Neisseria gonorrhoeae: vaginal clearance and its correlation with resistance to infection in subcutaneous chambers in orally immunized estradiol-primed mice.

The rate of clearance of Neisseria gonorrhoeae in vaginal specimens harvested from inbred Balb/c and outbred (Institute of Cancer Research [ICR]) white mice, previously primed with saline or beta-estradiol and then challenged with live gonococci, was determined. Survival of gonococci was significantly (P < 0.05) better in estradiol-primed mice from both inbred and outbred lines at 24 h after challenge. Vaginal clearance of gonococci from ICR mice parenterally primed with a gonococcal PI-B synthetic peptide and then orally immunized with gamma irradiated, protein-III deficient gonococcal cells was significantly (P < 0.001) faster than in corresponding nonimmunized, estradiol-primed controls. The more rapid vaginal clearance in the orally immunized mice was correlated with an elevated subcutaneous chamber ID50% (> 100,000 colony forming units) determined for the same groups of mice. Peptide-primed mice orally immunized with irradiated Escherichia coli cells demonstrated a chamber ID50% of < 100 CFU and were significantly (P < 0.01) slower to clear gonococci following vaginal challenge. High levels of specific IgA and IgG antibodies to gonococci were detected by an enzyme-linked immunosorbent assay in vaginal wash specimens from orally immunized mice. However, high antibody titers were not always predictive of an increased resistance to vaginal challenge. These models may be helpful in providing more economical and accessible in vivo methods for screening certain gonococcal vaccine candidates before more expensive testing in chimpanzees or humans.

Characterization of a catalase-deficient strain of Neisseria gonorrhoeae: evidence for the significance of catalase in the biology of N. gonorrhoeae.

We obtained a catalase-deficient (Kat-) strain of Neisseria gonorrhoeae isolated from a patient who had been unsuccessfully treated with penicillin. Quantitative enzyme assays and electrophoresis of cell extracts on native polyacrylamide gels subsequently stained for catalase and peroxidase activities failed to detect both enzymes. The strain exhibited no growth anomalies or unusual requirements when grown under ordinary laboratory conditions. However, the Kat- strain proved extremely sensitive to exogenous hydrogen peroxide, and analysis of the bacterial DNA after such exposure showed extensive single-strand breakage in both chromosomal and plasmid DNAs. Partial characterization of the gonococcal catalase from a Kat+ laboratory strain revealed that the enzyme had the physical and chemical properties of both catalase and peroxidase.

Influence of immunization on Porphyromonas gingivalis colonization and invasion in the mouse chamber model.

The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.

Mouse subcutaneous chamber model for in vivo growth of Haemophilus ducreyi.

The ability of Haemophilus ducreyi, the causative agent of chancroid, to grow in subcutaneous chambers implanted in mice was studied. All seven H. ducreyi strains tested were able to maintain a long-term infection in ICR mice; one mouse remained infected with strain Hd175 for more than 4 months. Growth curves obtained following the removal of chamber fluid at various time points from infected mice demonstrated that H. ducreyi was growing during the course of the infection. In addition, all three mouse strains tested (ICR, CBA and BALB/c) were able to maintain long-term H. ducreyi infections. This model will be valuable in studying the effects of in vivo growth on the antigenic composition of H. ducreyi as well as for the identification of virulence factors.

Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers.

Iron-uptake mutants of Neisseria gonorrhoeae strain 340 were obtained following treatment with streptonigrin, and one such mutant (Fud14) was characterized. N. gonorrhoeae strain Fud14 was unable to grow with human transferrin or haemoglobin as the sole source of iron, but grew normally with heat-inactivated normal human serum or haemin. Internalization of 55Fe from transferrin by strain Fud14 was only 25% of the parent level. Strain Fud14 (less than or equal to 1 x 10(8) c.f.u.) did not grow in subcutaneous chambers implanted in mice, whereas the parent strain was infective at an ID50 of 4.3 x 10(1) c.f.u. Supplementation of chambers with either normal human serum or haemin resulted in the establishment of strain Fud14 in vivo for at least 240 h post-inoculation. Electroporation of Fud14 with wild-type DNA and selection for growth on medium containing human transferrin resulted in a recombinant (Fud15) that was capable of utilizing haemoglobin, and was virulent in mice. These results suggest that a gonococcal strain defective in the ability to utilize in vivo iron sources is not capable of survival in vivo.

Binding of S protein by Neisseria gonorrhoeae and potential role in invasion.

An agglutination assay was used to examine the binding of purified human S protein (vitronectin, serum spreading factor) to 201 clinical isolates of Neisseria gonorrhoeae. Strains belonging to the protein IA serovars were significantly (P less than 0.001) more reactive in agglutination tests with human S protein and were more serum resistant than strains belonging to the protein IB serovars. The strains from patients with disseminated infections belonged predominantly to the IA serovar (19 of 23) and, with the exception of IA-4 and certain IB serovars, avidly agglutinated with S protein. The serovar IA-4 and IB strains isolated from joint or cerebrospinal fluid failed to agglutinate with S protein and appeared to be less serum resistant than most other IA isolates. Cysteine hydrochloride or 2-mercaptoethanol inhibited agglutination of S protein and a more than twofold increase in resistance to killing by fresh human serum following preincubation with S protein; the serum-sensitive parent strain did not agglutinate S protein, and serum resistance was not increased following preincubation with this protein. Binding of S protein by gonococci may represent a novel pathogenic mechanism that can contribute to serum resistance.

Characteristics of toxic shock syndrome toxin 1-induced lectin-like activity and inhibitor(s) in rabbit serum.

In adult rabbits intravenously injected with toxic shock syndrome toxin 1 (TSST-1) or staphylococcal enterotoxin B, serum lectin-like activity (detectable by cation-dependent agglutination of bacteria) developed. This activity was sensitive to heat, trypsin, and formaldehyde but resistant to neuraminidase or galactose oxidase. Formaldehyde-fixed Propionibacterium acnes or Escherichia coli cells reactive with plant lectins provided sensitive targets for titration of serum agglutination activity that was competitively blocked with D-galactose, D-glucose, D-mannose, and alpha-L-fucose. The lectin-like activity, partially purified by affinity chromatography, was a protein of approximately 76,000 Da with an isoelectric point of 5.4. Both lectin-positive and normal serum contained agglutination inhibitors that were absorbed by protein A-producing staphylococci. S protein may be the origin of this lectin-like activity. In vitro exposure of peripheral-blood mononuclear cells to TSST-1 (1.0 micrograms/mL) and to lectin-positive serum induced rapid cell clumping and subsequent "activation" to a larger blast form expressing receptors for buccal epithelial cells. The interaction of toxin/lymphokine-activated mononuclear cells with glycoproteins and/or other antigens selectively expressed by tissues in various organ systems may play a role in target cell pathology in rabbits dying with toxic shock syndrome.

Tissue fluid penetration and anti-treponemal activity of trospectomycin (a new spectinomycin analog) in the rabbit syphilis model.

The antitreponemal activity of trospectomycin (a novel 6'-propyl analog of spectinomycin) was correlated with concentrations in serum and tissue chamber fluid in a cutaneously-infected rabbit model of syphilis. After single-dose intramuscular (im) injections of trospectomycin, spectinomycin hydrochloride, and aqueous procaine penicillin G(APPG), concentrations of the drugs in paired specimens of serum and tissue fluid were determined and correlated with response of Treponema pallidum-infected lesions. Lesions responded most rapidly in APPG-treated animals. Rapid response correlated with more prolonged levels of APPG in both serum and tissue fluid. Trospectomycin, when given in single doses exceeding 40 mg/kg, surpassed penicillin in peak serum levels (greater than 60 micrograms/ml at 1 hr) but not in duration of activity. Higher and prolonged concentrations of trospectomycin in tissue fluid correlated with more effective clearance of treponemes from cutaneous lesions. Animals treated with less than 40 mg of trospectomycin/kg or with 20-80 mg/kg of the parent compound (spectinomycin hydrochloride) had cutaneous lesions that were persistently darkfield-positive, as confirmed by three or more consecutive smears.

Immune serum to protein molecular weight standards for calibrating Western blots.

A method for calibrating proteins by Western blot analysis is described. Rabbit immune serum, specific for seven protein molecular weight standards, and goat anti-rabbit horseradish peroxidase conjugate are used to visualize standards after electrotransfer onto nitrocellulose. This method can be used as a reagent and procedure control.

Affinity purification of staphylococcal toxic shock syndrome toxin 1 and its pathologic effects in rabbits.

Toxic shock syndrome toxin 1 (TSST-1) was purified to apparent homogeneity by chromatofocusing and affinity chromatography. The amino acid composition of the toxin was very similar to that reported for TSST-1 by other investigators. The amino-terminal amino acid was serine. A partial specific volume of 0.73 ml/g was calculated for the toxin from the amino acid data, and a molecular weight of 19,200 +/- 1,300 was determined by hydrodynamic methods. New Zealand white rabbits of both sexes were equally susceptible to the lethal effects of the toxin; however, older rabbits (greater than 12 months) were far more susceptible than young adults or weanlings. The 50% lethal dose of TSST-1 in older rabbits was 50 to 60 micrograms/kg when injected subcutaneously and 20 to 30 micrograms/kg when injected intravenously. Enhancement of lethal endotoxin shock by TSST-1 could not be demonstrated when both toxins were injected subcutaneously; however, lethal shock did occur when endotoxin (10 micrograms/kg) was injected intravenously after TSST-1 had been injected by either the subcutaneous (50 to 60 micrograms/kg) or the intravenous (20 to 30 micrograms/kg) route. Endotoxin alone was not lethal at a dose of 500 micrograms/kg of body weight when injected subcutaneously. When injected intravenously, endotoxin at a dose of 500 micrograms/kg was not lethal in weanling males or in females in any age group; however, young (6 to 7 months) and adult (greater than 12 months) males were killed by endotoxin doses as low as 45 to 50 micrograms/kg. Histopathologic studies of rabbits by both sexes which died as a result of TSST-1 alone or in combination with endotoxin showed extensive damage to organs rich in lymphoid and mononuclear phagocytic cells such as the thymus, mesenteric lymph nodes, liver, and spleen. Severe congestion of these organs as well as erythrophagocytosis and lymphoid depletion in the spleen and mesenteric lymph nodes were noted. Congestion and hemorrhage were also found in the heart, lungs, trachea, and thymus. The systemic pathology produced by TSST-1 was strikingly similar to that seen in humans who had died of toxic shock syndrome and in rabbits with subcutaneous chamber inoculated with toxic shock case strains of Staphylococcus aureus. Rabbits that were not killed by the toxin suffered a very rapid and severe leukopenia followed by leukocytosis with a left shift. Lymphopenia was also noted as was a mild but persistent anemia. With the exception of the early leukopenia, very similar hematologic findings have been noted in humans with toxic shock syndrome.

Staphylococcus aureus isolates from patients with nonmenstrual toxic shock syndrome. Evidence for additional toxins.

Although toxic shock syndrome toxin-1 (TSST-1) has been proposed as the toxin responsible for toxic shock syndrome, its role in this disease has not been proved. To study this question, we examined Staphylococcus aureus strains isolated from normally sterile sites in patients with nonmenstrual toxic shock syndrome for the presence of TSST-1 production. Only 20 (62.5%) of 32 produced TSST-1, compared with 41 (93%) of 44 vaginal isolates from patients with menstrual toxic shock syndrome. Of strains of S aureus from patients with nonmenstrual toxic shock syndrome, TSST-1-negative isolates were more likely to be associated with a fatal outcome and to not be phage group 1 than TSST-1-positive isolates. Seven of the TSST-1-negative strains were evaluated in a rabbit subcutaneous chamber model of toxic shock syndrome. Fifteen (60%) of 25 rabbits developed a toxic shock syndrome-like illness and nine died. Clinical signs and histopathologic findings in the rabbits were similar to those seen in rabbits inoculated with TSST-1-positive S aureus isolates. These results suggest that other, as yet unrecognized, toxins play a role in toxic shock syndrome, and that TSST-1 production may not be essential to the pathogenesis of toxic shock syndrome.

Acquired ability of Staphylococcus aureus to produce toxic shock-associated protein and resulting illness in a rabbit model.

Staphylococcus aureus from patients with toxic shock syndrome (TSS) produce TSS toxin 1. We transferred, by a bacteriophage, the ability to produce TSS toxin 1 from a TSS toxin 1-positive to a TSS toxin 1-negative strain of S. aureus. This recombinant strain produced TSS toxin 1 as confirmed by isoelectric focusing, immunodiffusion, radioimmunoassay, and autoradiography. The recombinant produced TSS-like illness in rabbits, and was significantly (P less than 0.001) more lethal than the recipient strain. Both strains produced fever and diarrhea, but, in addition, rabbits challenged with the recombinant also developed lowered blood pressure (P = 0.002), conjunctival hyperemia, erythroderma, and respiratory distress. Histopathological findings in rabbits challenged with the recombinant strain were remarkably similar to those described for humans with TSS, e.g., erythrophagocytosis, liver "triaditis," and vasodilatation. This study demonstrates that this protein may contribute to the pathogenesis of the TSS.

Evaluation of a C-reactive protein latex agglutination detection test with sera from patients with sexually transmitted diseases.

A total of 149 sera, including 79 pre- and posttreatment sera from 33 patients with disseminated gonococcal infections, 18 from patients with uncomplicated gonococcal infections, 6 from patients with pelvic inflammatory disease, 4 from patients with genital Chlamydia trachomatis infections, and 42 from normal volunteers, were examined for C-reactive protein with a latex agglutination C-reactive protein detection kit (Difco Laboratories, Detroit, Mich.). Results were quantitated with LC-Partigen C-reactive protein radial immuno-diffusion plates (Calbiochem-Behring, La Jolla, Calif.). Positive latex agglutination results were observed in all of the pretreatment sera and some of the posttreatment sera of patients with disseminated gonococcal infections and in two sera from patients with pelvic inflammatory disease, which corresponded to quantitative C-reactive protein levels in the radial immunodiffusion plates. C-reactive protein levels were not detectable in the serum samples from normal volunteers or patients with uncomplicated gonococcal infections or genital chlamydial infections. Positive latex agglutination occurred as early as 20 s in sera with high C-reactive protein levels, and all positive results were observed within 90 s of the 3-min test limit. Positive latex test results were obtained with C-reactive protein levels as low as 1 mg/dl (10 micrograms/ml).

Superoxol and amylase inhibition tests for distinguishing gonococcal and nongonococcal cultures growing on selective media.

Two inexpensive screening tests were evaluated singly and in tandem for distinguishing Neisseria gonorrhoeae from other oxidase-positive microorganisms growing on selective gonococcal media. In tests of 728 cultures, including 460 N. gonorrhoeae, 4 Neisseria lactamica, 257 Neisseria meningitidis, and 7 Branhamella catarrhalis, both Superoxol (30% H2O2; J. T. Baker Chemical Co., Phillipsburg, N.J.) and amylase inhibition tests were 100% sensitive (positive) for 20-h cultures of N. gonorrhoeae. Singly, the Superoxol test was 92.7% specific for N. gonorrhoeae, compared with a specificity of 82.3% for the amylase inhibition test. By using tandem screening tests to distinguish gonococci, we achieved an overall specificity of 98.6%. Group A meningococci were the primary source of error in the Superoxol test, with 97% (37 of 38) strains producing gonococcal like reactions for catalase. From 5 to 20% of N. meningitidis serogroups X, Y, Z, and Z' and nontypable strains, as well as about 50% of B. catarrhalis and N. lactamica strains, were also strong catalase producers.

A rabbit model of toxic shock syndrome: clinicopathological features.

The complete pathogenesis of toxic shock syndrome (TSS) has yet to be elucidated. Unmasking the complex interactions among bacterial products, host factors, and possibly tampon components requires a suitable in vivo model. For this purpose, subcutaneous chambers implanted in rabbits were inoculated with Staphylococcus aureus isolated from patients with TSS. Infected rabbits developed illness characterised by multisystem involvement that included periportal inflammation of the liver, erythrophagocytosis in the spleen and lymph nodes as well as extreme vascular dilatation and epithelial lesions similar to those described in patients with TSS. Concentrations of serum creatinine (P less than 0.03) and triglycerides (P less than 0.04) were significantly raised in rabbits infected with TSS strains compared with rabbits infected with non-TSS strains of S. aureus. Both TSS and non-TSS strains of S. aureus produced fever and diarrhoea, but TSS strains were significantly (P less than 0.05) more lethal and more likely to produce respiratory distress and lowered blood pressure. This model may help to prove or disprove proposed mechanisms for the development of TSS.

Antigonococcal effects of vaginal tampons.

Different brands of vaginal tampons varied significantly (p less than 0.0001) in their anti-bacterial effects when tested with 46 strains of Neisseria gonorrhoeae. Gonococcal strains recovered from patients with disseminated infections were substantially more sensitive to the anti-bacterial effects of tampons than were strains from patients with uncomplicated genital infections. Strains from patients with pelvic inflammatory disease were moderately sensitive. Tampons showing strong in-vitro antigonococcal effects were also generally effective in vivo in eliminating gonococcal infections from subcutaneous chambers in mice. Extracts of the Rely tampon showed no in-vitro antigonococcal effect, however, but did induce antibacterial activity when injected into subcutaneous chambers in mice. These results emphasise the importance of both in-vitro as well as in-vivo testing of tampon materials to elucidate more fully the nature of their antibacterial effects and their potential for affecting vaginal pathogens and disease processes.

Differentiation of Kingella denitrificans from Neisseria gonorrhoeae by growth on a semisolid medium and sensitivity to amylase.

All 239 strains of Neisseria gonorrhoeae tested for sensitivity to amylase-impregnated disks showed zones of growth inhibition ranging from 15 to 30 mm in diameter. None of 52 strains of Kingella denitrificans were inhibited by the same amylase test. In addition, all N. gonorrhoeae strains grew well on a gonococcal base medium supplemented with either IsoVitaleX (BBL Microbiology Systems) or Supplement B (Difco Laboratories), whereas K. denitrificans grew adequately only on the medium with IsoVitaleX. The addition of L-cysteine or cystine to the medium with Supplement B enhanced the growth of K. denitrificans.