Mechanism of penicillin killing in the absence of bacterial lysis

Abstract

Exposure of group A streptococci (a nonlytic-death phenotype) to benzylpenicillin (penicillin G) produced a dose-dependent, rapid, and extensive hydrolysis of total cellular RNA, with the subsequent loss of hydrolysis products from the cell. This loss of RNA correlated well with loss of viability and was not accompanied by solubilization of the cell wall or comparable losses of either protein or DNA. Simultaneous treatment with penicillin G and either chloramphenicol or rifampin resulted in reduced levels of killing and the complete inhibition of RNA loss. These findings define a new mechanism of penicillin G-induced killing in the absence of cell wall disruption and suggest a basis for drug-induced antagonism of penicillin G-mediated nonlytic death.A unified model for the action of inhibitors of cell wall synthesis (23,26) predicts that in all susceptible bacteria, cell wall antibiotics act by inhibiting the assembly of insoluble peptidoglycan (PG), leading to bacteriostasis. The secondary events are species dependent and can be related to growth rate (2) and medium composition (10). The widely accepted mechanism of penicillin-induced killing is that inhibition of synthesis results in a deregulation of the endogenous PG hydrolases (autolysins), which leads to the destruction of the structural integrity of the PG and ultimately death (23,26). It is clear, however, that a diverse group of bacteria which are sensitive to penicillin do not lyse, even after prolonged exposure to high concentrations of the drug (3,19,23). These nonlytic phenotypes can be grouped into two categories: tolerant strains, which are growth inhibited but lose viability slowly (3,6,19,23), and nonlytic-death strains, which die rapidly after treatment with relatively low doses of, for example, benzylpenicillin (penicillin G). Both classes of nonlytic phenotypes fail to express autolytic activities.A previously proposed mechanism for nonlytic death predicted that partial hydrolysis of the PG fabric (nicking) would be sufficient to alter the essential structural features of the cell wall and would lead to death (3). However, we were unable to find evidence to support this hypothesis (15).In this report, we describe results of studies of penicillininduced nonlytic death in group A streptococcus. Our findings clearly demonstrate that penicillin G induces a rapid, dose-dependent, specific loss of total cellular RNA in the absence of hydrolysis of the cell wall. In addition, we present evidence which suggests that the antagonistic effects of inhibitors of transcription or ribosomal function are associated with impairment of penicillin G-induced RNA hydrolysis. These observations define a mechanism of penicillin G action which can account for the bactericidal nature of the drug in the absence of cellular dissolution. MATERIALS AND METHODSOrganisms and growth conditions. Group A streptococcus strain 1224 was a clinical isolate obtained from St. Christopher's Hospital for Children, Philadelphia, Pa. Enterococcus hirae ATCC 9790 (Streptococcus faecium)...

Effects of medium composition on penicillin-induced hydrolysis and loss of RNA and culture turbidity in group A streptococci

“…However, in contrast to the previous studies with group A streptococci (3, 4), we observed significant reductions in culture turbidity and viability that were not associated with reductions in cell number (11). Results of radiolabeling studies demonstrated that these losses of culture turbidity were a direct reflection of penicillin G-induced destruction of RNA (11).…”

“…For these strains, the GIC50 of penicillin G was the same in both media: 0.05 ,ug/ml for strain 2443C and 0.1 ,ug/ml for strain 1224. [3H]leucine previously incorporated into RNA and protein, respectively, that was retained on membrane filters (11). Briefly, for these chase studies macromolecules were extensively labeled by growing cultures in CDM in the presence of 14C-and 3H-labeled precursors.…”

“…Susceptibility to the lethal effects of penicillin G was determined by previously described protocols (8,11), and all CFU values were corrected for drug-induced changes in streptococcal chain length (7). Potassium penicillin G (1,595 U/mg) used in these experiments was a gift from Wyeth Laboratories, Philadelphia, Pa.…”

Localization of penicillin-binding proteins to the splitting system of Staphylococcus aureus septa by using a mercury-penicillin V derivative

“…␤-Lactam antibiotics induce bacteriolysis by disrupting the fine control of endogenous autolytic enzymes (36), but cell death can occur in the absence of lysis. ␤-Lactam-induced killing of gram-positive bacteria in the absence of lysis, or nonlytic death, has been found in penicillin-sensitive staphylococci (13) and in group A streptococci (24). The mechanism of ␤-lactam action in nonlytic death is poorly understood in staphylococci, but in streptococci, hydrolysis and loss of RNA correlated with loss of viability (24).…”

“…␤-Lactam-induced killing of gram-positive bacteria in the absence of lysis, or nonlytic death, has been found in penicillin-sensitive staphylococci (13) and in group A streptococci (24). The mechanism of ␤-lactam action in nonlytic death is poorly understood in staphylococci, but in streptococci, hydrolysis and loss of RNA correlated with loss of viability (24). In our experiments, strains Col and BB270 experienced nonlytic death, whereas the methicillin-susceptible strain underwent moderate killing with moderate lysis.…”