Enzyme-linked immunosorbent assays for virulence properties of Campylobacter jejuni clinical isolates.

To evaluate the capacity of enzyme-linked immunosorbent assays (ELISAs) to identify pathogenic strains among clinical fecal isolates of Campylobacter jejuni, 40 consecutively obtained strains from 39 sick patients and 1 asymptomatic person were tested by respective ELISAs for enterotoxin production in culture filtrates and for the invasive virulence antigen of bacterial cells. Of the 40 strains, 14 produced the enterotoxin; 15 strains, two of which were also enterotoxigenic, were invasive; and 11 strains had no detectable virulence property. The presence or absence of these virulence properties was confirmed by the demonstration that viable cells of all 12 randomly selected enterotoxigenic or invasive strains tested, but none of 9 nonpathogenic strains tested, caused fluid secretion in rat ligated ileal loops. All 12 patients examined who were infected with an invasive strain had grossly or microscopically evident blood cells in their stools or both, whereas none of those infected with an enterotoxigenic strain had overtly bloody diarrhea, and only 1 of 8 patients examined had microscopically evident blood cells in the stool. Twelve of the invasive, five of the enterotoxigenic, and three of the nonpathogenic strains also produced small amounts of cytotoxin, but there was no correlation between cytotoxin production and an abnormal response in rat ligated ileal loops. These observations show that enterotoxin production or invasiveness or both can be detected by ELISAs in three-fourths of C. jejuni fecal isolates and that there is usually a relationship between the specific pathogenic property of the infecting strain and the clinical mainfestations.

Autoplaquing in Neisseria gonorrhoeae.

Irregularly shaped autoplaques were observed on a lawn of two different strains of Neisseria gonorrhoeae. Autoplaquing occurred on gonococcal genetic medium lacking arginine and was noninducible on complete gonococcal genetic medium. The cell density, incubation temperature, and agar base influenced autoplaquing. Single-colony suspensions varied in plaque morphology. We were unable to isolate a stable nonplaquing variant but separated strain RUN5287 into two plaquing phenotypes.

Anaerobic survival of clinical isolates and laboratory strains of Neisseria gonorrhoea: use in transfer and storage.

Eleven laboratory strains and 67 clinical isolates of Neisseria gonorrhoeae were tested for the ability to survive during anaerobic incubation. The survival of the laboratory strains was dependent on auxotype, temperature, and cell density on agar plates. For both the laboratory strains and the clinical isolates, anaerobic survival was better at lower temperatures. We concluded that anaerobic incubation, for as long as 7 days, is useful when transporting or storing N. gonorrhoeae.

Immunological cross-reactivity of heat-labile enterotoxins produced by enterotoxigenic and enteropathogenic strains of Escherichia coli.

Weanling rats were immunized with a heat-labile enterotoxin contained in whole cell lysate (WCL) ultrafiltrate preparations of enteropathogenic (EPEC) and enterotoxigenic (ETEC) strains or with a purified preparation of heat-labile toxin (LT) from the ETEC strain and then challenged either with viable bacteria of each strain or the purified ETEC LT by means of the ileal ligated loop technique. Immunization with the WCL toxin preparations of either the EPEC or ETEC strain conferred protection against challenge with viable organisms of both strains; immunization with a similar preparation from a nontoxigenic strain did not yield protection. Immunization with either the WCL or purified LT toxin from ETEC strain afforded protection against challenge with the ETEC LT toxin, but immunization with the EPEC WCL preparation did not. The antigenicity of all of the toxin preparations was destroyed by heat-treatment. Possible contributory protective effects of somatic or colonization factor (CFA) antigens present in the WCL were excluded by the findings that protection was afforded against a heterologous somatic serotype, ileal bacterial counts were not reduced in protected animals, and WCL preparations of strains containing or lacking CFA yielded equal protection. These observations indicate that the heat-labile enterotoxin of EPEC strains is antigenic and is immunologically related to a heat-labile toxin present in similarly prepared material from an ETEC strain but not to the conventional LT toxin of ETEC strains. They suggest that the WCL preparation of the ETEC strain contains two heat-labile enterotoxins, one of which is conventional LT and the other of which resembles the EPEC toxin.

Protective effect of immunization with heat-labile enterotoxin in gnotobiotic rats monocontaminated with enterotoxigenic Escherichia coli.

The protective effect of active immunization with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile enterotoxin (LT), administered using a parenteral prime and peroral boosts given after ablation of gastric secretion by means of cimetidine, was assessed in gnotobiotic rats which were challenged by monocontamination with enterotoxigenic strains of E. coli. Water transport was evaluated by the in vivo marker perfusion technique at weekly intervals over a 3-week period after contamination. Water transport in unimmunized control rats was consistently in absorption in those contaminated by a nontoxigenic strain, in secretion during only week 2 in those contaminated by an LT(+)/- strain, in secretion during weeks 2 and 3 in those contaminated by an LT(+)/ST(+) (heat-stable enterotoxin) strain, and consistently in absorption in those contaminated by an -/ST(+) strain. Rats immunized with a booster dosage of 250 mug had a significant increase (P < 0.001) in net water absorption as compared to unimmunized rats, with values in the borderline range of absorption, when challenged with either the LT(+)/- or LT(+)/ST(+) strains. Rats immunized with a 10-fold-higher boosting dosage had a significant increase (P < 0.001) in net water absorption as compared to those boosted at the lower dosage; water absorption was within the normal range. There was no difference between the ileal bacterial counts of unimmunized and immunized rats challenged by the various strains. These observations indicate that this immunization program provides complete protection in an animal model against challenge by intestinal contamination with enterotoxigenic strains of E. coli which produce LT, either alone or in combination with ST.

Comparison of assay of coliform enterotoxins by conventional techniques versus in vivo intestinal perfusion.

Thirty-six strains of coliform bacteria were tested for enterotoxigenicity both by conventional assays, including the Y-1 adrenal and Chinese hamster ovary cell assays for heat-labile toxin and the suckling mouse assay for heat-stable toxin, and by determining the ability of graded concentrations of ultrafiltrate high- or low-molecular-weight toxin preparations to induce water secretion during in vivo perfusion in the rat jejunum. The ultrafiltrates of all 18 strains isolated from persons with infectious diarrheal disease, including seven of Escherichia coli, seven of Klebsiella pneumoniae, and four of Enterobacter cloacae, contained one (nine strains) or two (nine strains) potent toxin fractions (resembling either heat-labile or heat-stable toxin in terms of apparent molecular weight and heat lability characteristics) that induced water secretion at perfusion concentrations of 10 ng/ml or less. Unconcentrated broth filtrates of five of the E. coli strains and two of Klebsiella reacted positively in one or more of the conventional assay systems. Concentrated ultrafiltrates from two strains that were negative in the in vitro assays for heat-labile toxin were tested and also proved to be inactive in these test systems. None of 18 strains isolated from control sources produced, in the ultrafiltrates, enterotoxins capable of inducing water secretion at low concentrations, and none reacted positively in the conventional assays. These results indicate that some strains of coliform bacteria elaborate potent toxin materials that are capable of inducing water secretion and can be detected by perfusion of concentrated ultrafiltrates but not by conventional assay systems for enterotoxigenicity. Whether this represents quantitative or qualitative differences between the toxin materials that stimulate these different test systems remains to be established.

Auxotypes and antibiotic susceptibility patterns of Neisseria gonorrhoeae from disseminated and local infections.

The arginine-hypoxanthine-uracil auxotype has been linked with the propensity of gonococci to cause disseminated infections. Gonococci recovered from 25 patients with disseminated gonococcal infections were compared with gonococci recovered from matched controls, patients with uncomplicated gonorrhea selected during the same month. Minimal inhibitory concentrations of penicillin, tetracycline, erythromycin, and ampicillin, and the nutritional requirements (auxotypes) for proline alone, arginine alone, arginine, hypoxanthine and uracil together, serine alone and cysteine-cystine (wild type) were analyzed by discriminant analysis. Significant susceptibility to penicillin characterized strains causing disseminated infection, and a proline requirement was the most common auxotype (48%) among strains isolated in Atlanta. Together the minimal inhibitory concentration of penicillin and the proline auxotype best separated the strains causing gonorrhea. The arginine-hypoxanthine-uracil auxotype was was found in only 24% of strains causing disseminated infections. A trait other than auxotype must determine the capacity of the organisms to disseminate.

Enterotoxigenicity of colonising coliform bacteria in tropical sprue and blind-loop syndrome.

The enterotoxigenicity of strains of Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli, which represented the predominant coliform species isolated from the jejunum of 12 patients with tropical sprue and 5 with the blind-loop syndrome, was quantitatively assessed in terms of the ability of toxin preparations to induce water secretion as assayed by in-vivo perfusion in the rat jejunum. All 12 patients with sprue harboured 1 or more highly toxigenic strains--14 of the 16 strains isolated from this group produced heat-labile and/or heat-stable toxins which were as potent as toxins derived from strains isolated from persons with acute diarrhoea and documented as toxigenic. None of the 9 strains isolated from patients with the blind-loop syndrome produced potent toxins. This difference between the coliform bacteria in sprue and the blind-loop syndrome probably accounts, at least partly, for the different intestinal response in these two disorders to contamination by these organisms.

Enterotoxigenicity of enteropathogenic serotypes of Escherichia coli isolated from infants with epidemic diarrhea.

Enteropathogenic serotypes of Escherichia coli which have been incriminated by epidemiological evidence as responsible for epidemics of acute diarrhea in infants are often found to be nontoxigenic when tested by conventional systems such as Y1-adrenal, Chinese hamster ovary, and suckling mouse assays. Twelve such strains, representing four different enteropathogenic serotypes, were examined for their capacity to elaborate toxic materials which alter water transport. Ultrafiltration fractions prepared to contain either a high-molecular-weight, heatlabile or a low-molecular-weight, heat-stable form of toxin from each strain were perfused through rat jejuna in graded concentrations ranging from 100 mug to 0.1 ng/ml. Ten of the twelve enteropathogenic strains produced one or both toxin forms that induced water secretion at concentrations of 1 to 10 ng/ml. Values in this range are considered indicative of clinically significant enterotoxigenicity in this assay system, and toxins from well-documented toxigenic strains examined in this study were active at these same concentrations. Similar preparations from ten control strains from healthy persons were either inactive or evoked water secretion only at concentrations of 10 to 100 mug/ml. These observations suggest that enteropathogenic serotypes of E. coli isolated from epidemics of infantile diarrhea produce diarrhea by elaborating potent heat-labile and heat-stable toxin forms which alter water transport but which are inactive in conventional assay systems. The manner in which these toxins differ either quantitatively or qualitatively from those which stimulate the conventional test systems is unknown.

Rapid method for auxotyping multiple strains of Neisseria gonorrhoeae.

A rapid method for auxotyping strains was developed that uses microtiter plates. This miniplate technique enables rapid identification of major auxotypes present in clinical strains. Additional growth requirements can be identified by adding individual amino acid supplements to complete gonococcal genetic medium. Analysis of 8 clones from 40 patients revealed that 10 had more than 1 auxotype. Deoxyribonucleic acid-mediated transformation can be used to establish whether the strains with apparently more than one auxotype are defective in the same locus in each of the involved biosynthetic pathways. Selection of more than one clone is required in precise epidemiological studies.

Relative enterotoxigenicity of coliform bacteria.

The enterotoxigenicity of 12 strains of coliform bacteria (Enterobacter cloacae, Klebsiella pneumoniae, and Escherichia coli) isolated from the gastrointestinal (GI) tract of persons with either acute diarrhea or tropical sprue and of 13 strains of the same species isolated from urine (GU) cultures was determined. Fractions of heat-labile (LT) and heat-stable (ST) toxins of each strain were separated by ultrafiltration, and the effect of graded concentrations (range, 100 microgram-10 pg/ml) on water transport was assessed by in vivo perfusion in the rat jejunum. Enterotoxigenic activity was defined as inducement of net secretion of water. All 12 of the GI strains and six of 13 GU strains elaborated enterotoxins, but there was a millionfold quantitative difference in the potency of the toxins produced. All of the GI strains produced one or both forms of toxin, which had a minimal effective concentration of as low as 0.1-10 ng/ml, whereas the GU strains produced toxins of weak or, rarely, of intermediate potency.

Contamination of the small intestine by enterotoxigenic coliform bacteria among the rural population of Haiti.

Coliform bacteria were isolated by either aerobic or anaerobic culture techniques from aspirates of the proximal small intestine of 4 of 5 Haitians with tropical sprue, but not from any of 10 well nourished Haitians who had milder gastrointestinal complaints and abnormalities. Klebsiella (Klebsiella pneumoniae and Klebsiella ozaenae) was cultured from the jejunal aspirates of 2 sprue patients and Escherichiae coli from the other 2. Fifteen colonies of coliform bacteria cultured from each aspirate were specifically identified by their biotype. In three instances, every colony in each aspirate was the same; In three instances, every colony in each aspirate was the same; in the fourth aspirate, two biotypes of E. coli were present, one of which grew under both aerobic and anaerobic culture conditions and another which grew only under anaerobic conditions on initial isolation. A randomly selected strain of each of the five coliform biotypes isolated was examined for enterotoxigenicity by determining the effect of variously prepared cell-free preparations on water transport in the rat jejunum using standard marker perfusion techniques. Every strain tested was toxigenic; one produced only a heat-stable toxin, one produced a heat-labile toxin only, and three elaborated both forms of enterotoxin. These observations indicate that most Haitians with tropical sprue have colonization of the proximal small intestine by a specific strain of enterotoxigenic coliform bacteria, but such is not the case among Haitians who have milder intestinal abnormalities.