Pre-treatment with cell-wall targeting antibiotics and aminoglycosides does not confer cross protection to cell-wall targeting antibiotics in Escherichia coli despite upregulation of rprA

09/26/2019

Josh Bowman, Robin Couput, Anthony Pookpun, Kendrew Wong

Volume 24
Fall 2018 / Winter 2019

The Rcs phosphorelay system is a signaling pathway in Escherichia coli involved in cell envelope stress response. RcsB, located in the cytoplasm, activates
numerous downstream targets including rprA upon disruption of the peptidoglycan layer. rprA is believed to confer greater resistance to cell-wall targeting antibiotics in Escherichia coli. Previous studies observed rprA upregulation in wild-type Escherichia coli (DH300) following exposure to aminoglycosides and cell-wall targeting antibiotics, but not in rcsB deletion mutants (DH311). A correlation was also shown between rprA expression and susceptibility to cell-wall targeting antibiotics where the absence of rprA expression was found to coincide with increased susceptibility. It is unknown whether elevated expression
of rprA results in increased resistance to antibiotics. We hypothesized that pre-treatment with cell-wall targeting antibiotics and aminoglycosides will result in rcsB-mediated upregulation of rprA, conferring increased resistance to subsequent treatments with cell wall-targeting antibiotics. A preliminary round of minimum inhibitory concentration (MIC) assays was conducted with penicillin, streptomycin and tetracycline to determine sub-MIC levels of each antibiotic for E. coli DH300 and DH311. Following this, rprA expression was quantified using a β-galactosidase activity assay based on expression of an rprA-lacZ promoter reporter fusion gene. Strains were then pre-treated at sub-MIC levels of each antibiotic, and another set of MIC assays was carried out to measure resistance to penicillin. Under all antibiotic pre-treatments, DH300 showed increased β-galactosidase activity indicating increased rprA expression compared to DH311. DH300 pre-treated with
penicillin and tetracycline also showed increased rprA expression compared to untreated DH300. Regardless of rprA expression levels, no differences in penicillin resistance were seen between DH300 and DH311 in MIC assays. Similarly, pre-treatment with antibiotics did not result in any difference in resistance to penicillin between DH300 and DH311 in subsequent MIC assays.