Macrolide Structures Can Confer Differential Susceptibility in Escherichia coli K30 Deletions of Group 1 Capsule Assembly Genes

The Wzy-dependent biosynthesis system in Escherichia coli K30 is responsible for the assembly and transport of extracellular capsule polysaccharides. The Wzy system has three key components: Wza, a transport channel located on the outer membrane; Wzc, an autokinase in the inner membrane coupled to Wza; and Wzb, a cytosolic phosphatase that regulates Wzc activity. Knock-outs of this assembly system have been observed to correlate with increased resistance to some macrolide antibiotics, including erythromycin, clarithromycin, and roxithromycin, but not to telithromycin, which is distinct from other macrolides with additional ketone groups and aromatic rings. We hypothesize that the route of entry of macrolides is partially dependent on the Wzy capsular transport system, and that distinct molecular structures of macrolides contribute to differences in bacterial susceptibility. Disc diffusion assays were conducted using Wza knock-outs, Wzc knock-outs, and Wza-Wzb-Wzc triple knock-outs of the E. coli K30 strain to test their resistance against erythromycin, clarithromycin, azithromycin, and telithromycin. We observed that a Wza deletion confers greatest resistance to erythromycin and clarithromycin, while the absence of Wzc resulted in mildly increased resistance against these two macrolides. All knock-out strains were observed to have increased but incomplete resistance to telithromycin, and only the Wza single knock-out strain was more resistant to azithromycin. We conclude that Wza is essential for outer membrane penetration of erythromycin and clarithromycin, while telithromycin entry is equally dependent on Wza and Wzc. Azithromycin entry is largely dependent on Wza. However, the antibiotic susceptibilities of the triple knockout results contrasted the Wza single knockout susceptibilities, indicating that there may be other factors involved in susceptibility.