Fig 2. The LptD and LptE mutants are resistant to various antimicrobial substances.

(A) The LptD WT, LptD G580S, LptE WT, and LptE T95I strains were incubated at 37˚C for 30 min in PBS, 1.7% or 3.3% silkworm hemolymph in which antimicrobial peptides were induced (AMP-induced Hemolymph), or 3.3% silkworm hemolymph in which antimicrobial peptides was not induced (Normal Hemolymph). After incubation, the number of live bacterial cells was determined. Data shown are the mean ± standard errors from two independent experiments performed in triplicate. The asterisk represents a p value less than 0.05 (Student’s t test). (B) The LptD WT, LptD G580S, LptE WT, and LptE T95I strains were incubated at 37˚C for 45 min in PBS, porcine serum (Serum), or heat-treated porcine serum (Inactivated Serum). After incubation, the number of live bacterial cells was determined. Data shown are the mean ± standard errors from two independent experiments performed in triplicate. The asterisk represents a p value less than 0.05 (Student’s t test). (C) Overnight cultures of LptD WT, LptD G580S, LptE WT, and LptE T95I strains were 5-fold serially diluted, spotted onto LB agar plates supplemented without or with vancomycin, levofloxacin, tetracycline, chloramphenicol, ampicillin, colistin, streptomycin, or cholic acid, and incubated at 37˚C. (D) The LptD WT, LptD G580S, LptE WT, and LptE T95I strains were aerobically cultured at 37˚C in LB broth, and n-hexane was added to the culture at 1 h after the incubation. The OD₆₀₀ of the bacterial cultures were measured every 1 h.