier interpretation of the results, strains were divided into the following categories:

• Non-biofilm producer = OD ≤ODc
• Strong biofilm producer = 4×ODc \

All strains were tested for antibiotic susceptibility to azithromycin and doxycycline The minimal inhibitory concentration (MIC) was evaluated by the broth microdilution method according to CLSI (2012). The minimal bactericidal concentration (MBC) was evaluated by using a modified flash microbicide method (Hernandes et al., 2013).

The MICs of the tested antimicrobial agents were determined by the broth microdilution method according to CLSI (2012) as above in the presence of 2% salicylic acid, 5% acetylcysteine and 0.05% isotretinoin