The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass-adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11-enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16-24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing.

A survey of the occurrence of the phosphoenolpyruvate-dependent glucose phosphotransferase system was carried out in a number of bacteria, representing both gram-positive and gram-negative facultative anaerobic and strictly aerobic types. The system was found to be present in representatives of genera that are characteristically facultative anaerobes, but the system was absent in members of those genera that are strictly aerobic. Thus, although the phosphoenolpyruvate phosphotransferase system is an important system for the transport of sugars in bacteria carrying out anaerobic glycolysis, it plays no role in sugar transport by those organisms having a strictly oxidative physiology. A fundamentally different system, probably not involving phosphorylation during transport, is indicated in this latter group.

Exposure of exponentially growing cultures of Streptococcus mutans strains FA-1 and GS-5 to various concentrations of benzylpenicillin (Pen G) resulted in inhibition of turbidity increases at low concentrations (0.02 to 0.04 ,tg/ml). In many bacterial species, addition of a sufficient concentration of benzylpenicillin (Pen G), or other antibiotic inhibitors of cell wall peptidoglycan (PG) assembly, to prevent further growth of cultures is rapidly followed by cell death, accompanied in many cases by cellular lysis (1,33,34). The availability of bacterial species that are tolerant to both the lethal and the lytic consequences of inhibition of cell wall PG assembly permits studies of the processes that lead to growth inhibition of intact cells without the complications of rapid losses of viability. Furthermore, comparisons of the events occurring in such tolerant species with those occurring in species that are killed by treatment with these drugs will help provide insights into the lytic and other possible mechanisms of lethality. Currently (34), it is clear that the lethal t Present address:

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The extent of sublytic autolysin activity (peptidoglycan [PG] nicking) after exposure of exponentially growing cultures of a group A streptococcus (GAS) to benzylpenicillin (PenG) was studied by determining changes in the glycan chain length of PG polymers. The average PG chain length in isolated cell walis was estimated by calculating the ratio of the total hexosamine content (Morgan-Elson-reactive material) A unified model for the action of inhibitors of cell wall synthesis (21, 26) predicts that in all susceptible organisms, cell wall antibiotics initially act by inhibiting the assembly of insoluble peptidoglycan (PG), which in turn leads to bacteriostasis. The subsequent (secondary) events are species dependent and, on the basis of recent reports, can also be related to growth rate (4) and medium composition (14). Apparently, some bacteria die and then lyse, whereas others lyse immediately, and still others undergo nonlytic death. An additional response, tolerance, is considered in the model as a continuation of stasis (21) and has been suggested to be associated with specific regulatory mechanisms (19,21). A central feature of lytic phenotypes is the secondary expression of endogenous PG hydrolases (autolysins) after exposure to antibiotics (21,26). It follows that a prerequisite for nonlytic phenotypes is an absent, weakly expressed, or tightly regulated autolytic system. PG hydrolase activities are considered to be essential for normal surface growth and cell division processes (5) and are therefore presumed to be present in all bacterial cells. Considering the following phenomena: (i) the demonstrated unity in the primary mechanism of action of, for example, P-lactams in the binding to penicillin-binding proteins and inhibition of their function(s) (2, 9), (ii) the requirement of autolytic activities for growth (5), ahd (iii) the documented relationship between inhibition of PG assembly and deregulation of autolysins in lytic phenotypes (21, 26), hydrolysis of a small number of bonds insufficient to cause cellular lysis (nicking) has been suggested as a possible secondary and potentially fatal outcome of inhibition of PG synthesis in bacteria which undergo nonlytic death (8).In this report, we present results of experiments designed to assess the role of PG nicking in penicillin-induced nonlytic death in a group A streptococcus (GAS). Preliminary characterization of the autolytic system of GAS is also presented.( Quantitative comparison of growth inhibition. Conventional methods for determining MICs were considered to be unsatisfactory for use in these studies, since they rely on small, physiologically poorly defined inocula and long incubation intervals. Therefore, 50% growth inhibitory concentrations (GIC50s) were determined for each strain as described previously (17). For these studies we used GIC50s obtained after exposure of exponentially growing cultures at the time that exponentially growing control cultures had increased fourfold in turbidity. Potassium PenG (1,595 U/mg) used in these experim...