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. 2021 May 7;12:2560. doi: 10.1038/s41467-021-22845-2

Fig. 3. Resistant bacteria release markedly less PGN and hyphae-inducing activity than sensitive strains in response to β-lactam antibiotics.

Fig. 3

a C. albicans was grown side-by-side with MSSA#1, MRSA#1, AmpS-Ec, or AmpR-Ec with or without Aug at 30 °C for 4 d. Scale bar, 250 µm. Antibiotic resistance of the resistant strains was confirmed (Supplementary Fig 1b). The experiment was repeated independently three times with similar results. b ELISA quantification of PGN in the supernatant of MSSA#1 or MRSA#1 with or without antibiotic treatment. Sa supernatnats were prepared as described in Fig. 2a, PGN was quantified by ELISA. Three samples (n = 3) for each treatment were analyzed. Authentic MDP was used to prepare the standard curve. Experiments were performed in triplicate. P values were determined by two-tailed unpaired t test. Source data are provided. c Amp-treated S. aureus culture supernatant was incubated with 5 mg/mL of 2E7 or a control antibody (cAb) at 37 °C for 1 h before testing the hypha-inducing activity in HBSS at 37 °C for 3 h. Percentage of hyphal growth is shown on each image (n = 50). Scale bar, 5 µm. d S. aureus (MSSA#1) was grown and treated with Cef or Amp, as described in Fig. 2a. In total, 100 mL supernatant was freeze-dried, resuspended into 10 mL sterile water, and fractionated by reversed-phase HPLC. Each fraction was freeze-dried and resuspended into 10 mL sterile water. Then, PGN content in each fraction was quantified by 2E7-ELISA, and the hypha-inducing activity of each fraction was tested at a final concentration of 5% (v/v) in HBSS. Hyphal induction was done at 37 °C for 2 h. Fraction 13 (F13) of both Amp and Cef-treated S. aureus cultures have the highest PGN content as well as a high hypha-inducing activity while F13 of untreated culture had low PGN levels and no activity. Fractions outside of the PGN peak areas had no significant activity. Scale bar, 5 µm. The experiment was repeated independently three times with similar results. e A muropeptide identified in F13 of Amp and Cef-treated S. aureus culture supernatants. Also, see compound e in Supplementary Fig 4.