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)...
The accumulation of ppGpp in three streptococci starved for isoleucine was studied via HPLC analysis of cell extracts prepared from mechanically disrupted bacteria. Starvation was achieved either by reduction of isoleucine in the growth medium or the addition of pseudomonic acid. The results indicate that while both treatments produced a physiological response similar to that described for stringent strains of other bacteria, in the streptococci, stringency was not necessarily coupled with ppGpp.
Exposure to penicillin G of exponentially growing cultures of group A streptococci growing in chemically defined medium (CDM) can lead to extensive loss of culture turbidity. Significant reductions in culture turbidity did not accompany comparable treatments of group A streptococci growing in Todd-Hewitt broth (THB). Studies with THB and a high-molecular-weight (>12,000) fraction of THB demonstrated that components in this complex medium inhibited the efflux of RNA hydrolysis products from otherwise intact cells. Hydrolysis products accumulated intracellularly and inhibited the extensive hydrolysis of RNA and consequently the loss of culture turbidity. Results of survival studies with cultures of group A streptococci exposed to penicillin G in THB demonstrated that this treatment protocol produces conditions of phenotypic tolerance relative to exposure in CDM. In combination, these findings provide further support for the hypothesis of RNA hydrolysis as the bactericidal mechanism of penicillin G action in this nonlytic death phenotype.Exposure of group A streptococci to antibiotics that inhibit cell wall synthesis leads to loss of viability without cellular lysis (nonlytic death) (3, 4, 10). The term nonlytic death originated with the observation that losses of viability were not accompanied by reductions in cell numbers or loss of culture turbidity. This phenomenon has been described for both gram-negative enteric bacilli (1) and group A streptococci (3, 4). 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).In this report, we present results of experiments demonstrating that medium composition dramatically affects penicillin G-induced losses of turbidity and viability in cultures of group A streptococci. In addition, we show that this effect is associated with the presence of high-molecular-weight components in the medium, which inhibits the release of RNA hydrolysis products from cells and consequently inhibits the extent of penicillin G-induced hydrolysis of RNA.(A preliminary report of this work has been presented
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