Activation of complement by serum-resistant Neisseria gonorrhoeae. Assembly of the membrane attack complex without subsequent cell death.

Interaction of the human complement system in normal human serum (NHS) with serum-resistant and -sensitive Neisseria gonorrhoeae was evaluated to better understand the mechanism of serum-resistance. Complement activity (CH50) was depleted from NHS in a dose-dependent fashion by both serum-resistant and -sensitive N. gonorrhoeae. No detectable CH50 remained in NHS incubated with 10(9) colony-forming units (CFU)/ml serum of either resistant or sensitive strains. When smaller numbers of bacteria were incubated with NHS, lesser, yet comparable, amounts of CH50 were depleted by both resistant and sensitive strains. Hemolytic C2 activity was diminished by 33% in the case of resistant N. gonorrhoeae (10(8) CFU/ml serum) and by 48% in the case of a sensitive strain. No detectable decreases in hemolytic C4 or C7 activities were found with either sensitive or resistant strains at this concentration. Both resistant and sensitive strains activated C1s in NHS. Resistant strains specifically activated 19-21% of radiolabeled C1s in NHS, whereas sensitive strains activated 18-32%. Both resistant and sensitive strains also activated C5 in NHS. In binding assays using radiolabeled C5 and C9 in NHS, resistant and sensitive strains bound comparable amounts of C5 and C9. The number of bound C5 and C9 molecules varied according to the number of bacteria or amount of serum used in the assay. The ratio of C9/C5 bound to a sensitive strain was 6.8, and to a resistant strain was 8.2, suggesting that C5 and C9 were incorporated into membrane attack complexes (MAC). Electron microscopic examination of resistant and sensitive strains incubated with NHS revealed that MAC is bound to the surfaces of the resistant strain as well as the sensitive strain.

The influence of bacteriocins on resistance to infection by gram-negative bacteria. II. Colicin action, transfer of colicinogeny, and transfer of antibiotic resistance in urinary infections.

Dual urinary infections were produced in rats with colicinogenic Escherichia coli CF1, elaborating colicin V in the urine, and colicine-sensitive E. coli 9224 by injecting each organism into the medulla of opposite kidneys. The colicin-sensitive organism was eradicated from the urine of 24.3% of rats and the degree of infection by E. coli 9224 reduced to less than half of the control group. Colicin-resistant mutants of E. coli 9224 were not inhibited in mixed infections with colicin producing E. coli CF1. No evidence of inhibitory activity by colicin V was found in the kidneys. The bladder urine, but not the kidney, was also the site for transfer of colicinogeny between homologous (E. coli) and heterologous (E. coli and Aerobacter aerogenes) species. Episomes controlling colicin V and J + I were transferred within 24 hr after establishing the mixed infection. Since E. coli 9224 was resistant to streptomycin and tetracycline, observations were also made on transmission of multiple drug resistance. Streptomycin and tetracycline resistance was readily transferred to E. coli CF1 within 48 hr in the bladder. These results demonstrate that in urinary infections colicins can kill susceptible bacteria and that bacterial genetic elements are transferred.

Human antiserum for prevention of the local shwartzman reaction and death from bacterial lipopolysaccharides.

Bacterial lipopolysaccharides from dead bacteria have been blamed for the continuing high mortality from gram-negative infections despite antibiotic treatment. Because animal antiserum against these lipopolysaccharides has been shown to protect against several of the effects of endotoxin, we undertook the development of antiserum in human subjects. 21 men were immunized with a single injection of Salmonclla typhimurium or Escherichia coli 0:111 heat-killed cells and immune serum was collected at 2 wk. Preimmune serum was obtained as a control in all animal experiments. 1 ml antiserum given intravenously protected mice against a lethal intravenous dose of homologous endotoxin (P < 0.005 for both antisera). E. coli antiserum reduced the incidence of positive local Shwartzman reactions with E. coli endotoxin from 100 to 38%; S. typhimurium antiserum reduced the incidence from 92 to 35%. (P < 0.0005 for both antisera). There was no protection against heterologous endotoxin in either animal model. These experiments demonstrate for the first time that human antiserum confers exceedingly potent passive immunity to the effects of endotoxin.

Prevention of chronic experimental pyelonephritis by suppression of acute suppuration.

In order to evaluate the importance of suppuration, persistent infection, and scar formation in the evolution of Escherichia coli chronic pyelonephritis, we treated rats with different antibiotic regimens at different stages of the disease. The results show that (a) if acute suppurative pyelonephritis is aborted with early antibiotic therapy, chronic pyelonephritis is prevented; (b) chronic pyelonephritis can develop even after eradication of infection if acute suppuration persists beyond 3 days; (c) persistent infection does not lead to chronic pyelonephritis, if the acute suppuration is suppressed; and (d) residual infection, antigen-load, antibody, and(or) cell-dependent autoimmune processes did not play a significant role. We interpret these results as evidence that the pathologic entity recognized as chronic pyelonephritis results from kidney damage, scarring and shrinkage secondary to acute suppuration.

Rapid identification of Candida albicans septicemia in man by gas-liquid chromatography.

Gas-liquid chromatography (GLC) was used to study normal serum and serum from patients with septicemia caused by a variety of bacteria and by Candida albicans. The gas chromatograms of seven sera from six patients with septicemia due to C. albicans were found to be significantly and reproducibly different from those of normal sera. Chromatograms of serum from 19 bacteremic patients were indistinguishable from normals. The major peaks present in chromatograms of normal sera were identified by GLC and mass spectroscopy as the methyl esters of palmitic, oleic, linoleic, and stearic acids. In addition to these peaks, serum from patients with candidemia contained abnormal peaks that were also present in cultures of C. albicans grown in normal serum and in washed C. albicans harvested from cultures in yeast nitrogen base broth. Chromatograms from 11 cases of mucosal candidates differed little from normal and were easily distinguished from those of fungemia patients. Chromatograms of serum from two of four patients with deep-invasive candidiasis were indistinguishable from those of fungemia and reverted to normal after infections were eradicated.

Thrombocytopenia in experimental trypanosomiasis.

The effect of experimental trypanosomiasis on coagulation was studied because a patient in this hospital with Rhodesian trypanosomiasis developed thrombocytopenia with disseminated intravascular coagulation. Rats injected intraperitoneally with this strain of Trypanosoma rhodesiense consistently developed trypanosomiasis and severe thrombocytopenia without changes in hematocrit or concentration of fibrinogen or fibrin split products. At the time of 50% mortality (4-5 days) mean platelet counts per cubic millimeter of infected rats were 18,000+/-9,000 (+/-2 SEM) compared to 1,091,000+/-128,000 in uninfected controls. In vitro, concentrated trypanosomes and trypanosomefree supernates of disrupted organisms added to normal rat, rabbit, or human blood produced platelet aggregation within 30 min. This platelet aggregation was not blocked by inhibitors of ADP, kinins, or early or late components of complement. In vivo thrombocytopenia also occurred in infected rabbits congenitally deficient in C6 and in infected, splenectomized rats. Although the aggregating substance obtained from disrupted trypanosomes is heat-labile, it is active in the presence of complement inhibitors, suggesting that this trypanosomal product may be a protein enzyme or toxin. Since the phenomenon is independent of immune complexes, complement, ADP, and kinins, it appears to represent a new mechanism of microbial injury of platelets and the induction of thrombocytopenia.

In vitro susceptibility of fungi to killing by neutrophil granulocytes discriminates between primary pathogenicity and opportunism.

Pathogenic fungi, according to their propensity to cause infection of apparently normal individuals, can be grouped into either primary pathogens (e.g., Coccidioides, Histoplasma, Paracoccidioides, Blastomyces, and Sporothrix) or opportunists (e.g., Candida, Mucoraceae, Aspergillus spp., Petriellidium, and Trichosporon). There is, however, no unifying concept explaining the difference between the virulence of the two fungal categories. Previously we have speculated that neutrophils are the common denominator of the high natural resistance to opportunistic fungi. Accordingly, we then compared the susceptibility to killing by neutrophil granulocytes of Histoplasma, Blastomyces, Paracoccidioides, and Sporothrix with that of 14 opportunistic fungi. We found the four virulent dimorphic yeasts, in contrast to opportunistic fungi, to be resistant to killing by neutrophils. Virulent dimorphic yeasts were ingested by neutrophils, and triggered a respiratory burst comparably to opportunists but were less susceptible to hydrogen peroxide, suggesting that differences in the susceptibility to microbicidal products of leukocytes may explain the difference in virulence.

Induction of immunity against lethal Haemophilus influenzae type b infection by Escherichia coli core lipopolysaccharide.

Efforts to prevent Haemophilus influenzae type b (HIB) infections in infancy have been hampered by the low immunogenicity of capsular polysaccharide vaccines in children younger than 18 mos. In searching for alternate immunogens, we have studied the protective potential of polysaccharide-poor, lipid-rich endotoxin (LPS) core in experimental HIB infections. Because all gram-negative bacteria have similar LPS core structures, we were able to use as vaccine the J5 mutant of Escherichia coli 0111, the LPS of which consists only of core components, and thus to avoid problems in interpretation arising from vaccine contamination with non-LPS HIB immunogens. Mice were given graded inocula of HIB and developed lethal infection analogous to human HIB disease when virulence was enhanced with mucin and hemoglobin. After active immunization with heat-killed E. coli J5, 40/50 (80%) of infected mice survived, compared with 14/50 (28%) of saline-immunized controls (P less than 0.005). Passive immunization with rabbit antiserum against E. coli J5 prevented lethal HIB infection when administered 24 or 72 h before or 3 h after infection. This protection was abolished by adsorption of antiserum with purified J5 LPS, with survival reduced from 14/24 to 0/24 (P less than 0.005). Furthermore, rabbit antiserum to purified J5 LPS gave just as potent protection against death as antiserum to whole J5 cells. These studies demonstrate that immunity to core LPS confers protection against experimental murine HIB infection and provide the framework for a new approach to prevention of human disease from HIB.

Immunization against nosocomial infection.

Overwhelming infection with gram-negative bacteremia has become the most serious nosocomial infection in compromised patients. Because gram-negative bacteria share a common core lipopolysaccharide, we tried to develop a single vaccine or antiserum that might control these infections regardless of species. We used a mutant of Escherichia coli 0111 (J5) deficient in uridine diphosphate-galactose (UDP-GAL) epimerase and thus unable to attach "0" side chains, so that core lipopolysaccharide was exposed. A vaccine composed of this mutant produced antibody that gave broad protection against lethal infections by different gram-negative bacteria in immunosuppressed animals. The J5 vaccine protected against 98 percent lethal doses of Pseudomonas aeruginosa, and J5 antiserum improved survival tenfold in animals dying of Esch. coli, Klebsiella and Pseudomonas bacteremia. The protection with vaccine or prophylactic antiserum was undiminished in animals challenged six weeks after immunization. Encouraged by these results, we conducted a double-blind trial in patients with gram-negative bacteremia. In those given J5 antiserum, the mortality rate was cut in half and survival from deep shock increased from 28 percent to 82 percent. Because of these preliminary results in 136 patients, the study has been extended to 300 patients and the double blind code will be examined again to see if the early favorable results are confirmed and extended.

Differentiation of Cryptococcus neoformans serotypes by isoenzyme electrophoresis.

Cryptococcus neoformans has been divided into four serotypes by specific agglutination in immune rabbit sera. Based on mating characteristics of the perfect state and epidemiologic and biochemical differences, the serotypes have been divided into two major pairs. In an attempt to characterize the serotypes further, the authors studied 22 strains of C. neoformans by the technic of horizontal starch-gel isoenzyme electrophoresis. The glucose-phosphate isomerase and phosphoglucomutase of serotypes A, C, D, and a subset of the serotype B strains migrated to distinguishable locations in this system. The activities of the remainder of the serotype B strains co-migrated with the serotype C strains. Thus, this technic distinguishes all the serotypes of C. neoformans except for a subset of serotype B and should be a useful adjunct for further elucidation of the epidemiologic and biochemical differences among serotypes.

Resistance to lysis by human serum of pathogenic Entamoeba histolytica.

A comparison was made of susceptibility to lysis by human sera among five non-pathogenic and 11 pathogenic strains of Entamoeba histolytica already characterized into zymodemes. The nonpathogenic strains were found to be uniformly susceptible to lysis. Nine of 11 pathogenic strains, including five strains isolated from liver abscesses, were found to be resistant to lysis by serum under identical conditions. Resistance to complement-mediated lysis may be an inherent property of most pathogenic strains and may prove to be a necessary virulence factor for dissemination.