Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis.

The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis. Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD50 of 10(3) colony forming units (CFU) or greater (low virulence) from those having an LD50 of approximately 10(1) or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica, a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD50 of 10(2) were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.

Immunogold labelling of Listeria monocytogenes virulence-related factors within Caco-2 cells.

We demonstrated by immunoelectron microscopy that listeriolysin O (LLO), phospholipases and other putative virulence-related proteins produced by Listeria monocytogenes were primarily cell-wall-associated when the bacterium infected Caco-2 tissue culture cell monolayers. Antibodies made to LLO, serogroup 1/2a reacted poorly with serogroup 4b cells and vice-versa, indicating fundamental structural differences in the two proteins. Finally, comet-tail pseudopod structures shown to be involved in cell-to-cell passage of Listeria in Caco-2 cells did not possess detectable Listeria antigens on their anterior surface or within their structure, suggesting that the phagocytic process is primarily host-cell-dependent once it is initiated by the bacterial cell.

A filamentous-like mutant of Listeria monocytogenes with reduced expression of a 60-kilodalton extracellular protein invades and grows in 3T6 and Caco-2 cells.

We describe a spontaneous rough mutant of Listeria monocytogenes that produces reduced amounts of a 60-kilodalton major extracellular polypeptide (p60) as shown by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot analysis. The cells of this mutant are filamentous, do not give rise to smooth wild-type colonies, and produce listeriolysin O in amounts equal to that of the wild-type cells, but they show a reduced virulence in the mouse LD50 model and in the Caco-2 tissue culture virulence assay. Light and electron microscopic studies show that this mutant invades and remains filamentous during in vivo growth in both Caco-2 and 3T6 tissue culture monolayers. The reduced virulence of the rough mutant is not due to the inability of its filamentous forms to invade or to grow in nonprofessional phagocytes since invasion and growth of the smooth wild-type and the rough mutants are comparable in both Caco-2 and 3T6 monolayers.

Cytopathogenic effects in enterocytelike Caco-2 cells differentiate virulent from avirulent Listeria strains.

We have developed a simple test that differentiates between virulent and avirulent Listeria species as defined by the mouse 50% lethal doses (LD50S). The assay is based on trypan blue-revealed cytopathogenic effects that are produced during the infection of the human enterocytelike cell line Caco-2. These effects were elicited only by Listeria strains that had an intraperitoneal mouse LD50 less than 10(8) and were not produced by nonhemolytic, avirulent strains of Listeria monocytogenes generated spontaneously or by Tn916 mutagenesis or by avirulent Listeria species. A negative test was also obtained with hemolysin-producing, avirulent L. monocyotogenes NCTC5105 and Listeria ivanovii KC1786. The test was negative with avirulent L. monocytogenes strains which are strong inducers of opacity in egg yolk agar. However, a strain which has a low LD50, such as 10(4), may show less severe cytopathogenic effects than a strain having a higher LD50, such as 10(6). The test has been effectively used to screen for virulent listerial isolates, spontaneous mutants, and transposon-induced mutants.

The type strain(s) of Listeria monocytogenes: a source of continuing difficulties.

The type strain of Listeria monocytogenes differs from wild-type L. monocytogenes strains in more characteristics than just the previously reported deficiency in hemolytic activity and virulence in the murine infection model. The type strain from the American Type Culture Collection (strain ATCC 15313) produces lecithinase, is hemolytic on rabbit (but not sheep) blood agar, lacks motility, and shows limited cytopathogenic effects on Caco-2 monolayers, whereas the type strain from the Special Listeria Culture Collection (strain SLCC 53) is unable to produce lecithinase, is nonhemolytic on rabbit or sheep blood agar, is motile, and shows no cytopathogenic effects on Caco-2 monolayers.

Nonhemolytic Listeria monocytogenes mutants that are also noninvasive for mammalian cells in culture: evidence for coordinate regulation of virulence.

We identified nonhemolytic mutants of Listeria monocytogenes that were severely deficient in their ability to invade mammalian nonprofessional phagocytes. These mutants were generated spontaneously or by means of transposon Tn916 mutagenesis. In terms of their extracellular proteins, the noninvasive mutants were deficient not only in the sulfhydryl-activated hemolysin (listeriolysin) but also in an antigenically unrelated extracellular protein with an apparent molecular weight of 32,000 which could induce opacity in egg yolk and is considered to be a phospholipase. Our results suggest the existence of a common genetic control between the expression of listeriolysin and that of other determinants, including a phospholipase and determinants involved in the ability of L. monocytogenes to enter mammalian cells.

Listeria monocytogenes intragastric and intraperitoneal approximate 50% lethal doses for mice are comparable, but death occurs earlier by intragastric feeding.

The intraperitoneal (i.p.) and intragastric (i.g.) mouse approximate 50% lethal dose values (ALD50S) were determined for 15 food and clinical isolates of Listeria monocytogenes. Although all strains gave i.g. ALD50S comparable to or less than their i.p. ALD50S, the i.g. feeding of most strains produced more deaths within the first 3 days of the 6-day test than did i.p. injection. ALD50S ranged from 50 to 4.4 x 10(5) cells with approximately 1-log 95% confidence intervals. Of five strains tested by suspension in milk or by growth in milk, none gave i.g. ALD50S that were lower than those of washed cells. Results with 10- to 21-g mice supported the use of 15-g mice for i.g. testing; 21-g mice were more resistant to i.g. infection. These results indicate that i.g. feeding permits an evaluation of the role of the carrier (such as milk) in the determination of listerial virulence, permits strain characterization by i.p. and i.g. ALD50S, and emphasizes a potentially more rapid infection when the bacterium is introduced i.g.

Analyses of fermentation products of Listeria species by frequency-pulsed electron-capture gas-liquid chromatography.

Aerobic fermentation broths of eight Listeria monocytogenes strains, two or more strains of the remaining six Listeria species, and one strain of Jonesia denitrificans were examined by frequency-pulsed electron-capture gas-liquid chromatography for carboxylic acids, alcohols, amines, and hydroxy acids. All species produced acetic, isobutyric, butyric, isovaleric, phenylacetic, lactic, 2-hydroxybutyric, 2-hydroxyvaleric, and 2-hydroxyisocaproic acids. Propionic acid was not formed, and traces of isocaproic acid were observed. Of the alcohol and amine derivatives observed, only acetylmethylcarbinol, butylamine, and putrecine were identified. Recognition of the products of glucose and amino acid metabolism serves to further characterize the members of the genus Listeria both taxonomically and physiologically.

Physiological studies on the growth and utilization of sugars by Listeria species.

Experiments, relevant to growth in milk, were done to delineate the aerobic and anaerobic growth of Listeria species on selected sugars in several media. All species grew on glucose aerobically, forming lactic acid and (or) acetic acid. Anaerobically, only lactic acid was formed; cell yields were 80% of those obtained aerobically. When incubated aerobically, small amounts (1.5 microns/mL) of isovaleric acid, 2-hydroxyisovaleric acid, and trace amounts of isobutyric acid were formed. These products were characteristically formed by 26 strains representing all the species of Listeria. Added leucine stimulated isovaleric acid formation. Anaerobic fermentations of glucose could be followed by 60 to 80% cell lysis; less lysis occurred in air. Anaerobically, only hexoses and pentoses supported growth; aerobically, maltose and lactose supported growth of some strains, but sucrose did not support growth of any strain tested. Listeria grayi and Listeria murrayi utilized the galactose and glucose moieties of lactose for growth; Listeria monocytogenes and Listeria innocua used only the glucose moiety. Glucosamine and N-acetylglucosamine supported aerobic and anaerobic growth as well as glucose, and their presence stimulated the utilization of lactose by "lactose-negative" strains. Analyses of cultures grown at 5 degrees C in sterile milk treated with glucose oxidase supported the conclusion that the glucose of the milk was the major, if not the limiting, substrate that supported growth.

The Swedish medical care programs: an interim assessment.

The medical care program (MCP) concept emerged from a conviction that it would be possible to combine biomedical knowledge about a certain disease, principles of care and an efficient organization into a holistic approach to care. The purpose of the present review of nine MCPs was to: (1)provide and overview of MCP development and and evaluation in the Stockholm County; (2) present different perspectives regarding the current status of the MCP policy and future developments; and (3) contribute to a discussion of factors which enhance or block the effectiveness of MCPs. Information was gathered during interviews with 32 representatives of professionals and interest groups. The majority of MCPs were initiated by medical professionals while two, the program for alcohol disorders and that for rheumatoid diseases, were initiated by politicians or the rheumatoid patients. Three central problems were identified: (1) the original desire for standardization and the emergent demand for local variation; (2) ambiguities about specific roles of the newly developing general practitioners; and (3) lack of resources to develop, implement and evaluate MCPs to the standards of the original concept. The experience of the MCPs certainly has increased understanding of the policy-program-implementation-outcome process and inevitable gaps that materialize as policy struggles towards implementation.

Listeria monocytogenes ATCC 35152 and NCTC 7973 contain a nonhemolytic, nonvirulent variant.

Listeria monocytogenes NCTC 7973 and this same strain deposited as ATCC 35152 contain two phenotypes: hemolytic virulent colonies and nonvirulent colonies that show no zones of hemolysis when streaked on heart infusion agar containing 5% rabbit blood. Results of examinations of these virulent and nonvirulent strains by investigators at the Centers for Disease Control, Atlanta, Ga., the Pasteur Institute, Paris, France, and the University of Würzburg, Federal Republic of Germany, support the conclusion that the avirulent strain is a nonhemolytic mutant of the virulent strain and that hemolysin is a virulence factor for L. monocytogenes.

Role of keto acids and reduced-oxygen-scavenging enzymes in the growth of Legionella species.

Keto acids and reduced-oxygen-scavenging enzymes were examined for their roles in supporting the growth of Legionella species and for their potential reactions between the chemical components of the media. When grown in an experimental ACES (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid)-buffered chemically defined (ABCD) broth, the presence of keto acids shortened the lag periods, increased the rates of growth, and gave maximum cell yields. In addition, keto acids affected the specific activities of reduced-oxygen-scavenging enzymes determined during growth. The specific activities of superoxide dismutase of Legionella pneumophila (Knoxville) and L. dumoffii (TEX-KL) were increased three- to eightfold, while that of L. bozemanii (WIGA) was not affected. All strains appeared to be equally sensitive to the effects of superoxide anion (O2-) generated by light-activated riboflavin, and all were equally protected by the presence of keto acids in the ABCD broth. Production of trace amounts of acetate and succinate in pyruvate- and alpha-ketoglutarate-containing media exposed to light suggested that hydrogen peroxide was formed. Pyruvate and alpha-ketoglutarate were products of growth on amino acids, and there was no quantitative evidence that these keto acids were metabolized when they were added to the medium. The rate of cysteine oxidation in ABCD broth was increased by the presence of ferric ion or by exposure to light or by both, and keto acids reduced the rate of this oxidation. ACES buffer was a substrate for the production of O2- in the presence of light, and the combined addition of Fe2+ ions, cysteine, and either keto acid to the medium strongly inhibited the production of O2-. Thus, keto acids inhibited the rate of cysteine oxidation, they stimulated rapid growth by an unknown process, and, in combination with added Fe2+ ions and cysteine, they reversed the toxic effects of light by inhibiting O2- production.

Guanine is a growth factor for Legionella species.

Evaluation of previously described chemically defined media for the growth of Legionella pneumophila showed that these media supported poor growth of several strains of L. pneumophila and did not support growth of certain of the Legionella species described later. Growth was stimulated by the dialysate from yeast extract but not by the nondialyzable fraction. Further investigations indicated that the active factors from the yeast extract dialysate were purine or pyrimidine derivatives, and certain known purines and pyrimidines were found to stimulate growth. Of these, guanine universally stimulated growth of all Legionella strains and was a growth requirement for several of the species tested. A balanced, N-(2-acetamido)-2-aminoethanesulfonic acid-buffered, chemically defined medium having guanine or a purine-pyrimidine mix is presented for the general growth of Legionella species.

Actinomyces and the intrauterine contraceptive device: aspects of the fluorescent antibody stain.

Using fluorescein-conjugated globulins specific for Actinomyces israelii and Arachnia propionica, we observed large dispersed actinomycete populations in vaginal smears of several asymptomatic women. Mycelial granules, commonly revealed by the Papanicolaou stain, were not seen. These observations are discussed in regard to the threat of infection and sensitivity of the fluorescent stain.

PIP: Using fluorescein-conjugated globulins specific for Actinomyces israelii and Arachnia propionica, the authors sobserved large dispersed actinomycete populations in vaginal smears of several asymptomatic women. Mycelial granules, commonly revealed by the Papanicolaou stain, were not seen. These observations are discussed in regard to the threat of infection and sensitivity of the fluorescent stain.

Presence of toxic shock toxin in toxic shock and other clinical strains of Staphylococcus aureus.

Toxic shock toxin (TST), also known as pyrogenic exotoxin C (Schlievert et al., J. Infect. Dis. 143:509-516, 1981) and staphylococcal enterotoxin F (Bergdoll et al., Lancet i:1017-1021, 1981), was purified from toxic shock strains of Staphylococcus aureus by preparative isoelectric focusing and by chromatofocusing. Neither method produced an absolutely pure protein as determined by silver staining of sodium dodecyl sulfate-acrylamide gels, although chromatofocusing was the better method of the two. Three molecular weight variants of the protein were found in the two toxic shock syndrome strains that were studied, regardless of the purification method that was used. An isoelectric point of 7.15 and molecular weights of 21,400, 22,100, and 23,200 were determined for the different forms of the protein from electrophoresis data. A sedimentation coefficient of 2.3S was determined by sucrose gradient centrifugation, and a Stokes radius of 2 X 10(-7) cm was determined by gel filtration. An average molecular weight of 18,900 for all of the TST forms was calculated from these data by the Stokes-Einstein equation. A survey for TST in 32 control and 46 toxic shock strains of S. aureus by isoelectric focusing and by agarose gel double immunodiffusion with specific rabbit antiserum revealed that the isoelectric focusing method tends to overestimate the number of TST-positive strains because of the detection of non-TST, neutral staphylococcal proteins. Based on immunodiffusion data, the association of TST with toxic shock strains was found to be 100% in vaginal isolates and 62% in non-vaginal isolates. In the control strains, TST was found in 16% of the vaginal strains and 23% of the non-vaginal strains. The value of this toxin as a marker for toxic shock and its relationship to the pathogenesis of this disease are discussed.

Determination of catalase, peroxidase, and superoxide dismutase within the genus Legionella.

We examined 40 strains of Legionella for reduced-oxygen scavenging enzymes. Using a simple reaction chamber with a Swinney filter for the Beers and Sizer assay, we determined the catalase activity of live cells grown on buffered charcoal-yeast extract agar. For 29 strains of Legionella pneumophila, the apparent first-order rate constants for catalase ranged from 0.000 to 0.005. Similarly, low values ranging from 0.001 to 0.005 were observed for Legionella wadsworthii, Legionella oakridgensis, and Legionella gormanii. High catalase activities were found for Legionella jordanis, Legionella longbeachae, Legionella micdadei, and Legionella bozemanii, with first-order rate constant values of 0.010 to 0.035. Cell-free extracts were analyzed for catalase, peroxidase, and superoxide dismutase. Cell-free extracts of all strains had superoxide dismutase levels ranging from 8.2 to 30.5 U per mg of protein. The species could be characterized by their catalase and peroxidase since L. pneumophila and L. gormanii had only peroxidase (relative molecular weight [Mr], 150,000); L. dumoffii had a peroxidase (Mr, 150,000) plus a catalase (Mr, 174,000); and all remaining species had catalase only (Mr, 300,000, 220,000, or 150,000).