Defensins are smalL cysteine-rich antimicrobial peptides that are abundant in human, rabbit, and guinea pig neutrophils (PMN). Three defensins (human neutrophil peptide defensin constitute between 30 and 50% of the total protein in azurophil granules of human PMN. We examined the mechanism of HNP-mediated bactericidal activity against Escherichia coli ML-35 (i-, y-, z+) and its pBR322-transformed derivative, E. coli ML-35p. Under conditions that supported bactericidal activity, HNP-1 sequentially permeabilized the outer membrane (OM) and inner membrane (IM) of E. coli. Coincident with these events, bacterial synthesis of DNA, RNA, and protein ceased and the colony count fell. Although these events were closely coupled under standard assay conditions, OM permeabilization was partially dissociated from IM permeabilization when experiments were performed with E. coli that had been plasmolyzed by mannitol. Under such conditions, the rate and extent of bacterial death more closely paralleled loss of IM integrity than OM permeabilization. Electron microscopy of E. coli that had been killed by defensins revealed the presence of striking electron-dense deposits in the periplasmic space and affixed to the OM.Overall, these studies show that HNP-mediated bactericidal activity against E. coli ML-35 is associated with sequential permeabilization of the OM and IM, and that inner membrane permeabilization appears to be the lethal event.

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We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.

A rapid method for the detection of methicillin-resistant Staphylococcus aureus is described. A bioluminescent technique was used to compare the amounts of extracellular adenosine triphosphate released by methicillin-resistant and methicillin-sensitive strains. When tested by this method 29 Staph. aureus strains of known methicillin susceptibility were correctly identified.

SynopsisIn this study bacterial luminescent toxicity tests (LTT) were used to monitor the residual toxicity concentration of a biocide-inhibitor after several months storage. The study was completed in two parts. The first study investigated the relationship between the chemical and toxicity concentrations of a biocide-inhibitor stored in mild steel containers and glass containers. After three months storage in the mild steel containers there was a significant difference (p < 0.01) between the chemically assayed level of the biocide-inhibitor and the toxicity level determined by the LTT. The results of the laboratory studies were confirmed in the second part of the study when the biocide-inhibitor was incorporated in the sea water used to flood a large undersea pipeline during the pressure testing programme. The disparity between the chemical and toxic assayed concentrations was not as great when the biocide-inhibitor was stored in glass containers. The influence ofthe container used for biocide testing plus the failure of the chemical analysis to reflect the toxicity level of this biocide-inhibitor demonstrates a different application of LTT. The results of this study also illustrate the usefulness and relevance of L' M' in testing the effectiveness of some biocides and indicate that LTT could be useful in monitoring the efficiency of biocides during their industrial application.