Glycosidase activities of Bacillus anthracis.

Bacillus anthracis could be distinguished from the taxonomically related species B. cereus, B. mycoides, and B. thuringiensis by a comparison of glycosidase activities. All the bacilli tested possessed alpha-glucosidase activity, as evidenced by the hydrolysis of p-nitrophenyl-alpha-D-glucoside. In B. anthracis, the glucosidase activity could be enhanced by the addition of agents which damage cellular surface structures. Treatment of B. anthracis strains with toluene. Triton X-100, or mutanolysin or cellular disruption by sonication resulted in higher rates of alpha-glucoside hydrolysis than were accomplished by cells suspended in buffer. It is suggested that intact B. anthracis cells have a limited permeability to the glucosidase substrate. In contrast to the results obtained for B. anthracis, Triton X-100 markedly diminished the enzymatic hydrolysis of p-nitrophenyl-alpha-D-glucoside by strains of B. cereus, B. mycoides, and B. thuringiensis. Triton X-100 also enhanced the alpha-maltosidase activity of B. anthracis but not that of the other bacilli. B. mycoides possessed an apparently inducible N-acetylglucosaminidase although the enzyme was absent in B. anthracis. The glucosaminidase was inducible in the presence of p-nitrophenyl-N-acetylglucosamine in the absence of conventional nitrogen sources. Chloramphenicol prevented the induction of the glucosaminidase in B. mycoides. In several B. cereus and all B. thuringiensis strains, the glucosaminidase was constitutive. The results suggest a means for the rapid laboratory differentiation of B. anthracis from other closely related bacilli. Assays for alpha-glucosidase and alpha-maltosidase, in the presence and absence of Triton X-100, can be used to distinguish B. anthracis from B. cereus, B. mycoides, and B. thuringiensis. Similarly, the hydrolysis of p-nitrophenyl-beta-N-acetylglucosamine induced by B. mycoides but not by B. anthracis provides an additional means for differentiating these similar bacilli.

Diagnostic value of interactions between members of the family Neisseriaceae and lectins.

The lectin slide agglutination test for Neisseria gonorrhoeae has been modified and improved. Results show that wheat germ agglutinin and soybean lectin agglutinate 100% (193 of 193 tested) of clinical isolates of N. gonorrhoeae. Lectin-reactive meningococci can be readily identified by the hydrolysis of gamma-glutamyl-beta-naphthylamide. Branhamella catarrhalis, Neisseria lactamica, Neisseria sicca, Neisseria subflava, Neisseria perflava, and meningococcal serogroups A, B, C, X, Y, and Z do not interfere with the positive identification of N. gonorrhoeae. The frequently encountered problem of autoagglutination of members of the family Neisseriaceae may be circumvented by a short treatment of cellular suspensions with DNase. Based on agglutination assays, the enzyme treatment did not result in a loss of wheat germ agglutinin receptors from the bacteria. The lectin agglutination test, coupled with the gamma-glutamyl aminopeptidase assay, is proposed as a rapid and accurate means of identifying clinical isolates of gonococci.

Differentiation of Bacillus anthracis and other Bacillus species by lectins.

Bacillus anthracis was agglutinated by several lectins, including those from Griffonia simplicifolia, Glycine max, Abrus precatorius, and Ricinus communis. Some strains of Bacillus cereus var. mycoides (B. mycoides) were strongly reactive with the lectin from Helix pomatia and weakly reactive with the G. max lectin. The differential interactions between Bacillus species and lectins afforded a means of distinguishing B. anthracis from other bacilli. B. cereus strains exhibited heterogeneity with respect to agglutination patterns by lectins but could readily be differentiated from B. anthracis and the related B. mycoides. Spores of B. anthracis and B. mycoides retained lectin receptors, although the heating of spores or vegetative cells at 100 degrees C resulted in a decrease in their ability to be specifically agglutinated. Fluorescein-conjugated lectin of G. max stained vegetative cells of B. anthracis uniformly, suggesting that the distribution of lectin receptors was continuous over the entire cellular surface. B. anthracis cells grown under conditions to promote the production of capsular poly(D-glutamyl peptide) were also readily agglutinated by the lectins, suggesting that the lectin reactive sites penetrate the polypeptide layer. Trypsin, subtilisin, lysozyme, and mutanolysin did not modify the reactivity of B. anthracis with the G. max agglutinin, although the same enzymes markedly diminished the interaction between the lectin and B. mycoides. Because the lectins which interact with B. anthracis are specific for alpha-D-galactose or 2-acetamido-2-deoxy-alpha-D-galactose residues, it is likely that the bacteria possess cell surface polymers which contain these sugars. Lectins may prove useful in the laboratory identification of B. anthracis and possibly other pathogenic Bacillus species, such as B. cereus.

Interaction between plant agglutinins and Legionella species.

Serogroups of Legionella pneumophila exhibited differential reactivities with plant agglutinin. Agglutination patterns were modified by growing the organisms in different media. The passage of four strains through guinea pigs did not result in altered reactivities with lectins or with plant agglutinins.

Differentiation of coagulase-positive and coagulase-negative staphylococci by lectins and plant agglutinins.

The screening of staphylococci with a panel of 14 lectins and extracts demonstrating lectin-like activity led to the development of a rapid agglutination slide test for the differentiation of certain coagulase-negative staphylococci and human strains of Staphylococcus aureus. The coagulase-negative staphylococci were agglutinated by agglutinins from Mangifera indica, Triticum vulgaris, and crude Limulus polyphemus. The test is rapid, requiring only 5 to 15 min to identify an unknown strain of staphylococci, as opposed to the 4 to 16 h required to perform the conventional tube coagulase test.

Strongyloidiasis in immunosuppressed patients.

An increasing number of severe, even fatal cases of strongyloidiasis in patients on immunosuppressive drugs are being reported. During a 26 month period, three severe cases of Strongyloides stercoralis infestation were diagnosed in patients who were on steroid therapy. These cases are of special interest because of the following factors. Firstly, all the patients lived in a nonendemic metropolitan area. Secondly, a single stool specimen from one patient contained several distinct developmental stages of the parasite's life cycle. This finding appears to be unique considering the fact that the patient had not been administered a purgative or anthelminthic. Lastly, a surviving renal transplant patient continues to have persistent strongyloidiasis despite repeated Mintezol (thiabendazole) therapy.

Lectins in diagnostic microbiology: use of wheat germ agglutinin for laboratory identification of Neisseria gonorrhoeae.

A lectin slide agglutination test has been developed for the confirmatory identification of Neisseria gonorrhoeae. With wheat germ lectin as an agglutinin, 164 of 165 clinical isolates of N. gonorrhoeae gave a 3 to 4+ reaction within 6 to 8 min. Four gonococcal isolates, even though negative by the fluoresecent-antibody method, gave strong positive reactions with the wheat germ lectin. Among 23 isolates of Neisseria meningitidis tested, which included representatives of sero-groups A, B, C,D, X, Y, and Z, only one strain in group X gave a false-positive reaction. The nonpathogenic species of Neisseria, as well as Branhamella catarrhalis, all showed negative reactions with the wheat germ agglutinin. The novel method provides a simple, rapid, and inexpensive means for the laboratory diagnosis of gonorrhea and obviates the need for performing second-stage sugar fermentation studies or utilizing the more expensive fluorescent-antibody techniques.

"Ghost" forms of Giardia lamblia cysts initially misdiagnosed as Isospora.

Because of their distinctive morphology the cysts of Giardia lamblia usually are not mistaken for cysts or eggs of other intestinal parasites. At a hospital laboratory in Kentucky a case of giardiasis was encountered in which the initial fecal examination revealed only degenerated cysts of Giardia which were mistaken for the oocysts of Isospora at the one-sporoblast stage.

Protective effects of passively transferred immune T- or B-lymphocytes in mice infected with Salmonella typhimurium.

Purified populations of bone marrow-derived (B-) lymphocytes and thymus-derived (T-) lymphocytes were obtained from C3D2F1 hybrid mice shown to be immune to Salmonella typhimurium. These subpopulations of lymphocytes were injected into normal mice; four days later the animals were challenged with 50 50% lethal doses of S. typhimurium, and viable bacteria in livers, spleens, and blood were counted at various intervals after challenge. On day 8 after challenge, the mice supplemented with B-lymphocytes showed a significant decrease in the number of organisms recovered from all three sites, compared with that seen in recipients of T-lymphocytes and in controls. The mice given B-lymphocytes showed a better rate of survival (65%) than mice that received only T-lymphocytes (21%) or T-lymphocyte fractions contaminated 10%-30% with B-lymphocytes (49%). These data indicate that, although the humoral response is not totally protective, it does play an important role in the suppression of the infection during its early stages.

A mechanism for tobacco smoke-induced allergy.

Normal CFW mice, when exposed to tobacco smoke, showed a significantly increased susceptibility to the lethal effects of histamine. The LD50 for mice subjected to smoke was 45 mg/kg of histamine, whereas in normal CFW mice the LD50 was 1,100 mg/kg. The histamine susceptibility of smoked mice was markedly diminished by injecting the animals with isoproterenol. Normal CFW mice, as well as sham control mice, exhibited an epinephrine-induced hyperglycemia, whereas the blood glucose values for smoked mice given epinephrine were essentially the same as those for sham mice given only saline. This observation indicates that tobacco smoke may contain a component which causes an autonomic imbalance, hence rendering the mice more susceptible to histamine. This tobacco smoke-induced allergy is probably related to a blockade of adrenergic receptors and not to an immunologic phenomenon.

In vivo and in vitro manifestations of adrenergic blockade in Bordetella pertussis-vaccinated mice.

The in vivo metabolic events which follow the administration of epinephrine, norepinephrine, or isoproterenol were examined in normal, Bordetella pertussis-vaccinated, and alpha and beta adrenergically blocked mice. The normal hyperglycemic response to epinephrine was suppressed in all experimental groups. The pertussis-sensitized and beta-blocked animals produced similar split patterns of altered response not duplicated by the alpha-blocking compounds. Those catecholamines that normally increase free fatty acids and lactic acid in the circulation failed to do so in the pertussis-sensitized and beta-blocked animals; the inhibition of free fatty acid mobilization was also demonstrated with adipose tissue incubated in vitro. An extract of the pertussis organism added to incubation media prevented the catecholamine-induced free fatty acid response. The epinephrine-stabilizing effect of bovine serum albumin (Cohn-fraction V) was observed. The results of these studies further emphasize a correlation between pertussis-sensitized and beta-adrenergically blocked mice.