Survival of group A streptococci in dried human blood.

The resurgence of streptococcal infections in the USA and Europe and their high incidence in other parts of the world prompted an examination of the survival and maintenance of virulence of group A streptococci. Human blood containing group A streptococci was placed on small pieces of sterile paper towelling and allowed to dry at room temperature. At periods of 2, 8, 15 and 20 weeks later, the paper with the dried blood was placed in Todd-Hewitt broth and incubated at 37 degrees C overnight. All the samples tested at 2 weeks grew in broth, and with only one exception, grew in fresh human blood provided by five donors. At 8 weeks only two of the 10 strains failed to grow in broth; seven of the eight viable cultures also grew in blood. At 15 and 20 weeks after drying the eight cultures were still viable. Since seven were able to grow in fresh blood as well as in broth it is assumed that their virulence factor(s) had been retained.

Comparison of skin changes induced on mice by either group A type 12 or group G streptococci.

Adult Swiss webster mice were injected with 3 x 10(6) colony-forming units (cfu) of group G or 2.5 x 10(6) cfu of group A streptococci at intradermal injection sites on the right and left paralumbar areas of the back. The mice were sacrificed at intervals between 4 hours and 14 days post-injection (p.i.) and full thickness biopsies of skin 10 mm in diameter encompassing the sites of injection were taken. One tissue specimen was homogenized in PBS and plated to determine the number of cfu, while another was used for histopathological studies. The number of viable group A and group G streptococci in the tissue increased to 3 x 10(9) cfu by 96 hours p.i.: after 192 hours p.i. the group A cells had declined to 2.7 x 10(6) cfu compared to 1.1 x 10(8) cfu for group G cells. No streptococci of either group were detected at 336 hours (14 days p.i.). Gross edematous lesions induced by either streptococcus group were evident on all animals at 24 hours (p.i.). Group G streptococci lesions were larger and persisted longer than lesions induced by group A. Histological examination consistently revealed more inflammation and necrosis in tissue sections from mice injected with group G streptococci.

Biochemical properties of group G streptococci isolated from cats and man.

The biochemical characteristics of group G streptococci isolated from cats were markedly similar to the characteristics of group G streptococci from man. Both cat and human isolates of group G streptococci were also very similar in biochemical characteristics to group A streptococci so that to identify the source of group G streptococci by biochemical reactions is not a reliable procedure. The group G streptococci found in many cats could be pathogenic to man, since their physiological and biological characteristics are very similar to those of group A streptococci.

Protein synthesis is required for cholera toxin-induced stimulation of arachidonic acid metabolism.

The molecular events in the mechanism of action of cholera toxin were analyzed using Chinese hamster ovary (CHO) cells. Cholera toxin stimulated both 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and arachidonic acid metabolism in these cells. The turnover of phospholipid by cholera toxin-induced stimulation of phospholipase activity evoked the synthesis of PGE2 and other prostaglandins. Cholera toxin-induced release of both [3H]arachidonic acid and PGE2 was blocked by addition of either cycloheximide or actinomycin D. In contrast, accumulation of cAMP in cholera toxin-treated CHO cells was unaffected by adding these drugs. Further, dibutyryl cAMP or forskolin caused [3H]arachidonic acid release, which also was blocked by cycloheximide and actinomycin D. We concluded that the sequence of molecular events in cholera toxin-treated CHO cells first involved activation of adenylate cyclase, which caused an increase in cAMP. In turn, cAMP promoted transcription of mRNA that encoded either a specific phospholipase or a phospholipase-activating protein. The emerging arachidonic acid metabolites (e.g., PGE2 and PGF2 alpha) might be important mediators of cholera toxin's stimulatory effects on vascular permeability and smooth muscle contraction in the intestine during cholera.

Chloroquine inhibition of cholera toxin.

Cholera toxin (CT) stimulated adenylate cyclase and a phospholipase which elevated cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid (AA). The AA was quickly converted to prostaglandins (PGs) via the cyclo-oxygenase pathway. Chloroquine exerted minimal inhibition of cAMP levels in CT-treated cells, although CT-induced release of [3H]AA and PGs was blocked completely when the drug was added in concentrations as low as 0.1 mM (50 micrograms/ml). Inhibition of [3H]AA release was complete when chloroquine was added before or within 30 min after CT. The capacity of chloroquine to inhibit either phospholipase C (PLC) or phospholipase A2 (PLA2) could explain the antisecretory activity of this drug.

Synthesis of prostaglandins in cholera toxin-treated Chinese hamster ovary cells.

The prostaglandin (PG) and adenosine 3',5'-cyclic monophosphate (cAMP) responses of Chinese hamster ovary (CHO) cells were measured after cholera toxin (CT) exposure to evaluate dose and kinetic relationships. Release of prostaglandin E2 (PGE2) and the accumulation of cAMP were dependent on the dose of CT, with an effective dose of approximately 10-100 ng/ml within 4 h; the PGE2 response was about four- to six-fold more than that of PGE1. CHO cells exposed to CT also released increased amounts of thromboxane B2 (TxB2), PGF2 gamma, and 6-keto PGF1 gamma (a non-enzymatic degradation product of prostacyclin). Kinetic analysis of CT-treated cells revealed that small peaks of cAMP accumulation and of PGE1 and PGE2 release were detected at approximately 30 min, but larger, progressive PG and cAMP responses were measured 2-4 h later. Exposure of the cells to relatively high doses of membrane-permeable derivatives of cAMP (1 mM) and forskolin (10 microM) caused PGE2 release. Concomitantly, exogenous PGE2 (100 microM) increased intracellular levels of cAMP. We have considered the interrelationship of the cyclo-oxygenase and the cyclic nucleotide pathways relative to the molecular mechanism of CT.

Inhibitory effect of aspirin on cholera toxin-induced phospholipase and cyclo-oxygenase activity.

Cholera toxin (CT) stimulated phospholipase activity and caused [3H]arachidonic acid (3H-AA) release in a murine macrophage/monocyte cell line. Pretreatment of cells with dexamethasone, a phospholipase A2 (PLA2) inhibitor, did not affect CT-induced 3H-AA release. In contrast, aspirin, which is an inhibitor of phospholipase C (PLC), blocked CT-induced 3H-AA release and subsequent prostaglandin (PC) synthesis. The inhibitory effect of aspirin was dose dependent, with 4 mM reducing the CT response by approximately 50%. Similarly, inhibition was time dependent, occurring when the drug was added to the culture medium as late as 30 min after CT. Brief exposure (30 min) of the cells to aspirin did not alter their subsequent response to CT, but 3H-AA release from cells exposed to aspirin for 2.5 h was irreversibly inhibited. The data suggested that CT stimulation of AA metabolism may involve increased PLC activity.

Stimulatory effects of cholera toxin on arachidonic acid metabolism in Chinese hamster ovary cells.

Cholera toxin (CT) stimulated the release of arachidonic acid (AA) from Chinese hamster ovary cells with no apparent lag period. CT-induced release of [3H]AA or its metabolites was dose dependent during a 4-hr period of toxin exposure with a minimum effective dose of 0.1 ng/ml. CT-induced release of [3H]AA metabolites began within 15 min of toxin addition and became maximal after approximately 5 hr. Neither CT-A subunit nor CT-B subunit alone caused [3H]AA release. Furthermore, [3H]AA release was not caused by addition of dibutyryl cAMP to the culture medium, indicating that the observed effect of CT on arachidonate metabolism appeared to be independent of cAMP. The effect of CT on AA metabolism is proposed as a possible mechanism leading to the synthesis of prostaglandin E and fluid secretion during cholera.

Multiplication in human blood and deoxyribonuclease production by group G streptococci isolated from animals and humans.

An M protein or an M protein-like substance was found to be present in a large proportion of group G streptococci isolated from animals and humans. Forty-seven percent of the isolates from cat throats and 38% of the isolates from the vagina of cats were able to multiply in human blood. Only 14% of the human isolates of group G isolated from various anatomical sites and sources were able to multiply in fresh human blood. Deoxyribonuclease was produced by 81% of cat vagina isolates, by 80% of cat throat isolates and by only 27% of the group G isolates from humans. Thirty-five percent of the cat isolates but only 5% of the human isolates were able to both grow in blood and produce DNase.

The occurrence of nicotinamide adenine dinucleotide glycohydrolase and the serum opacity reaction in groups B, C, F and G streptococci.

The frequency of the serum opacity reaction (SOR) and nicotinamide adenine dinucleotide glycohydrolase (NADase) production in non-A groups of beta-haemolytic streptococci isolated from humans and animals was surveyed. The SOR was positive with five of eighteen group B isolates (28%), four of fifteen group C (27%), two of five group F (40%), and thirty-seven of sixty-eight group G (54%) isolates. NADase activity, in addition to SOR, was found in three of eighteen group B isolates (17%), three of fifteen group C (20%), forty of sixty-eight group G (59%), and none of the group F isolates. The SOR was produced by forty-eight (45%) and NADase was produced by fifty-one (48%) of the one hundred and six isolates of non-group A beta-haemolytic streptococci. Twenty-five percent of the isolates were both NADase and SOR positive.

Salmonella cytotoxin: a component of the bacterial outer membrane.

Salmonella cytotoxin present in cell-free sonic lysates causes rounding and detachment of Chinese hamster ovary (CHO) cells. Although the precise role of this toxin in the pathogenesis of salmonellosis is unclear, cytotoxin production by Salmonella could account for tissue damage or possibly, facilitate invasion. A variety of other bacteria (e.g. Shigella, Escherichia, Legionella) have been shown to form soluble cytotoxins, many of which may be involved in pathogenesis. The data in this report indicate that the Salmonella cytotoxin in cell-free sonic lysates is firmly associated with cell membrane fragments that can be pelleted by ultracentrifugation (270,000 g for 2.4 h). Furthermore, lysozyme treatment of filter-sterilized sonic extracts of Salmonella species followed by isopycnic sucrose gradient ultracentrifugation allowed separation of the outer and inner membrane components. The outer membrane (OM) peak contained the cytotoxic activity when assayed for detachment of CHO cells. The importance of these data resides in the observation that the Salmonella cytotoxin is an outer membrane component. Its mere location places it in a position of direct contact with host cells and suggests a possible role in cell damage and/or invasion. Furthermore, ultracentrifugation provides a method by which much of the Salmonella cytotoxin in sonic extracts can be removed allowing expression of the Salmonella enterotoxin, whose CHO cell elongation effect is usually obscured by the presence of the cytotoxin causing cell rounding and detachment.

Experimental necrotic dermatosis induced by group G streptococci in mice.

Self healing necrotic lesions were produced on the backs of laboratory mice by injecting group G streptococci into the skin. The incidence and severity of necrotic dermatosis was dose related. When 1 x 10(1) colony forming units (cfu) were injected subcutaneously, lesions developed on three of 16 mice 4 days post inoculation. Injection of 1 x 10(3) cfu produced lesions on five of 16 mice and 1 x 10(5) cfu produced lesions on seven of 15 mice 3 days post inoculation. An inoculation of 1 x 10(7) cfu produced lesions on all of 16 mice 2 days post inoculation. Lesions produced by the 1 x 10(1) inoculum were smaller and had healed by the 15th day post inoculation, whereas lesions produced by the 1 x 10(7) inoculum persisted until the 24th day post inoculation. No mortality could be attributed to experimental design and all lesions healed without the use of medication or antibiotics.

Reappearance of Sporothrix schenckii lesions after administration of Solu-Medrol R to infected cats.

In 10 of 34 adult cats infected with Sporothrix schenckii, demonstrable lesions healed spontaneously in 31 to 88 days after inoculation of the organism. None of 5 untreated cats developed additional visible lesions during the period of observation, nor were they positive for S. schenckii by culture at necropsy. Five animals were treated with Solu-Medrol R to determine if the apparent clinical cure was also accompanied by a mycological cure. Lesions reappeared at, or near, the site of the original lesions in 3 of the 5 treated cats from 4 to 6 months after the initial lesions healed. Fungi were cultured both from the regional lymph node and from exudate of the skin lesions. Thus, it appears that viable S. schenckii may be sequestered in tissues for at least 6 months without showing clinical evidence of their presence. Moreover, apparently "healed" lesions may be reactivated and progress to typical demonstrable skin lesions following immuno-suppression.

Chronic infection of cats with Brugia malayi and streptococcus.

An elephantoid condition was seen in the affected limbs of 5 of 6 cats at necropsy 12 to 18 months after initial infection with Brugia malayi. Repeated infection with Brugia and exposure to an opportunistic streptococcus appeared to enhance the production and persistence of edematous and fibrotic tissues surrounding the affected lymphatics.