Genetic and molecular studies of plasmids coding for colonization factor antigen I and heat-stable enterotoxin in several escherichia coli serotypes.

Plasmids coding for colonization factor antigen I (CFA/I) and heat-stable enterotoxin (ST) were identified in 10 strains of human enterotoxigenic Escherichia coli. The strains, which belonged to serogroups O63, O114, O128, and O153, were isolated in Bangladesh, Latin America, Spain, and South Africa. Two strains produced heat-labile enterotoxin in addition to ST. CFA/I-ST plasmids were mobilized from two O128 strains into E. coli K-12 with the R factor R1-19K-. Like the prototype CFA/I-ST plasmid NTP113, mobilized previously from an E. coli O78 strain into K-12, these two plasmids were non-autotransferring. All 10 CFA/I-ST plasmids were incompatible with NTP113 and had molecular weights ranging from 59 X 10(6) to 72 X 10(6). The molecular properties of seven of these plasmids were compared with those of six CFA/I-ST plasmids previously mobilized from O78 strains from Ethiopia, South Africa, and Bangladesh and with those of one plasmid coding for CFA/I, ST and heat-labile enterotoxin from a South African strain of serogroup O63. Digestion with the restriction endonuclease HindIII showed that several plasmids had very similar fragment patterns and two were identical. Generally, a larger proportion of HindIII fragments were of common size in digests of plasmids identified in strains from related geographical areas, regardless of serogroup. However, all except one plasmid shared five or six HindIII fragments of the same size, one of which had been shown previously to be involved in CFA/I production. There was at least 90% DNA homology between CFA/I-ST plasmids with a molecular weight of about 58 X 10(6) from O78 strains from different sources. Most of the DNA sequences of these plasmids were present in a larger CFA/I-ST plasmid (72 X 10(6) from an O128 strain. The results of genetic and molecular studies suggest that CFA/I and ST production is determined by very similar plasmids in different serogroups of human enterotoxigenic E. coli from several sources.     

Transfer of a CFA/I-ST plasmid promoted by a conjugative plasmid in a strain of Escherichia coli of serotype O128ac:H12.

Escherichia coli strains belonging to serotype O128ac:H12 and producing heat-stable enterotoxin (ST) and colonization factor CFA/I were found in Sao Paulo in children with diarrhea, but not in normal children. Segregants occurred in such strains with a frequency of about 10%, which have lost the ability to produce ST and CFA/I at the same time. From one strain, both properties were transformed jointly in matings to an E. coli K-12 strain. All such ST+ CFA/I+ progeny had received two plasmids of length 97 and 64 kilobases in the matings. Insertion of a transposon, Tn5, carrying a gene for kanamycin resistance, into the two plasmids enabled us to select for kanamycin-resistant progeny in further matings. Analysis of such progeny strains in terms of plasmid content and production of ST and CFA/I revealed that the larger plasmid carries the genes for St and CFA/I and is not self-transmissible, whereas the smaller plasmid does not carry any recognizable phenotypic traits, but is conjugative and promotes cotransfer of the larger plasmid with a frequency of about 30%.     

Biotypes, antibiotic resistance and plasmids coding for CFA/I and STa in enterotoxigenic Escherichia coli strains of serotype O153:H45 isolated in Spain

Enterotoxigenic Escherichia coli (ETEC) strains of serotype O153:H45 have been found recently to be a frequent cause of sporadic cases and outbreaks of neonatal diarrhoea in Spain and the most important cause of infant diarrhoea in Chile. Relationships between sugar fermentation patterns, resistance to antibiotics and plasmid profiles were analysed in nine E. coli O153:H45 strains isolated in Spain that synthesised CFA/I antigen and STa enterotoxin. Derivative strains obtained by curing with acridine orange, and transconjugants rendered antibiotic resistant, were characterised phenotypically and analysed for plasmid content. Two fermentation patterns were recognised: rhamnose fermenters (four strains) and rhamnose non-fermenters (five strains). The ability to ferment rhamnose was the only differential characteristic found among 49 carbohydrate fermentation tests used to establish fermentation patterns. All nine strains possessed similar plasmid profiles of three or four plasmids of 52-87 Mda. A non-conjugative large plasmid of 82 Mda or 87 Mda, depending upon the strain, was identified as that responsible for production of both CFA/I and STa. Resistance to antibiotics was determined by plasmids other than those coding for CFA/I and STa. Two conjugative resistance factors were identified: a 52-Mda plasmid coding for resistance to ampicillin, streptomycin and sulphonamide in rhamnose-fermenting strains, and a 77-Mda plasmid coding for resistance to ampicillin, streptomycin, kanamycin, tetracycline and sulphonamide in rhamnose non-fermenting strains. Our results support the hypothesis that the prevalence and distribution of ETEC strains belonging to serotype O153:H45 in Spain and Chile could be due to the extensive cultural relations between Spain and South American from the past.     

Analysis of the plasmids of Escherichia coli O148:H28 from travellers with diarrhea

98 Escherichia coli strains of serotype O148:H28 isolated from diarrheal patients from 10 Asian countries and Mexico at Osaka Airport Quarantine were analyzed for enterotoxigenicity and plasmid profile. They were classified into three groups. The first group contained 44 strains that were non-enterotoxigenic and carried 3.9 kb and 50 kb non-enterotoxin plasmids. The second group contained 9 strains that produced LT and ST. They carried a 45 kb enterotoxin plasmid, and 4.6 kb and 9.2 kb non-enterotoxin plasmids. The third group contained 45 strains that produced ST. They carried a 40 kb enterotoxin plasmid, and non-enterotoxin plasmids other than 3.9 kb, 4.6 kb, 9.2 kb and 50 kb. Southern blot hybridization demonstrated that all the non-enterotoxin or enterotoxin plasmids carried by the strains of the same group were identical or similar. These results suggested that the 98 E. coli strains with O148:H28 serotype were derived from three clones, and that the individual strains among each group were derived from a single clonal strain.     

Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt

Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.     

Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina.

A prospective study was performed to evaluate the presence of colonization factor antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) strains isolated from 1,211 children with diarrhea in Argentina. One hundred nine ETEC strains that were isolated from seven different laboratories in various regions of the country were tested for CFAs by using monoclonal antibodies against CFA/I and E. coli surface antigens CS1, CS2, and CS3 of CFA/II and CS4 and CS5 of CFA/IV; a polyclonal antiserum against CS6 was used. The CFAs searched for were found in 52% of the ETEC strains: 23% of the strains carried CFA/I, 17% carried CFA/IV, and 12% carried CFA/II. All of the CFA/I strains produced heat-stable enterotoxin, and several of them were of the prevalent serotypes O153:H45 and O78:H12. Among the 19 strains expressing CFA/IV, 16 expressed CS5 and CS6 and produced the heat-stable enterotoxin and most were of serotype O128:H21; the remaining 3 strains produced CS6 only. No ETEC strains expressing CS4 were found. Most (11 of 13) of the CFA/II-carrying ETEC strains expressed CS1 and CS3, and 10 of them were of the O6:K15:H16 serotype and produced both heat-labile and heat-stable toxins. As many as 24 of the 109 CFA-negative ETEC strains gave mannose-resistant hemagglutination with erythrocytes from different species; 4 strains had high surface hydrophobicity, suggesting the presence of additional, as yet undefined, colonization factors in up to 25% of the ETEC isolates.     

Relationship among enterotoxigenic phenotypes, serotypes, and sources of strains in enterotoxigenic Escherichia coli.

The relationship among O groups, O:H serotypes and enterotoxigenic phenotypes was examined in 76 Escherichia coli strains isolated in Brazil from different sources. Of the 17 heat-labile and -stable enterotoxin (LT/ST)-producing strains whose O antigens were identified, 15 belonged to serotypes O6:H16 (7 strains), O63:H- (5 strains), and O139:H28 (3 strains). All 11 ST strains were in group OO128PAC, which was represented by four O:H serotypes. The 23 LT strains with the O antigen identified were distributed among serotypes of 14 O groups. Colonization factor CFA/I was not found in any of the LT strains, but it was found in six LT/ST and three ST strains. On the whole, each E. coli O:H serotype had a particular fermentation pattern. LT/ST as well as ST strains were all isolated from patients with diarrhea, whereas LT strains were isolated from patients with diarrhea, normal children, food, and river water.     

Enterotoxigenic Escherichia coli in a population of infants with diarrhea in Chile.

The incidence of enterotoxigenic Escherichia coli (ETEC) was investigated in 95 E. coli strains isolated from 48 infants with diarrhea in Santiago, Chile. By using standard biological assays and DNA-DNA hybridization procedures, ETEC was found in 31.2% of the cases: 14 strains produced heat-stable enterotoxin (ST) only, three strains produced heat-labile enterotoxin (LT) and ST, and two strains produced LT only. DNA probes detected all enterotoxin producers except one ST-producing strain. The ST strains hybridized with one or both of the human ST probes (ST Ib and ST A2). Two of the LT-ST strains hybridized with the ST Ia and ST Ib probes, and the third strain did not hybridize with any of the ST probes. Only the ST group expressed multiple resistance (85.7%) and colonization factor antigen I (CFA I) (92.8%); CFA II was found in two of three LT-ST strains. The O153:H45 serotype was found in 10 of 14 ST strains, and O6:K15:H16 was found in one LT strain and in two LT-ST strains. These findings suggest that ETEC, especially strains that produce ST, may be an important cause of diarrhea among Chilean infants.     

Plasmids coding for drug resistance and localized adherence to HeLa cells in enteropathogenic Escherichia coli O55:H- and O55:H6.

Plasmids coding for drug resistance and localized adherence (LA) to HeLa cells were found in two enteropathogenic Escherichia coli strains belonging to serotypes O55:H- and O55:H6. Strain 49-81 HSJ (O55:H-) carries two plasmids, one coding for both ampicillin resistance (Apr) and LA (pMS49). Strain 71-82 HSJ (O55:H6) harbors only one plasmid, coding for resistance to sulfadiazine, chloramphenicol, kanamycin, ampicillin, and LA (pMS71). Plasmids pMS49 and pMS71 were transferred to E. coli K-12 711 and from this strain to E. coli K-12 J53. Curing with acridine orange of an Apr LA+ transconjugant showed that both characteristics were lost simultaneously. The plasmids have a molecular weight of approximately 55 X 10(6) and are the first naturally recombinant plasmids coding for adherence and drug resistance described in enteropathogenic E. coli.     

Prevalence of enterotoxigenicEscherichia coli strains in outbreaks and sporadic cases of diarrhoea in Spain

Escherichia coli strains isolated 1985–1988 in Spain from patients with diarrhoea were examined; 1170 strains were isolated from 582 sporadic cases of diarrhoea in children, and seven strains were associated with seven outbreaks of diarrhoea. Strains positive for STa enterotoxin production in the infant mouse test were also assayed for production of LT enterotoxin on Vero cells and by a coagglutination test. Thirty-one strains were STa positive: 28 were isolated from 16 (2.7 %) sporadic cases of diarrhoea and three were responsible for outbreaks. The majority of STa⁺LT^– strains from both outbreaks and sporadic cases were serotype O153.H45 and expressed the CFA/I colonization factor antigen. Enterotoxigenic STa⁺LT^– strains of serotype O27:H7 and STa⁺LT⁺ CFA/II⁺ strains of serotype O6:H15: H16 were also isolated frequently from sporadic cases.     

Characterization of enterotoxigenic Escherichia coli isolated from U.S. troops deployed to the Middle East.

Enterotoxigenic Escherichia coli (ETEC) was a common cause of traveler's diarrhea in U.S. soldiers in the Middle East in 1989 and 1990. To determine which bacterial components would be useful in a vaccine, potential protective antigens (toxin, colonization factor antigen [CFA], and serotype) from 189 ETEC isolates were examined. Nearly half of the isolates expressed both ETEC toxins, 39% had only heat-stable enterotoxin (ST), and 17% had heat-labile enterotoxin (LT). CFA/I was the least common colonization factor antigen (11%), CFA/II was common (34%), as was CFA/IV (31%), and 24% expressed none of these CFAs. Fifty-seven O:H serotypes were found. Serotype O6:H16 was the most common, occurring in 29% of the ETEC isolates, usually with LT-ST and CFA/II. Generally, CFA/II was associated with expression of both toxins, CFA/IV was associated with expression of ST, and none of the CFAs was routinely found with LT. We conclude that ETEC from soldiers in the Middle East expressed a variety of antigens and that an effective vaccine will require multiple protective antigens.     

Genetic control and properties of coli surface antigens of colonization factor antigen IV (PCF8775) of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli producing coli surface antigen 4 (CS4), CS5, and CS6 of colonization factor antigen IV were examined. This factor was originally called putative colonization factor 8775 (PCF8775). All of the coli surface antigens were plasmid coded and were usually carried on the same plasmid as the genes coding for heat-stable toxin (ST) or heat-labile toxin (LT); thus, CS5-CS6-ST, CS6-ST, and CS6-LT plasmids were found. In strains of serotype O25:H42, the genes coding for CS4 and CS6 were on a plasmid separate from that containing the genes coding for ST and LT. CS4 and CS5 were fimbrial antigens with a subunit molecular mass of about 17.0 and 21.0 kilodaltons (kDa), respectively. CS6 was found as a single polypeptide with a molecular mass of about 14.5 kDa in strains of serotypes O25:H42, O27:H7, and O27:H20 when heated extracts were run on sodium dodecyl sulfate-polyacrylamide gels. CS6-positive extracts of strains of serogroups O148, O159, and O167 showed two bands with molecular masses between 14.5 and 16.0 kDa.     

Relationship between enterotoxin production and serotype in enterotoxigenic Escherichia coli.

We examined the relationship between serotype and enterotoxin production in 109 enterotoxigenic Escherichia coli strains isolated from 109 patients with severe cholera-like diarrhea in Dacca, Bangladesh. Of 69 strains producing both heat-labile and heat-stable toxins, 59 (86%) belonged to the one of four O serogroups, and 56 (81%) of these strains belonged to one of six O:K:H serotypes. In contrast, 34 strains producing only heat-stable toxin were distributed among 15 O serogroups, and six strains producing only heat-labile toxin were distributed among six O serogroups. Twelve strains producing heat-labile and heat-stable toxins and five strains producing heat-stable toxin were found which had the same serotype (O78:K-:H12) and biotype. It appears that at least in one geographic setting E. coli strains producing both heat-labile and heat-stable toxins are more restricted in their O groups and O:K:H serotypes than E. coli that produce only heat-stable toxin and that certain serobiotypes may commonly include strains which produce both toxin types.     

Molecular analysis of enterotoxin plasmids of enterotoxigenic Escherichia coli of 14 different O serotypes

A total of 104 isolates of enterotoxigenic Escherichia coli derived from diarrheal patients from more than 10 countries were examined for serotype and toxigenicity. The transferability and molecular structure of the enterotoxin plasmids from each isolate were also examined. Enterotoxin plasmids from serotypes such as O6, O25, O27, O126, O128, and O159, which are frequently associated with E. coli diarrhea (classical strains) generally did not transfer by conjugation from clinical isolates, whereas those from serotypes such as O7, O17, O80, O98, O139, O150, and O153, which are rarely associated with diarrhea (rare strains) transferred almost always from the clinical isolates by conjugation. Analyses of enterotoxin plasmids by restriction endonucleases and DNA-DNA hybridization with the enterotoxin probes revealed that the strains with the same O serotype and toxigenicity carry closely related enterotoxin plasmids. These results suggest that classical strains resulted from the dissemination of ancestral clones which received enterotoxin plasmids long ago, while the rare strains acquired the enterotoxin plasmids recently by conjugation and have not yet been spread to the same degree as the ancestral clones.     

Pathogenicity and immune response measured in mice following intranasal challenge with enterotoxigenic Escherichia coli strains H10407 and B7A

The pathogenicity and immunogenicity induced in BALB/c mice by intranasal (i.n.) inoculation of enterotoxigenic Escherichia coli (ETEC) strains H10407 (O78:H11:CFA/I:LT(+):ST(+)) and B7A (O148:H28:CS6:LT(+):ST(+)) (two ETEC strains previously used in human challenge trials) were studied. The i.n. inoculation of BALB/c mice with large doses of ETEC strains H10407 and B7A caused illness and death. The H10407 strain was found to be consistently more virulent than the B7A strain. Following i.n. challenge with nonlethal doses of H10407 and B7A, the bacteria were cleared from the lungs of the mice at a steady rate over a 2-week period. Macrophages and neutrophils were observed in the alveoli and bronchioles, and lymphocytes were observed in the septa, around vessels, and in the pleura of the lungs in mice challenged with H10407 and B7A. In mice i.n. challenged with H10407, serum immunoglobulin G (IgG) and IgM antibodies were measured at high titers to the CFA/I and O78 lipopolysaccharide (LPS) antigens. In mice i.n. challenged with B7A, low serum IgG antibody titers were detected against CS6, and low serum IgG and IgM antibody titers were detected against O148 LPS. The serum IgG and IgM antibody titers against the heat-labile enterotoxin were equivalent in the H10407- and B7A-challenged mice. The CFA/I and O78 LPS antigens gave mixed T-helper cell 1-T-helper cell 2 (Th1-Th2) responses in which the Th2 response was greater than the Th1 response (i.e., stimulated primarily an antibody response). These studies indicate that the i.n. challenge of BALB/c mice with ETEC strains may provide a useful animal model to better understand the immunogenicity and pathogenicity of ETEC and its virulence determinants. This model may also be useful in providing selection criteria for vaccine candidates for use in primate and human trials.     

Comparative analysis of plasmids and some metabolic characteristics of Escherichia coli K1 from diseased and healthy individuals.

All 62 Escherichia coli strains possessing the K1 capsular polysaccharide contained plasmid deoxyribonucleic acid, and most (51 of 62) had multiple plasmid species. The incidence of hemolysins, colicins, hemagglutinins for human erythrocytes, and plasmids did not differ among K1 strains isolated from the cerebrospinal fluids of neonates with meningitis or among those strains isolated from the stools of healthy individuals of all ages. There was an association between E. coli serotype and the distribution of plasmids, hemolysins, and colicins among the K1 strains. A common plasmid of about 65 megadaltons was found in all of the O18 serotypes; the similarity of these plasmids was confirmed by analysis with the restriction endonuclease EcoRI. Plasmids of similar molecular weight were also present in E. coli strains of the O7:K1 and O75:K100 serogroups. These data are consistent with the hypothesis that E. coli strains of the same serotype may be descendents of a single bacterial clone.     

Plasmids coding for colonization factor antigens I and II, heat-labile enterotoxin, and heat-stable enterotoxin A2 in Escherichia coli.

Colonization factor antigens I and II (CFA/I and CFA/II) are important in the pathogenesis of diarrhea in humans caused by some enterotoxigenic Escherichia coli (ETEC). Plasmid DNA from 16 CFA/I+ and five CFA/II+ ETEC were examined by Southern blot analysis with enterotoxin gene probes and were compared with plasmid DNA from derivatives of the same ETEC that had lost the ability to produce these colonization factors. Among the 16 CFA/I+ ETEC strains, the loss of CFA/I was accompanied by the loss of a plasmid of between 34 and 68 megadaltons (MDa) coding for heat-stable enterotoxin A2 (ST-A2) in 12 strains, by the loss of a 60-MDa plasmid coding for heat-labile enterotoxin (LT) and ST-A2 in one strain, or by deletions of a segment of DNA encoding for ST-A2 in three strains. Among five CFA/II+ ETEC strains, the loss of CFA/II was associated with the loss of a plasmid of 75 MDa coding for LT and ST-A2 in three strains, with the loss of genes coding for LT and ST-A2 from a 68-MDa plasmid in one strain, or with no discernible loss of a plasmid or DNA sequences coding for enterotoxins in the remaining strain. The loss of CFA/I and CFA/II production was associated with the loss of DNA sequences encoding for ST-A2 in 20 of 21 ETEC examined.     

Enterotoxigenic Escherichia coli strains belonging to a new serogroup, Escherichia coli O166.

Thirty-two strains of Escherichia coli belonging to a new O group, O166, were examined. Twenty-one strains had the flagella antigen H27, five had the H15 antigen, five had the H7 antigen, and one was nonmotile. All the H27 strains and the nonmotile strain produced heat-stable enterotoxin but not heat-labile enterotoxin. All the H7 strains produced heat-labile enterotoxin but not heat-stable enterotoxin. The remaining strains were nonenterotoxigenic. None of the strains possessed colonization factor antigens CFA/I, CFA/II, or PCF8775.     

Beyond Serotypes and Virulence-Associated Factors: Detection of Genetic Diversity among O153:H45 CFA/I Heat-Stable Enterotoxigenic Escherichia coli Strains

Characterization of enterotoxigenic Escherichia coli (ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence-associated factors: heat-labile (LT) and heat-stable (ST) toxins and colonization factors (CFs). In the present work we show that the analysis of band patterns generated by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNA can be used to detect genetic diversity among ETEC strains expressing identical phenotypic traits. The study included 29 ETEC isolates from Latin America and Spain expressing the phenotype O153:H45 CFA/I ST plus 1 rough derivative, 2 nonmotile derivatives, and 1 O78:H12 CFA/I ST isolate, and a representative of a genetically distinct ETEC group. The results showed that the O153:H45 CFA/I ST ETEC isolates belong to a single clonal cluster whose isolates share on average, 84% of the RAPD bands and 77% of the PFGE restriction fragments, while the O78:H12 isolate shared only 44 and 4% of the RAPD bands and PFGE fragments, respectively, with the isolates of the O153:H45 group. More relevantly, RAPD and PFGE fingerprints disclosed the presence of different clonal lineages among the isolates of the O153:H45 cluster. Some of the genetic variants were isolated from defined geographic areas, while places like S?o Paulo City in Brazil and the middle-eastern part of Argentina were populated by several genetic variants of related, but not identical, ETEC strains. These results show that molecular biology-based typing methods can disclose strain diversity, which is usually missed in studies restricted to phenotypic typing of ETEC.     

Colonization factor antigens I and II and type 1 somatic pili in enterotoxigenic Escherichia coli: relation to enterotoxin type.

Enterotoxigenic Escherichia coli (ETEC) isolates from 36 persons with acute traveler's diarrhea from whom no other pathogens were recovered were tested (after no more than three subcultures) for the presence of colonization factor antigens I and II (CFA/I and CFA/II) and type 1 somatic pili. CFA/I or CFA/II was identified in 7 of 10 strains with heat-labile and heat-stable enterotoxins (LT+/ST+), but in only 2 of 12 LT-/ST+ (P less than 0.05) and 0 of 14 LT+/ST- (P less than 0.02) strains. CFA pili were not found among 74 non-enterotoxigenic E. coli strains. Type 1 somatic pili were demonstrable in 42% of the 36 ETEC and in 49% of the 74 non-enterotoxigenic E. coli isolates. The nine ETEC isolates bearing a CFA were serially subcultured on 10 consecutive days and retested for CFA and toxin. After five subcultures only one strain had lost a CFA, but after 10 passages three strains were negative: two lost CFA/I and one lost CFA/II. The strain that lost CFA/II became negative for both LT and ST as well and was found to lack a 48- and a 60-megadalton plasmid. The two strains that lost CFA/I also became negative for ST, but plasmid analysis revealed no plasmid loss. Disappearance of the CFA/I phenotype without loss of a plasmid can be explained by phase variation, as exhibited by type 1 somatic pili, or by rearrangement of base sequences in the CFA/I plasmid genome. If purified pili vaccines are to provide broad-spectrum protection against ETEC diarrhea, the search must be intensified to identify the antigens responsible for adhesion to intestinal mucosa in the many ETEC strains that lack CFA/I and CFA/II.