Eight pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect genes for staphylococcal enterotoxins A to E, exfoliative toxins A and B, and toxic shock syndrome toxin 1 in Staphylococcus aureus strains isolated from clinical specimens and contaminated foods. Primers were targeted to internal regions of the toxin genes, and amplification fragments were detected after the PCR by agarose gel electrophoresis. Unequivocal discrimination of toxin genes was obtained by the PCR by using nucleic acids extracted from 88 strains of S. aureus whose toxigenicity was established biologically and immunologically. In immunological assays, two strains of S. aureus produced equivocal results for production of enterotoxin C or toxic shock syndrome toxin 1, giving an overall concordance between phenotypic and genotypic identification of 97.7%. Primer specificity was established in the PCR by using nucleic acids from known toxin-producing bacterial pathogens and from nontoxigenic S. aureus. Strains of Streptococcus spp., including some producers of pyrogenic exotoxin A carrying the speA gene, were negative by the PCR designed to detect staphylococcal toxins. The detection limits were established for all the staphylococcal toxin genes within their respective PCR protocols. The identification of staphylococcal toxin genes in strains of S. aureus by the PCR offers a very specific, sensitive, relatively rapid, and inexpensive alternative to traditional immunological assays which depend on adequate gene expression for reliability and sensitivity.

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 VTÎ 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.

Multilocus enzyme electrophoresis and ribotyping were used to characterize 83 strains of Pseudomonas cepacia, mostly isolated from cystic fibrosis (CF) patients, although a number of isolates from non-CF nosocomial infections and reference environmental strains were represented. Twenty enzyme electrophoretic types (ETs) were determined; of these, one clone (ET12) was associated with six of nine ribotypes (RTs) said to be geographically representative of the United Kingdom and all of the Ontario (Canada) isolates from CF patients. This clone was not associated with nosocomial infections or environmental strains and was never found in CF isolates from British Columbia or Nova Scotia, Canada, or a center in the eastern United States. Individual isolate EcoRI RT signatures did not cluster geographically as did the ET signatures by clonal analysis. Frequently RTs occurred in more than a single ET. Known point source focal nosocomial outbreaks were typified by single ETs and stable RTs. Dendrographic analysis of the strains grouped those strains from CF patients, nosocomial outbreaks, and environmental sources into separate ET families, and diversity analysis indicated that, with the exception of ET17, CF isolates clustered in unique and closely related ETs different from those from nosocomial and environmental sources. This study has also shown the potential of multilocus enzyme electrophoresis to monitor the intercontinental spread of P. cepacia strains in CF patients, and this may have a significant impact on plans for CF patient summer camps and design of infection control practices. Whether the intercontinental ET12 clone, which predominates in the United Kingdom and the province of Ontario, linked by summer camp acquisition, has increased virulence for CF patients remains to be established.

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli 091:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coil, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per ,ug of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per ,ug of protein in strains carrying only the VT2 variant target (TCDss is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype.