Special O:K:H serotypes among enterotoxigenicE. coli strains from diarrhea in adults and children

O:H serotypes previously found to be prevalent among a number of toxigenic strains from several geographic areas were examined for polysaccharide K antigen type. Members of each O:H serotype had the same type of K antigen and were found to be characterized by a certain fermentation pattern. Some O:H serotypes had no K antigen. The serofermentative types defined were: 06:K15:H16, 08:K40:H9, 015:H11, 025:K7:H42, 025:K98:H^}-, 078:H11, 078:H12, and 0149:H10. Some strains of the last-mentioned serotype, which were suspected of having caused a food-borne infection, had K88. This serotype belongs to the group of strains causing diarrhea in swine.The surface antigen (CF) described as a colonization factor [5] was demonstrated in 078:H^–, 078:H11, and 078:H12 strains; but not in any strain of the other serotypes nor in any of 248 strains belonging to 078 but not isolated from cases of human diarrhea. Presence of the CF antigen was correlated with presence of a mannose-resistant ability to cause agglutination of human red cells. Behavior of the other serotypes as regards hemagglutinating abilities was examined and 025:K7:H42 strains were found to be very similar to the 078 strains in this respect.     

New fimbrial antigen F165 from Escherichia coli serogroup O115 strains isolated from piglets with diarrhea

Sixteen strains of Escherichia coli serogroup O115 isolated from piglets with diarrhea were examined for mannose-sensitive or mannose-resistant hemagglutination (MSHA or MRHA, respectively) for the presence of fimbriae by electron microscopy and for enterotoxigenicity by the ligated gut loop technique in 10-day-old piglets. Four strains demonstrated MRHA of sheep, goat, pig, dog, cat, chicken, and human erythrocytes but no MRHA of calf, horse, guinea pig, and rabbit erythrocytes. They were divided into pattern I (MSHA negative) and pattern II (MSHA positive). The remaining 12 strains were classified as pattern III (MRHA negative, MSHA positive) and pattern IV (hemagglutination negative). An antiserum produced against the MRHA-positive, MSHA-negative strain 4787 and absorbed by the same strain grown at 15 degrees C agglutinated all of the MRHA-positive strains but none of the MRHA-negative strains and completely inhibited the MRHA of these strains. The surface antigen against which this absorbed antiserum was directed was designated "F165." Fimbriae (pili) purified from strain 4787 hemagglutinated erythrocytes in the same mannose-resistant pattern as the strain itself and reacted with the anti-F165 antiserum in an enzyme-linked immunosorbent assay, thus demonstrating the fimbrial nature of the hemagglutinating F165 antigen. The F165 antigen showed no serological relationship with the fimbrial antigens F4, F5, F6, and "F41". A positive correlation between the presence of F165 and the lack of enterotoxigenicity was demonstrated. Thus, we found a new mannose-resistant, hemagglutinating fimbrial antigen, F165, which is produced only by nonenterotoxigenic strains of E. coli serogroup O115. The possible role of F165 as a virulence attribute of E. coli strains causing extraintestinal disease is discussed.     

Fimbrial serotypes of Escherichia coli strains isolated from extra-intestinal infections

Strains of Escherichia coli isolated from urinary tract infections and meningitis were characterised by their O:K serotype, haemolysin production, mannose-resistant haemagglutination, and the serotype of the P-fimbriae. The P-fimbriae of 71% of the mannose-resistant haemagglutination-positive strains from urinary tract infection and meningitis could be determined with specific monoclonal antibodies. Many strains expressed multiple P-fimbriae serotypes. The serotypes of P-fimbriae found most frequently among mannose-resistant haemagglutination-positive E. coli from urinary tract infections were the F11, F7 and F8 fimbriae, and among meningitic strains, F11, F8 and F9 fimbriae. The expression of certain F-serotypes did not correlate with O:K antigens.     

Virulence factors in Escherichia coli strains isolated from Swedish piglets with diarrhea.

Parenteral vaccination of sows against Escherichia coli diarrhea in their newborn piglets has become more common during the last decade in Sweden, and the vaccination has generally had positive effects. For more than 20 years we have investigated E. coli strains isolated from piglets and weaned pigs with enteric disorders, noting the presence of O groups, enterotoxins, and adhesins. There has been a continuous change in the frequency of these virulence factors. The present study was performed during 1983 and 1984 to follow this change, since such information is essential for the proper choice of vaccines. A total of 856 E. coli strains were obtained from 683 herds divided into three age groups: 1 to 6 days old, 1 to 6 weeks old, and weaned pigs. O group 149 still dominated in the last two age groups, while O group 101 was, for the first time, the most frequent O group in neonatal piglets. All but four O149 strains carried the K88 antigen, which was found in only one other strain (O group 8). K99 antigen was most often found in O groups 101 and 64, and among all the K99 strains ST mouse was the most common (44 of 57), followed by ST mouse-ST pig strains (12 of 57). The 987P antigen was demonstrated in 26 strains belonging to O groups 141 and OX46 and nontypable strains. Two strains belonging to O group 101 were positive for F41 antigen; one of them also carried the K99 antigen. Among all non-O149 strains, ST mouse was the most common type of enterotoxigenic E. coli ( n = 88), followed in decreasing order by ST mouse-ST pig strains ( n = 69) and ST pig strains ( n = 33). In 114 strains producing enterotoxins no adhesive factor was found. Thus, vaccination of the Swedish sow population for more than 5 years with vaccines containing O149 and K88 antigens has apparently changed the pattern of enterotoxigenic E. coli in neonatal diarrhea. The frequency of O149:K88 strains has been reduced, and O101:K99:ST mouse strains now dominate. However, O149 strains remain the dominant O group in piglets older than 1 week. In spite of all our diagnostic efforts, no virulence factors were detected in about one third of the piglets and weaned pigs with enteric disorders.     

Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.     

Characterization of enteroinvasive Escherichia coli strains isolated from children with diarrhea in Chile.

Analysis of stool samples from 912 cases of diarrhea among Chilean infants and 1,112 controls resulted in the isolation of 17 enteroinvasive Escherichia coli (EIEC) strains from diarrhea cases (1.9%) and 3 EIEC from the asymptomatic controls (0.3%). Biochemical analysis of the 20 isolates showed variability among them. However, the majority were lysine decarboxylase negative and nonmotile and utilized sodium acetate. The strains belonged to the O groups 28ac, 124, 143, or 144 or were untypable with the antisera used. Most of them had conjugative plasmids which mediated multiple antibiotic resistance. There was a strong correlation in this group of strains between a positive Sereny test, the presence of a plasmid of 120 megadaltons, and hybridization with the invasiveness probe, an HindIII fragment derived from the plasmid pPS15A. The isolates had a wide range of plasmid profiles. Bioassays and colony and Southern hybridization tests with iron uptake DNA probes indicated that 80% of the EIEC strains produced aerobactin and expressed its receptor, the genes for which are known to be chromosomally located.     

Contact hemolysin production by strains of enteroaggregative Escherichia coli isolated from children with diarrhea.

Forty-five strains of enteroaggregative Escherichia coli were tested for hemolytic activity with different culture media and erythrocytes from different species. Thirty-seven strains showed proteinase K-sensitive contact hemolysin activity with sheep erythrocytes when cultured in Casamino Acids-yeast extract broth supplemented with 1 mM calcium chloride and when cultured in nutrient broth media. The production of contact hemolysin was dependent on temperature, pH, and culture conditions.     

Genes coding for enterotoxins and verotoxins in porcine Escherichia coli strains belonging to different O:K:H serotypes: relationship with toxic phenotypes.

Seventy-four E. coli strains isolated from piglets with diarrhea or edema disease in Spain were serotyped and examined for production of heat-labile (LT) and heat-stable (ST) enterotoxins (LT-I, LT-II, STaH, STaP, and STb) and verotoxins (VT1, VT2, and VT2v = VTe) by phenotypic (Vero cell assay and infant mouse test) and genotypic (colony hybridization and PCR) methods. In general, an excellent correlation was found between the results obtained with a PCR approach and those determined with biological assays. DNA probes used in the hybridization also showed a very good agreement with phenotypic results, with the exception of a VT1 probe that initially produced 10 false-positive reactions. The gene coding for STb (58 strains) was the most prevalent gene detected by PCR, followed by those coding for STa (46 strains), LT (19 strains), VT2v (11 strains), and VT1 (1 strain). Apparently, in Spain three seropathotypes are predominant: (i) O149:K91:H10 K88+ LT-I+ STb+, (ii) O141:K85ab:H- P987+ STaP+, and (iii) O138:K81:H14 or H- STaP+ VT2v+. We conclude that PCR is a fast, specific, and practical method for the identification of enterotoxin and VT genes in clinical and epidemiological studies.     

Virulence factors of lactose-negative Escherichia coli strains isolated from children with diarrhea in Somalia

Lactose-negative Escherichia coli strains were isolated at high frequency from children with diarrhea in Somalia during a 2-year study on diarrheal diseases. Sixty-four of these strains, considered to be a representative sample, were characterized for virulence factors, plasmid profiles, and antibiotic resistance. Of these strains, 5 were recognized as enteroinvasive E. coli (they were serotyped as O135:K-:H-), 6 belonged to classical enteropathogenic E. coli serotypes, 9 were able to adhere to tissue culture cells (of these, 4 showed a pattern of localized adherence and 1 was an enteropathogenic strain), 18 were both adherent and hemolytic, and 8 were simply hemolytic. None hybridized with 32P-labeled heat-labile or heat-stable (a and b) enterotoxin gene probes or produced moderate or high-level cytotoxic effects on HeLa cells. Of the 64 strains examined, 24 produced mannose-resistant hemagglutination with human, chicken, and monkey erythrocytes. One of these was serotyped as O4:K-:H8, and a rabbit O antiserum raised against this strain allowed us to establish that 23 strains had the same O antigen. The 23 O4 strains were hemolytic and were not enterotoxic for rabbit ileal loops, and intact bacteria were able to destroy tissue culture cell monolayers very rapidly. The uniformity of the antibiotic resistance pattern and of the plasmid DNA content, together with the fact that they were isolated in different years and in different children, suggests that the O4 strains must be epidemiologically relevant in Somalia. A possible diarrheagenic role for the adherent-hemolytic E. coli strains is also discussed.     

Heat-stable-enterotoxin-producing Escherichia coli strains isolated from dogs.

Five strains of hemolytic Escherichia coli isolated from dogs suffering from diarrhea were shown by radioactive and enzyme-labeled oligonucleotide probes to possess genes coding for heat-stable enterotoxin (STIa). Four of the strains were shown by immunoassay (enzyme-linked immunosorbent assay) and bioassay (infant mouse test) to produce STI in vitro. All five strains, however, were able to induce fluid accumulation in ligated dog intestinal loops. The four STI-producing strains all possessed the K99 fimbrial antigen (F5) and belonged to serotype O42:H37. In these strains, genes encoding STI were located on a 98-megadalton plasmid. In the fifth strain, which produced STI in vitro only after several subcultivations, the STI gene was located on an 80-megadalton plasmid. This strain was nontypable.     

Coli surface antigens associated with enterotoxigenic Escherichia coli strains isolated from persons with traveler's diarrhea in Asia.

Enterotoxigenic Escherichia coli (ETEC) strains were isolated from travelers or military personnel who developed diarrhea after visiting Nepal or who were deployed to Thailand, Indonesia, or the Philippines. ETEC isolates were examined for colonization factor antigen (CFA). CFAs were identified on 59% (40 of 68) of the isolates examined. The lack of a detectable CFA on 41% (28 of 68) of the isolates is of concern for the development of an effective ETEC vaccine.     

Isolation of verotoxin-producing Escherichia coli O-rough:K1:H7 from two patients with traveler's diarrhea.

Two Escherichia coli O-rough:K1:H7 strains producing verotoxin 1 that were isolated from stool samples of two travelers with diarrhea who consulted our clinic after trips to the Indian Subcontinent and Central America were characterized. Both strains were sorbitol negative, the same phenotype presented by E. coli O157:H7, but in contrast they were beta-glucuronidase positive. Low-frequency restriction analysis of chromosomal DNA and pulsed-field gel electrophoresis and repetitive extragenic palindrome-PCR showed that both strains were epidemiologically related. The illness was self-limited in both cases but involved long-duration, watery diarrhea (10 to 50 days) accompanied by abdominal cramps and flatulence. This serotype should be taken into account as a possible cause of traveler's diarrhea.     

Virulence properties and clonal structures of strains of Escherichia coli O119 serotypes.

A total of 110 Escherichia coli strains of serogroup O119 were examined for the presence of virulence properties characteristic of enteropathogenic E. coli (EPEC). Three virulence patterns were distinguished based on the detection of a chromosomal gene mediating intimate attachment (eaeA) and plasmid DNA involved in localized adherence (EAF and bfpA). The first pattern, represented by strains which hybridized with three gene probes, was the most common (68%) and, with a single exception, included only O119:H6 strains. Of these strains, 90% showed a typical localized adherence (LA) pattern in HEp-2 cells and 96% were positive for intimate attachment in a fluorescent-actin staining test with a 3-h incubation period. The second pattern was represented by strains which hybridized with the eaeA gene only. Most (89.5%) of these strains showed the LA phenotype but only after 6 h of incubation (LA-like phenotype). The third pattern consisted of strains which were positive for eaeA and bfpA but did not hybridize with the EAF probe. Most (80%) of these strains exhibited the LA-like phenotype. Analysis of several eaeA+ bfpA+ strains for the expression of the pilin subunit (BfpA) of the bundle-forming pili demonstrated that all LA strains expressed BfpA whereas the LA-like strains did not. The study of the clonal relationships, carried out by multilocus enzyme electrophoresis in 79 representative strains, defined 11 distinct electrophoretic types (ETs). ET1 included 66% of the strains, most of which displayed the eaeA+ bfpA+ EAF+ pattern and were serotyped as O119:H6 or O119:H-. The remaining 10 ETs were each represented by no more than five strains and, with the exception of ET8, included strains of a single serotype. The genetic relatedness of the ETs revealed two main clusters, with most strains in cluster A having the eaeA+ bfpA+ EAF+ combination and a O119:H6 serotype. Cluster B was represented by atypical EPEC strains with only the eaeA+ and the eaeA+ bfpA+ virulence pattern.     

Hemolysis determinant common to Escherichia coli strains of different O serotypes and origins.

Ten Escherichia coli hemolytic strains, isolated from various types of human infection and belonging to at least six different O serotypes were analyzed. In all these strains, our results are consistent with the hly genetic determinant being located on the bacterial chromosome. An hly-specific probe (plasmid pAN215) was used to detect homologous fragments in the strains analyzed by the Southern blotting technique. It was found that all 10 strains contain sequences that hybridize against the probe (greater than 85% homology) and thus contain hly genetic determinants very similar to those found on Hly plasmids. These results strongly suggest that there is a unique ubiquitous genetic determinant responsible for the hemolytic phenotype in E. coli.     

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.     

Characterization of Escherichia coli strains producing heat-stable enterotoxin b (STb) isolated from humans with diarrhea.

Two of 49 cytolethal distending toxin-producing strains of Escherichia coli isolated from human stools contained the gene coding for heat-stable enterotoxin b (STb), as detected by a colony hybridization assay. The STb gene was found to be on a 70-kb plasmid also coding for heat-labile enterotoxin (pLT-I). Restriction endonuclease analysis showed the STb gene from human isolates to be similar to the STb gene found in porcine strains. Moreover, by enzymatic amplification based on oligonucleotide primers designed from a porcine STb sequence, the expected portion of the STb gene was amplified for the human E. coli strains. The STb enterotoxin from these strains was bioactive in rat jejunal loops and was detected with an enzyme-linked immunosorbent assay by using polyclonal antiserum raised against purified porcine STb toxin.     

High-pathogenicity island of Yersinia spp. in Escherichia coli strains isolated from diarrhea patients in China

The high-pathogenicity island (HPI) of Yersinia has been observed in 93% of 60 enteroadhesive Escherichia coli strains and 80% of E. coli strains isolated from blood samples. In the present study we investigated 671 fecal samples from patients with diarrhea in Shandong Province, China, and isolated HPI-harboring E. coli from 6. 26% of the samples. The isolation rates for patients with diarrhea in three age groups, 10 to 20, 30 to 40, and 50 to 60 years, were 6. 70, 12.35, and 10.81%, respectively. Therefore, HPI-harboring E. coli is the third most frequently isolated enteric pathogen from patients with diarrhea. Vomiting and abdominal pain were recorded for 33.33 and 66.67% of the patients, respectively. Stools with blood were observed for 9.52% of the patients. Twenty-four of 42 (57%) patients experienced a temperature over 37.4 degrees C. These observations indicate that HPI-harboring E. coli is one of the major causes of diarrheal disease in China and that the clinical symptoms caused by HPI-harboring E. coli differ from those caused by enteroadhesive E. coli.     

Characterization of nonfimbrial mannose-resistant protein hemagglutinins of two Escherichia coli strains isolated from infants with enteritis.

Escherichia coli strains 444-3 and 469-3, isolated from patients with severe infantile enteritis, are able to adhere to and penetrate human epithelial cells in culture. In addition to type 1 fimbriae and glycocalyces , both strains elaborate mannose-resistant nonfimbrial protein hemagglutinins specific for human erythrocytes. Purified agglutinins are aggregates (greater than 4 X 10(6) daltons) of a single protein subunit of apparent Mr 14,000 (469-3) to 14,500 (444-3). The optimal temperature for expression of the agglutinins is 37 degrees C. Bacteria grown at 22 degrees C, which show 1% or less of maximal activity, and mutants deficient in the ability to agglutinate human erythrocytes do not synthesize detectable levels of these surface proteins and, moreover, do not adhere to cultured epithelial cells. Coupled with the observation that purified agglutinins competitively inhibit bacterial adherence to cultured cells, these data indicate that the nonfimbrial surface proteins expressed by strains 444-3 and 469-3 are essential for adherence both to erythrocytes and to cultured epithelial cells.