Kaye Wachsmuth

Studies in Gnotobiotic Piglets on Non-0157:H7 Escherichia coli Serotypes Isolated From Patients With Hemorrhagic Colitis

A number of verotoxin-producing Escherichia coli strains isolated from sporadic cases of hemorrhagic colitis in the United States over the last 5 yr were shown to belong to serogroups other than O157:H7-the serotype originally implicated in this disease. Experimental infection of gnotobiotic piglets with five such strains (0111:NM, 0145:NM, 045:H2, 04:NM, and Ound:NM) caused diarrhea resulting from mucosal lesions in the cecum and colon that were indistinguishable from those previously described in piglets infected with E. coli O157:H7. This suggests that, as with other categories of pathogenic E. coli, several serotypes cause hemorrhagic colitis in humans. The five E. coli strains that were compared with one O157:H7 strain and with an enteropathogenic calf strain (serotype 05:NM) caused a spectrum of disease ranging from moderate diarrhea (O157:H7) to severe illness (including septicemia and death) (0111:NM). Characteristic lesions, which were identical for all seven pathogenic strains, included bacterial attachment, effacement of the microvillus border, and dissolution of the cell membranes of surface and glandular epithelium, resulting in complete cell destruction. Some piglets exhibited neurologic signs of convulsions and ataxia. It is concluded that a number of E. coli serotypes, in addition to O157:H7, fulfill the present limited criteria for enterohemorrhagic E. coli, which include association with hemorrhagic colitis, production of one or more verotoxins, possession of a large plasmid (50-70 megadaltons), and induction of distinct mucosal lesions in the large bowel of gnotobiotic piglets.

Cholera in the Americas: Foodborne Aspects

Over 100 serotypes of Vibrio cholerae exist, but generally the toxigenic strains of the serogroup O1 cause cholera and possess documented epidemic potential. The main symptom of cholera is a profuse diarrhea resulting in dehydration, that if untreated, leads to death. Seven pandemics of this contagious disease have been recorded during the last 200 years. A sick person secrets in his stool billions of organisms daily, and water and food contaminated with such a stool are the primary sources of infection during the epidemics. With the increase of the international food trade, food is often shipped from countries with endemic or epidemic cholera. With one exception, no documented cases of cholera have been reported, as a result of the internationally regulated food trade. However, during the present Latin American epidemic, inadequately cooked seafood has been implicated as a source of cholera. As a result of the epidemic, over 100 cases of cholera have occurred in the United States related to seafood consumed during a visit to Latin America or after its noncommercial transport into the country. Furthermore, V. cholerae persists as a free-living organism in environmental reservoirs in Australia and the U.S. Gulf Coast; there have been 65 domestically acquired cases of cholera in the United States since 1973. Molecular typing methods have enabled us to identify and characterize endemic and epidemic strains, and to document transmission of cholera when food was implicated epidemiologically as a vehicle of transmission.

Genetic transfer of antimicrobial resistance and enterotoxigenicity among Escherichia coli strains.

To understand the role of enterotoxin (Ent) plasmids in epidemics of enterotoxigenic (ET) Escherichia coli diarrhea in the United States, we studied the genetics of Ent plasmids in relation to E. coli serotypes and R plasmids. Twenty-nine ET E. coli strains, including all epidemic isolates available at the Centers for Disease Control, Atlanta, Ga. (CDC), were assessed for the ability to transfer antimicrobial resistances (if present) by conjugation, to mobilize a nonconjugative R plasmid, and to cotransfer enterotoxigenicity with R determinants. Of the 12 ET E. coli strains isolated in the United States, 5 were able to transfer R plasmids; one strain cotransferred detectable enterotoxigenicity. Another four U.S. isolates were able to mobilize plasmid DNA, but no toxin production was detected in transconjugants. Of 17 resistant ET E. coli from South Asia, 13 were able to transfer R plasmids; 5 of those 13 cotransferred detectable Ent plasmids. In all, 22 ET E. coli strains (76%) were able to initiate conjugation and genetic transfers. Six of these strains (20%) were able to cotransfer enterotoxigenicity with a conjugative R plasmid at a detectable frequency. One of the six strains transferred R and Ent determinants on a single plasmid. These data are addressed in relation to the observed immobility of Ent and R during outbreaks of ET E. coli, the efficacy of prophylactic tetracycline, and the worldwide occurrence of a limited number of ET E. coli serotypes.

Diagnostic value of plasmid analyses and assays for virulence in Yersinia enterocolitica

The possession of a 42- to 48-megadalton plasmid alone does not appear to be predictive of virulence in Yersinia species. Twelve of 100 Yersinia enterocolitica strains contained a 42 to 48-megadalton plasmid, and 4 of 30 Y. enterocolitica-like strains contained a 42- to 48-megadalton plasmid. Seven strains of Y. enterocolitica contained the 42- to 48-megadalton plasmid plus an 82-megadalton plasmid, and these were the only study strains lethal for mice. Based on restriction endonuclease digestion, the 42- to 48-megadalton plasmid DNA from these seven strains were similar and were not similar to the 42- to 48-megadalton plasmids present in the other nine strains. The ability to invade guinea pig eye tissues, calcium dependency, autoagglutination, and colonial morphology at 37 degrees C were also associated with plasmid DNA, but the relationships were either variable or not reciprocal. Neither tissue culture invasiveness nor heat-stable toxin production was associated with plasmid DNA. It was concluded that biochemical speciation and a total plasmid profile in combination with enzyme digests are predictive of virulence in Y. enterocolitica as it is measured by mouse lethality.

Enteropathogenicity of non-toxigenic Vibrio cholerae O1 for adult mice

The enteropathogenic potential of 32 Vibrio cholerae O1 isolates that do not produce cholera toxin was examined in the orally inoculated, sealed adult mouse model. Live cultures (2 x 10(10) cfu/ml) of 7/16 clinical and 6/16 environmental isolates produced a positive intestinal fluid accumulation (FA) ratio that reached near maximum at approximately 5 h post-inoculation. Colony hybridization did not detect genes for cholera toxin, Escherichia coli heat-labile and heat-stable toxins, or shiga-like toxins. FA activity did not correlate precisely with cytotoxic activities on Chinese hamster ovary (28/32 positive), Vero (29/32) or HeLa (25/32) cells. Certain clinical and environmental isolates of non-toxigenic V. cholerae O1 appear to be enteropathogenic for the mouse, providing evidence that they may have pathogenic potential for humans through an as yet undefined mechanism(s).

Plasmids and Serotypes of Hemolytic Fecal Escherichia coli

Twelve of 21 human, hemolytic, fecal isolates ofEscherichia coli produced type 1 hemolysin (HLY1), an extracellular, heat-labile molecule (alpha-hemolysin). Although no common plasmid species was apparent, 11 of 12 HLY1 strains possessed a plasmid≥60 megadaltons (Mdal); 5 of 9 strains with other hemolysins possessed a plasmid of comparable molecular mass (Fishers exact probability=0.0805). One derivative of an HLY1+strain, which contained a 125 Mdal plasmid, no longer expressed HLY1 and contained a single 102 Mdal plasmid. The presence of large plasmids of varying size and an apparent deletion mutation in HLY1 strains suggest that HLY1 determinants are located on a small, unstable genetic element. In an initial survey of 224 human fecal isolates ofE. coli, the predominant hemolytic serotype was 06:H-, and conversely most (85%) 06:H-isolates were HLY1+. Serotype appears to play an important role in HLY1 expression.

Potential pathogenic factors produced by a clinical nontoxigenicVibrio cholerae O1

A clinical isolate of nontoxigenicVibrio cholerae O1 that caused intestinal fluid accumulation (FA) in adult mice produced proteolytic, hemolytic, and cytotoxic activities in in vitro assays. The linkage of these secreted factors to the FA activity was studied by transposon (TnphoA) mutagenesis. Ten of the 12 TnphoA insertion mutants that were defective for proteolytic activity produced FA, hemolytic and cytotoxic activities; the remaining two mutants lost these latter three activities. These results indicate that FA activity is independent of proteolytic activity but closely associated with cytotoxic and hemolytic activities. Our results with the adult mouse model and a nontoxigenicV. cholerae O1 are in general agreement with previous studies that demonstrated linkage of cytotoxin and hemolysin of toxigenicV. cholerae O1 and non-O1 with FA activity in rabbit ileal loops.