Figure 1: PGH construct schematics, resistance development and eradicating S. aureus in a rat colonization model.

(A) Schematic of PGH constructs. Domains: CHAP endopeptidase (CHAP, red box); N-acetylmuramoyl-L-alanine amidase (AMID, green box); M23 endopeptidase (PEP, blue oval); CBDs (LysK SH3b, gold diamond; lysostaphin SH3b, gold diamond with dot); protein transduction domain (PTD, blue circle); hexahistidine purification tag (His₆). Domains not to scale. Specific PG cut sites are illustrated in Supplementary Fig. 2B. B. In vitro antimicrobial resistance development. Engineered triple fusions K-L and L-K suppress antimicrobial resistance development compared to LysK (K), lysostaphin (L), or a combination of equimolar concentrations of both (L+K). Changes in MIC are depicted as a fold-change at the tenth round of sublethal exposure compared to the first exposure, with the average fold-change of 4 replicates in red. Error bars = SEM. First exposure MICs: Lysostaphin, 0.77 μg/ml (27 nM); LysK, 47 μg/ml (840 nM); Lysostaphin and LysK (L+K) in combination 0.2 μg/ml (7 nM and 3 nM respectively) ;triple fusion K-L, 7 μg/ml (97 nM); triple fusion L-K, 7.8 μg/ml (107 nM). C. Colonization reduction in a rat nasal carriage model. Rats were inoculated with S. aureus strain ALR on day 1. After 5 days, the rats were treated twice daily for 3 days with 20 μl of a 10 mg/ml solution of each enzyme. The rat noses were excised on day 10, homogenized, and quantitative cultures were performed. Each point represents the CFU recovered from an individual rat. Bars indicate the median CFU/nose recovered from treated rats. Triple fusion L-K showed a significant reduction in colonization (98%) of treated rats compared with rats treated with buffer alone. Data were compiled from five independent experiments. Lyso = commercially purchased lysostaphin (AMBI, Tarrytown, NY). Statistical comparisons were made with the Mann-Whitney test.