Roles of different putative colonization factor antigens in colonization of human enterotoxigenic Escherichia coli in rabbits.

The role of some well-characterized putative colonization factors (PCFs) in enterotoxigenic Escherichia coli (ETEC), i.e. PCFO159, PCFO166, CS7, CS17 and CFA/III, for colonization of the bacteria in the intestine was studied in a non-ligated rabbit intestine model (RITARD). Intestinal administration of 10(11) organisms of the various strains only resulted in very mild symptoms with loose stools during a few days in most of the animals. Strains expressing PCFO159, CS7, CS17 and CFA/III were shed in the stool for a significantly longer period than PCF/CS-negative ETEC. However, the mean time of shedding PCFO166 positive organisms did not significantly exceed that of non-fimbriated E. coli. All strains that colonized rabbit intestine, as assessed by prolonged fecal excretion, also gave rise to high serum antibody responses against the homologous fimbriae whereas non-colonizing strains failed to induce such responses. This study strongly suggests that several of the recently described PCFs, e.g. PCFO159, CS7, CS17 and CFA/III are colonizing factors and strong immunogens.     

PCR-based identification of common colonization factor antigens of enterotoxigenic Escherichia coli

Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli (ETEC) have been classified into several groups based on their distinct antigenicity. We describe here a PCR-based method to detect common CFAs of ETEC, which were characterized using conventional serology. This PCR assay is simple and sensitive for the detection of expressed CFA genes.     

Comparison of methods for detection of colonization factor antigens on enterotoxigenic Escherichia coli.

Fecal Escherichia coli isolates from 196 patients with watery diarrhea and 68 healthy individuals (controls) were analyzed in Bangladesh immediately after isolation for the presence of colonization factor antigen (CFA) I or II (CFA/I or CFA/II, respectively) by a mannose-resistant hemagglutination (MRHA) test with six species of erythrocytes and by a slide agglutination test with absorbed CFA/I or CFA/II antisera. The presence of CFAs was confirmed by immunodiffusion analyses done in Sweden. By these methods, it was found that 49 of 69 enterotoxin-producing E. coli strains isolated from patients carried CFA/I or CFA/II, whereas none of the nonenterotoxigenic E. coli isolates or the three toxin-positive strains isolated from healthy individuals carried these adhesins. All E. coli strains retained their MRHA ability after transportation to Sweden followed by one subculture and after storage at -70 degrees C (but not at room temperature) for 1 to 2 years without further subculturing. After 5 to 10 subcultures of the fresh isolates, however, 70% of the initially CFA/I- and 80% of the initially CFA/II-carrying strains analyzed did not hemagglutinate. The efficacy of different methods for detecting CFAs on the fresh isolates was compared with that of immunodiffusion. The sensitivity of MRHA with human blood group A erythrocytes for the detection of CFA/I was high (97%), but the specificity was only 69%. The sensitivity of MRHA with bovine erythrocytes for the detection of CFA/II in Bangladesh was very low but increased considerably when chicken erythrocytes were also used. Whereas both false-positive and false-negative reactions were obtained when absorbed CFA antisera were used for agglutination, antisera against purified CFAs were equally effective as immunodiffusion in identifying CFA/I and CFA/II-carrying strains.     

Synergistic protective effect of antibodies against Escherichia coli enterotoxin and colonization factor antigens.

We studied the ability of antisera against different Escherichia coli surface antigens, both alone and in combination with anti-enterotoxin, to decrease fluid secretion induced by intestinal challenge with enterotoxigenic E. coli in rabbits. Antiserum against lipopolysaccharide protected significantly against O group homologous bacteria. Monospecific antisera against pilus-associated colonization factor antigens CFA/I and CFA/II were also effective, giving highly significant protection against enterotoxigenic E. coli strains bearing the corresponding colonization factor antigens. Protection was also observed with Fab fragments of the CFA/I antibodies. Addition of the anti-lipopolysaccharide serum to a protective antiserum against purified heat-labile enterotoxin resulted in an antisecretory effect which slightly exceeded the sum of the effects obtained with each preparation alone. The combination of antiserum against CFA/I or CFA/II with anti-enterotoxin gave protection that equaled the product of the effects obtained with each antiserum alone; i.e. the antisera cooperated synergistically.     

Evaluation of conventional media for detection of colonization factor antigens of enterotoxigenic Escherichia coli.

Strains of enterotoxigenic Escherichia coli producing either colonization factor antigen (CFA) I or II were tested for expression of CFA when grown on 16 different agar media by using agglutination and coagglutination with monoclonal antibodies, mannose-resistant hemagglutination, and a salt aggregation assay. CFA was detected from the CFA-positive strains when CFA agar was used, and it was also detected when other commercially available media were used, notably nutrient agar. CFA was not detected when other commercial media such as MacConkey agar were used. The use of nutrient agar with monoclonal antibody-based coagglutination reagents offers a potentially simple and rapid method for detecting E. coli which express CFA I or II.     

Roles of different coli surface antigens of colonization factor antigen II in colonization by and protective immunogenicity of enterotoxigenic Escherichia coli in rabbits.

The roles of the subcomponents of colonization factor antigen II, the coli surface antigens CS1, CS2, and CS3, as colonization factors and protective antigens was studied in a nonligated rabbit intestine model (RITARD). Infection with enterotoxigenic Escherichia coli (ETEC) carrying CS3 alone or CS1 plus CS3 induced diarrhea in most (80%) of the rabbits, whereas nonenterotoxigenic strains expressing CS1 or CS2 rarely induced diarrhea. Strains carrying CS1, CS2, or CS3 alone were all shed in stools for a significantly longer period than normal fecal flora-type E. coli. Initial infection with ETEC positive for CS1 plus CS3 induced significant protection against disease caused by reinfection with a highly diarrheagenic dose of the homologous strain; rabbits previously infected with serotype-heterologous, nontoxigenic bacteria carrying CS1 only were also protected against this challenge, whereas no such protection was induced by serogroup-homologous E. coli carrying CS2 only. Animals previously infected with CS1-, CS3-, or CS1-plus-CS3-positive bacteria excreted the CS1-plus-CS3 challenge strain for a significantly shorter period than did "nonimmunized" rabbits, whereas initial infection with bacteria carrying CS2 only did not result in such reduced shedding. Monoclonal antibodies against CS1, CS2, or CS3 all protected against experimental infection with ETEC carrying the corresponding CS factor. These results suggest that all the subcomponents of colonization factor antigen II are colonization factors and may induce anticolonization immunity.     

Adhesion and ultrastructural properties of human enterotoxigenic Escherichia coli producing colonization factor antigens III and IV.

Human enterotoxigenic Escherichia coli (ETEC) producing colonization factor antigen III (CFA/III) and coli surface antigens 4, 5, and 6 (CS4, CS5, and CS6) of CFA/IV were examined ultrastructurally and for ability to adhere to human small intestinal enterocytes and to cultured human intestinal mucosa. Strains of serotypes O25:H-, O25:H42, and O167:H5 producing CFA/III plus CS6, CS4 plus CS6, and CS5 plus CS6, respectively, showed good adhesion to human enterocytes (1.8 to 4.2 bacteria per brush border) and cultured human intestinal mucosa, whereas variants lacking these antigens or producing only CS6 were nonadherent (0 to 0.03 bacterium per brush border). By electron microscopy, CFA/III, CS4, and CS5 appeared as morphologically distinct rodlike fimbriae: CFA/III was 7 to 8 nm in diameter, CS4 was 6 to 7 nm in diameter, and CS5 was 5 to 6 nm in diameter. CS5 was unusual in that it appeared to be composed of two fine fibrils arranged in a double-helical structure. CS6 was difficult to characterize morphologically but possibly has a very fine fibrillar structure. By specific fimbrial staining and immunoelectron microscopy. CS4 and CS5 were shown to promote mucosal adhesion of ETEC; a similar adhesion role for the CS6 antigen could not be confirmed. ETEC strains of serotypes O27:H7, O27:H20, O148:H28, and O159:H20 which produced CS6 showed good adhesion to human enterocytes (1.6 to 3.0 bacteria per brush border), whereas variants which lacked CS6 were nonadherent (0 to 0.01 bacterium per brush border). These strains, however, also produced fimbrial or fibrillar surface antigens, in addition to CS6, which probably represent additional coli surface antigens responsible for the observed adhesive properties of these ETEC serotypes.     

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.     

Colicin V production by clinical isolates of Escherichia coli.

Strains of Escherichia coli isolated from random fecal samples, urines of hospitalized and nonhospitalized patients who had urinary tract infections, and blood of patients with septicemia were examined for colicin V production. The percentage of ColV+ strains isolated from blood (31.6%) or from urines of hospitalized patients with urinary tract infections (26.2%) was significantly greater (P less than 0.01) than the percentage isolated from feces (13.6%). The colicin V immunity determinant of ColV,I-K94 conferred immunity to 26% of the type V colicins produced by clinical isolates. Of the ColV+ strains studied, 63.6% produced at least one other type of colicin.     

Urease production from clinical isolates of beta-hemolytic Escherichia coli.

Twenty-four lactose-fermenting, urease-producing strains of beta-hemolytic Escherichia coli were isolated from a variety of clinical material. All isolates were indole positive, citrate negative, and produced the characteristic green metallic sheen on eosin-methylene blue agar.     

Isopycnic separation of Escherichia coli cultures possessing colonization factor antigen I.

A culture of Escherichia coli possessing colonization factor antigen I was subjected to isopycnic separation on Percoll gradients. The results demonstrated successful division of the culture into two populations: (i) bacteria which cause mannose-resistant hemagglutination and (ii) bacteria which lack the ability to hemagglutinate in the presence of mannose.     

E.coli临床分离株的基因组结构分型

目的：通过基因组结构分析对临床分离的Escherichia coli(E.coli)进行分型，并探讨分型与临床疾病的关系。方法：取临床不同疾病病人的痰、尿、血、分泌物等标本，分离E.coli。用I-Ceu Ⅰ酶切全基因组DNA，用脉冲场凝胶电泳分离DNA片段后，根据酶切图谱的异同进行分型。结果：从临床上分离的64株E.coli中，62株有7个I-Ceu Ⅰ酶切位点，2株有8个I-Ceu Ⅰ酶切位点。菌株间I-Ceu Ⅰ酶切图谱差异明显。这些菌株根据基因组结构的差异分为32个型，分型与疾病之间的对应关系分散。结论：临床分离的E.coli基因组结构存在多样性，其与临床疾病之间的关系有待进一步探讨。     

Hemagglutination activity and colonization factor antigens I and II in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli isolated from humans.

We examined 205 enterotoxigenic strains of Escherichia coli for colonization factor antigens (CFA) I and II, using an immunodiffusion technique with specific antisera. A total of 36 strains of serogroups O63, O78, O114, O128, and O153 and 1 rough strain possessed CFA/I and gave a single precipitin line; 47 strains of serogroups O6, O8, O80, and O115 possessed CFA/II. The latter strains gave a major precipitin line (component 3) when tested with specific antisera prepared against strain E1392 or PB-176 (both E. coli O6.H16; biotype A). However, all 16 strains of E. coli O6.H16 belonging to biotype A gave a second precipitin line (component 1) when tested with both antisera. When CFA/II-positive strains were tested with a specific antiserum prepared against E. coli O6.H16 strains of biotype B or C, all strains gave component 3, but 16 of 17 strains of E. coli O6.H16 belonging to biotype B, C, or F gave a second precipitin line (component 2) not given by strains of biotype A. CFA/II-positive strains of serogroups other than O6 gave only component 3 in tests with all specific antisera. Nine enterotoxigenic strains of serotypes O7, O15, O25, O115, and O128 gave mannose-resistant hemagglutination of human or calf erythrocytes but lacked CFA/I or CFA/II. Although mannose-resistant hemagglutination was common in non-enterotoxigenic strains of E. coli, none of the non-enterotoxigenic strains possessed CFA/I or CFA/II; these strains included fecal strains of serogroups O6, O8, O63, and O78, fecal strains of enteropathogenic serogroups, and strains from extraintestinal sources.     

Detection of colonization factor antigen I-positive enterotoxigenic Escherichia coli with a cloned polynucleotide probe.

We compared a new colony hybridization assay with an established enzyme-linked immunosorbent assay for detection of enterotoxigenic Escherichia coli (ETEC) expressing colonization factor antigen I (CFA/I). The tests were applied to 135 human ETEC strains. Of these isolates, 30 had previously been characterized for CFAs. A strain harboring the plasmid vector of the polynucleotide gene probe, nine non-ETEC strains from healthy infants, and eight ETEC strains of animal origin were included for further evaluation of probe specificity. The two assays showed a high level of concordance in the specific detection of ETEC strains expressing CFA/I. A total of 24 strains tested positive in the CFA/I hybridization assay, while 23 of those strains were positive in the CFA/I enzyme-linked immunosorbent assay. The single discrepant result could be explained by the loss of a regulatory gene. The strain harboring the plasmid vector of the probe, the non-ETEC E. coli strains, and the ETEC strains of animal origin were all negative in the CFA/I probe assay.     

Genetic relationship of putative colonization factor O166 to colonization factor antigen I and coli surface antigen 4 of enterotoxigenic Escherichia coli.

Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E. coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon. Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA. Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes. The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression. A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy. The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.     

Mannose-resistant hemagglutination of human erythrocytes by enterotoxigenic Escherichia coli with colonization factor antigen II.

The human erythrocyte receptor which mediates mannose-resistant hemagglutination by enterotoxigenic Escherichia coli possessing colonization factor antigen II is not universally distributed among donors. Mannose-resistant hemagglutination-positive erythrocytes are more common among black donors than nonblack donors; tests with erythrocytes of known antigenic makeup confirm this correlation. Colonization factor antigen II receptor activity of mannose-resistant hemagglutination-positive erythrocytes is unstable when whole blood is stored at 4 degrees C. Also, screening of donors is best performed with enterotoxigenic E. coli possessing colonization factor antigen II composed of the coli surface antigen 1 (CS1) plus CS3, since these consistently produce stronger hemagglutination reactions than strains with colonization factor antigen II composed of either CS2 plus CS3 or CS3 only.     

Evidence for two types of cytotoxic necrotizing factor in human and animal clinical isolates of Escherichia coli.

We have characterized the in vitro and in vivo toxic properties of cell sonic extracts from 22 animal and human clinical isolates of Escherichia coli that caused both necrosis in the rabbit skin and multinucleation in tissue cultures, two toxic properties previously reported as being specific for E. coli cytotoxic necrotizing factor (CNF). Two distinct toxic phenotypes were observed. Type 1, which was displayed by originally described CNF strains, was characterized by extensive multinucleation and rounding of cells in HeLa cell culture assays, moderate necrosis in the rabbit skin test, and absence of necrosis in the mouse footpad test. Type 2, which has recently been shown to be associated with E. coli Vir plasmid, was characterized by moderate multinucleation, by polymorphism and elongation of HeLa cells, and by an intense necrotic response in both the rabbit skin test and the mouse footpad test. The distinction between the two cytotoxins accounting for these effects (CNF 1 and CNF 2), together with their partial relatedness, was confirmed by seroneutralization studies of both cytopathic effects and necrosis in the rabbit skin test. In addition, type 2 extracts were more lethal in the mouse intraperitoneal test and induced a moderate, although not totally repetitive, fluid accumulation in the ileal loop test. The original toxic properties of these recently recognized categories of E. coli strains, together with their association with enteritis and septicemia, suggest that these strains may play a significant role in pathology.     

Detection and characterization of colonization factor of enterotoxigenic Escherichia coli isolated from adults with diarrhea.

The fimbriate colonization factor antigen (CEA) of Escherichia coli strain H-1047 was isolated and used to prepare anti-CFA antiserum. Enterotoxigenic E. coli (ETEC) isolated from 29 adults with diarrhea acquired in Mexico were examined for CFA by using this serum. Retrospectively, it was found that ETEC possessing the H-10407-type CFA were isolated from 25 (86%) of these diarrhea cases as compared with 2 of 11 (18%) from asymptomatic controls from whom ETEC had been isolated. CFA was found onE. coli of various serotypes, as demonstrated by bacterial agglutination by the anti-CFA serum. Heat treating the cells at 65 degress C for 1 h prevented the agglutination. CFA-positive strains did not react with anti-CFA serum when the cultures were grown at a low incubation temperature (18 degrees C). E. coli isolates identified serologically as CFA positive were shown to adhere to the intestinal villous surfaces of infant rabbits. By the indirect immunofluorescence technique, it was found that adhesion occurred preferentially in the upper 20 cm of the small intestine. Also, the ability or inability of various isolates to adhere to intestinal mucosa in vivo correlated with the presence or absence of fimbriae on the cells when grown in vitro. Agglutinability with anti-CFA serum, fimbriae, and adhesiveness were spontaneously lost by many isolates after laboratory passage in a manner previously described with E. coli H-10407. These observations suggest that the H-10407-type CFA plays a role in the virulence of ETEC possessing this antigen.     

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types.     

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.