Affinity of cefotiam for the alternative penicillin binding protein PBP3(SAL) used by Salmonella inside host eukaryotic cells

BACKGROUND: Following the invasion of eukaryotic cells, Salmonella enterica serovar Typhimurium replaces PBP2/PBP3, main targets of β-lactam antibiotics, with PBP2(SAL)/PBP3(SAL), two homologue peptidoglycan synthases absent in Escherichia coli. PBP3(SAL) promotes pathogen cell division in acidic environments independently of PBP3 and shows low affinity for β-lactams that bind to PBP3 such as aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefalotin. OBJECTIVES: To find compounds with high affinity for PBP3(SAL) to control Salmonella intracellular infections. METHODS: An S. Typhimurium ΔPBP3 mutant that divides using PBP3(SAL) and its parental wild-type strain, were exposed to a library of 1520 approved drugs in acidified (pH 4.6) nutrient-rich LB medium. Changes in optical density associated with cell filamentation, a read-out of blockage in cell division, were monitored. Compounds causing filamentation in the ΔPBP3 mutant but not in wild-type strain—the latter strain expressing both PBP3 and PBP3(SAL) in LB pH 4.6—were selected for further study. The bactericidal effect due to PBP3(SAL) inhibition was evaluated in vitro using a bacterial infection model of cultured fibroblasts. RESULTS: The cephalosporin cefotiam exhibited higher affinity for PBP3(SAL) than for PBP3 in bacteria growing in acidified LB pH 4.6 medium. Cefotiam also proved to be effective against intracellular Salmonella in a PBP3(SAL)-dependent manner. Conversely, cefuroxime, which has higher affinity for PBP3, showed decreased effectiveness in killing intracellular Salmonella. CONCLUSIONS: Antibiotics with affinity for PBP3(SAL), like the cephalosporin cefotiam, have therapeutic value for treating Salmonella intracellular infections.