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A multiplex PCR assay for detection of genes for staphylococcal enterotoxins A to E (entA, entB, entC, entD, and entE), toxic shock syndrome toxin 1 (tst), exfoliative toxins A and B (etaA and etaB), and intrinsic methicillin resistance (mecA) was developed. Detection of femA was used as an internal positive control. The multiplex PCR assay combined the primers for sea to see and femA in one set and those for eta, etb, tst, mecA, and femA in the other set. Validation of the assay was performed using 176 human isolates of Staphylococcus aureus. This assay offers a very specific, quick, reliable, and inexpensive alternative to conventional PCR assays used in clinical laboratories to identify various staphylococcal toxin genes.

A set of four synthetic oligonucleotide probes derived from sequences of the VT1 (Shiga-like toxin I [SLT-I]) and VT2 (SLT-II) genes were used in a polymerase chain reaction (PCR) amplification procedure to detect these genes in some enteric pathogens. A total of 40 verotoxin-producing Escherichia coli strains and 43 isolates of other recognized enteric pathogens were studied. PCR amplification products identifying the VT1 and VT2 gene sequences were observed only in nucleic acid extracted from strains found to be VT positive in traditional tissue culture assays. Template nucleic acid extracted from other gram-negative bacteria was found to be negative with the exception of five isolates of Shigella dysenteriae type 1 in which good amplification with the VT1 probe was observed. The oligonucleotide probes clearly distinguished VT1 and VT2 strains of E. coli and did not give specific amplification with nucleic acid from VTe (a SLT-II variant)-producing E. coli. VT1 or VT2 genes or both were not detected in E. coli K-12 strain C600 or HB101 or in strains known to express other virulence factors, such as enterotoxins, adhesins, hemolysins, or unrelated cytotoxins. The sensitivity of the PCR procedure for detection of both VT1 and VT2 genes was determined to be 1 ng of total nucleic acid. Furthermore, the VT1 gene was easily detected when only 100 pg of nucleic acid was used as the template in the PCR procedure.

Human isolates of Aeromonas hydrophila and Plesiomonas shigelloides were tested for their ability to produce cytotoxins and (or) enterotoxins. The incidence of cytotoxin production by A. hydrophila was 81% for isolates from stool and 44% for extraintestinal isolates. Chinese hamster ovary (CHO) cells were more sensitive to A. hydrophila cytotoxin than either Vero or Y-1 mouse adrenal tumor cells (y-1). There was no evidence of cytotonic enterotoxin production by any of the isolates tested. Cytotoxin-containing filtrates from A. hydrophila were found to provoke a positive reaction in suckling mice. The response in mice was heat labile, and data supporting correlation of this activity with the cytotoxin produced by these organisms are presented. Following concentration, cytotoxin and material reactive in suckling mice were found to coelute from Sephadex G-25 and dose-response was demonstrated in mice. Antitoxin prepared to this material effectively neutralized cytotoxin, mouse reactivity, and rabbit ileal loop response. No evidence was obtained for either cytotoxin or enterotoxin production by P. shigelloides strains tested in this study.

Six enterotoxigenic strains of Escherichia coli produced variable levels of heat-stable enterotoxin (ST) when grown under pH control at 8.5 in a simple synthetic medium containing neither amino acids nor vitamins. Bacterial growth and ST production were at levels as high as or higher than those observed in complex media. ST elaboration was detectable in the early logarithmic phase of growth and appeared to be related to disappearance of glucose in the growth medium. The results of this study did not suggest pH-dependent release of ST. Imposition of pH control in complex media resulted in increased growth rates, earlier detectable ST synthesis, and elevated levels of ST. In synthetic medium, attainment of the stationary growth phase was followed by a significant decrease in culture density and a concomitant increase in ST. Cellular autolysis experiments revealed that as much as 20% of the total ST activity was present in a cell-associated form.

The 45-kilobase tetracycline resistance plasmids pMAK175 and pUA466 from Campylobacter jejuni were examined using curing and mating experiments. However, these plasmids encoded neither cytotoxin production, as determined in Vero cells, nor enterotoxin activity, as determined in Chinese hamster ovary cells.

Ninety-nine nonclassical serogroups isolated from newborn pigs with neonatal diarrhea were tested for fimbrial antigens F4(K88), F5(K99), F6(987P), F41, and F165, and for heat-labile enterotoxin, heat-stable enterotoxin a (STa), heat-stable enterotoxin b, verocytotoxin, and cytolethal-distending toxin. Thirty-two strains, belonging mostly to serogroups O64:K"V142,", O9:K103, and O20:K101, were F5-positive and usually produced STa, although 5 strains produced only heat-stable enterotoxin b. Fifteen strains, belonging mostly to serogroups O64:K"V142" and O20:K101, were F41 positive and usually produced STa. Twenty-three stains, belonging mostly to serogroups O64:K"V142," O9:K103, and O20:K101, were F6-positive and usually produced STa. Several strains produced more than one fimbrial antigen, either F5 and F41, F5 and F6, F6 and F41, F6 and F165, or F5, F6, F41, and F165. None of the strains produced heat-labile enterotoxin, verocytotoxin, or cytolethal-distending toxin. The indirect immunofluorescence test was much more sensitive than was the slide agglutination test for the detection of each of the fimbrial antigens F5, F6, F41, and F165 on strains grown in vitro. The production of F6 by certain strains for which only a low proportion of cells were F6-positive in vitro, as demonstrated by immunofluorescence, was confirmed by experimental infection of newborn pigs.

Twenty-two enterotoxigenic Escherichia coli (ETEC) O8:KX105 strains isolated from 1- to 7-week-old diarrheic piglets were examined for virulence properties. Thirteen strains caused acute watery diarrhea in orally infected, colostrum-deprived newborn piglets, whereas the remaining nine did not. The enteropathogenic strains colonized the small intestine, albeit with lower intensity than classical porcine ETEC. They produced the heat-stable STb and heat-labile LT enterotoxins, whereas the nonenteropathogenic strains produced the STb enterotoxin alone. None of the E. coli O8:KX105 strains exhibited mannose-resistant hemagglutination with erythrocytes from 12 species. Ten of the enteropathogenic and two of the nonenteropathogenic strains were positive for mannose-sensitive hemagglutination. These strains produced rodlike fimbriae 3 to 5 nm in diameter, whereas no fimbriae were detected on the other strains. None of the 22 strains produced the fimbrial antigens F4, F5, F41, F2, F3, FY(Att 25), and F165. Of the 13 enteropathogenic strains, 10 expressed the F6 antigen in the intestines of infected piglets but not in cultures. The other three enteropathogenic strains apparently lacked all of the known fimbrial antigens from porcine ETEC.