New fimbrial antigenic type (E8775) that may represent a colonization factor in enterotoxigenic Escherichia coli in humans.

An enterotoxigenic strain of Escherichia coli O25:H42 (strain E8775), isolated from a patient in Bangladesh with diarrhea, caused mannose-resistant hemagglutination (MRHA) of human and bovine erythrocytes. The strain did not show slide agglutination or immunodiffusion precipitin lines with antiserum specific for the colonization factor antigen CFA/I or CFA/II. A variant E. coli strain, E8775-B, did not cause MRHA or produce enterotoxin. Electron microscopy revealed the presence of fimbriae on the surface of strain E8775 but not strain E8775-B. When strain E8775 was grown at 22 degrees C, it became MRHA negative and fimbriae were absent. An antiserum prepared against strain E8775 was absorbed with strain E8775-B to make an antiserum specific for the fimbrial antigen. Using this absorbed antiserum, we found the fimbrial antigen in 48 of 742 enterotoxigenic E. coli strains. The 48 strains belonged to serogroups O25, O115, and O167. It is suggested by analogy to the properties of previously described colonization factors that these fimbriae may play a part in the colonization of the intestinal epithelium.     

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.     

Hemagglutination patterns of enterotoxigenic and enteropathogenic Escherichia coli determined with human, bovine, chicken, and guinea pig erythrocytes in the presence and absence of mannose.

A hemagglutination (HA)-typing system has been developed for the presumptive identification of enterotoxigenic Escherichia coli (ETEC) possessing the colonization factor antigens (CFA) CFA/I or CFA/II. E. coli isolates are grown on CFA agar and tested for mannose-sensitive (MS) or mannose-resistant (MR) HA of human, bovine, chicken, and guinea pig erythrocytes. CFA/I-positive ETEC exhibit MRHA with human, bovine, and chicken erythrocytes, but no HA with guinea pig erythrocytes. CFA/II-positive ETEC produce HA (MRHA) only with bovine and chicken erythrocytes. Common pili appear to be the primary MS-hemagglutinin of E. coli because the prototype strain K-12 exhibits HA (MSHA) with all but bovine erythrocytes. However, only 6.6% (23 of 351) of E. coli belonging to the classical enteropathogenic E. coli serogroups (EPEC) possessed the same HA pattern as strain K-12; 42% of the EPEC cultures (146 of 351) were similar to K-12 in producing MSHA with chicken and guinea pig erythrocytes and no HA with bovine erythrocytes, but different in that these produced either no HA or MRHA with human erythrocytes. These EPEC-associated HA patterns were assigned to a separate category, termed HA type III. Non-EPEC serogroups associated with sporadic diarrhea (i.e., the facultatively enteropathogenic E. coli, or FEEC) and 41% (19 of 46) of available Salmonella isolates also produced HA type III patterns. This observation is of considerable interest because many FEEC possess somatic antigens cross-reactive with Salmonella. Although the biochemical basis for this result has not been established, the data reported herein suggest a relationship between the HA type III phenotype and virulence (enteropathogenicity) in both the EPEC and FEEC serogroups. We propose that HA typing be used in conjunction with serotyping of E. coli to determine the degree of association of HA type III E. coli with sporadic diarrhea in infants and young children.     

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.     

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.     

Binding Specificity of Piliated Strains of Escherichia coli and Salmonella typhimurium to Epithelial Cells, Saccharomyces cerevisiae Cells, and Erythrocytes

The binding to mammalian cells of piliated enteric bacteria and the inhibition of the binding by antibodies to purified pili were studied. The target cells were epithelial cells from human bucca and human and rat urinary tracts, erythrocytes from various species, and Saccharomyces cerevisiae cells. The strains were selected to represent the two main agglutination patterns of enteric bacteria: mannose-resistant agglutination of human and other erythrocytes and mannose-sensitive agglutination of guinea pig and other erythrocytes. Escherichia coli 3669 caused only mannose-resistant agglutination, E. coli 6013 caused only mannose-sensitive agglutination, and E. coli 3048 caused both types of agglutination simultaneously. Salmonella typhimurium SH6749 exhibited only mannose-sensitive hemagglutination and was included to allow comparison of its pili with those of E. coli strains. The range of epithelial cells to which the bacteria adhered was related to their agglutination patterns. All four strains attached to human buccal cells. Only E. coli strains 3669 and 3048, which caused mannose-resistant agglutination, adhered to human urinary tract epithelial cells, and only those strains that caused mannose-sensitive agglutination adhered to rat urinary tract epithelial cells. The binding of S. typhimurium SH6749, but not of the other strains with mannose-sensitive agglutination, was significantly inhibited by d-mannose. Globotetraosylceramide, a glycolipid present in the human urinary tract epithelium, inhibited attachment to human uroepithelial cells of the two strains with mannose-resistant hemagglutination. As tested by the enzyme-linked immunosorbent assay, cross-reactions between type 1 pili of the E. coli strains were strong, but those between S. typhimurium and E. coli mannose-sensitive pili were weak. The two pili that induced mannose-resistant hemagglutination on E. coli did not cross-react. Significant inhibition of adhesion of all four strains was obtained with the homologous anti-pilus antiserum. The binding of bacteria to mammalian cells may thus be mediated by several types of bacterial pili reacting with different receptors on mammalian cells.     

Haemagglutination and siderophore production as the urovirulence markers of uropathogenic Escherichia coli

A total of 160 strains of Escherichia coli isolated from urine of patients with clinically diagnosed urinary tract infection were included in the study and 50 faecal isolates of E. coli were studied. They were studied for virulence factors, namely mannose-resistant and mannose-sensitive haemagglutination (MRHA, MSHA) and siderophore production. Among 160 urinary isolates of E. coli , 40 (25%) showed MRHA, siderophore production was seen in 156 (97.5%). In 50 faecal isolates, two (4%) were MRHA, four (8%) MSHA and siderophore production in two (4%). The results suggest that MRHA and siderophore production positive strains can be considered as UPEC.     

Hemagglutinating and hemolytic activities of Enterococcus faecalis strains isolated from different human clinical sources

A total of 95 Enterococcus faecalis strains isolated from different human clinical sources were investigated for hemagglutinating activities and hemolysin (Hly) production in the presence of erythrocytes from a wide range of species. MRHA (mannose-resistant hemagglutination) activity was found in all clinical strains tested in this study. MRHA of E. faecalis strains isolated from different sources was most frequently observed with human (both group O and A) and guinea pig erythrocytes. None of the strains agglutinated horse erythrocytes in the presence of 1% alpha-D-mannose. It should be emphasized that our data indicate the absence of a relationship between sources and MRHA. In contrast, all 95 strains investigated in this report were negative for MSHA (mannose-sensitive hemagglutination) activity. Regarding hemolysin production, it was seen that E. faecalis, and particularly urinary strains, preferably lysed horse erythrocytes. On the other hand, none of the 95 clinical strains tested in this study showed hemolytic activity against bovine and sheep erythrocytes. In general, these results show that E. faecalis strains isolated from different clinical sources possessed a diversity of hemagglutinins and a limited repertoire of hemolysin activities.     

Hemagglutinating ability and fimbrial antigens of Escherichia coli strains isolated from urine

The close connection between mannose-resistant hemagglutination (MRHA) and adhesion to uroepithelial cells of urinary E. coli with regard to the pathogenesis of urinary tract infection (UTI) prompted us to examine the hemagglutinating ability of 1499 E. coli strains from urine using human blood group OP1 erythrocytes. In 317 strains (21.2%), an MRHA was found. There were no significant differences in the prevalence of MRHA related to the isolation time and admitting hospital. A correlation was found between MRHA and the presence of P fimbriae in the strains investigated. Another association appears to exist between certain O:K:H serovars and distinct fimbrial antigens which had been serologically identified. The F11 antigen was detected most frequently and proved to be present in strains of serovars O1:K1:H-, O1::K1:H7, O2:K1:H-, O2:K1:H4, O2:K1:H7, and O15:K1:H7. The F8 antigen was strongly associated with serovar O18:K5:H-. O18:K5:H1 and O6:K5:H1 were apparently related to cross-reacting F14 antigens.     

K-antigen identification, hemolysin production, and hemagglutination types of Escherichia coli O6 strains isolated from patients with urinary tract infections

Serotyping of 1918 Escherichia coli strains isolated in significant cell numbers from the urine of patients with urinary tract infections (UTI) revealed the presence of 117 O6 strains. The K antigens were identified by means of K-specific phages and serological methods. The phages used included a K1 phage pool (phi 1, A-E) and the separate phages phi 2, phi 5, phi 7, phi 12 and phi 13. The presence of H antigens, type 1 fimbriae formation, hemolysin production and mannose-resistant hemagglutination (MRHA) ability with human A, sheep, calf and pig erythrocytes were also analyzed. Six different MRHA types were defined and discussed in relation to the O6:K:H serotype. Remarkably, E. coli O6 strains were found to possess a whole arsenal of virulence factors (K antigens, MRHA, hemolysin). The most common serotypes - O6:K2:H1/H- (26), O6:K5:H1/H- (35) and O6:K13:H1/H- (20) - differed from each other in some cases in both MRHA type and hemolysin production.     

Adhesion, hemagglutination, and virulence of Escherichia coli causing urinary tract infections.

The capacity of 453 Escherichia coli strains to agglutinate erythrocytes and yeast cells and to attach to human urinary tract epithelial cells was tested. The strains were isolated from the urine of patients with acute pyelonephritis, acute cystitis, or asymptomatic bacteriuria and from the stools of healthy school children. Three main patterns of hemagglutination were found: (i) mannose-resistant agglutination of human erythrocytes alone or simultaneously with mannose-sensitive agglutination of guinea pig erythrocytes; (ii) only mannose-sensitive agglutination of guinea pig and other erythrocytes; and (iii) no agglutination. Strains with mannose-resistant agglutination of human erythrocytes alone or in combination with mannose-sensitive hemagglutination attached in high numbers to human urinary tract epithelial cells. Bacteria inducing only mannose-sensitive hemagglutination attached in low numbers, and non-agglutinating strains did not bind to the urinary tract epithelial cells. The bacterial surface antigen(s) mediating mannose-resistant hemagglutination of human erythrocytes and attachment to human urinary tract epithelial cells may be one factor selecting for E. coli from among the fecal flora which infect the urinary tract. The highest proportion of strains with this property was found among acute pyelonephritis isolates (77%), and the lowest proportion of strains with this property was found among normal fecal E. coli (16%).     

Colonization antigens and haemagglutination patterns of humanEscherichia coli

The haemagglutinating properties of 223 (35 enterotoxigenic and 188 non-enterotoxigenic)Escherichia coli strains with nine erythrocyte types were investigated; 153 strains were also tested for beta-haemolysis and colicin production and for the presence of CFA/I, CFA/II, K88 and K99 antigens. A selected group of strains was also examined by electron microscopy to determine the presence of fimbriae or fibrils and to establish the relationship between these, the haemagglutinating properties and the presence of colonization antigens. Generally, the haemagglutinating patterns yielded by the same strains grown in Mueller Hinton broth and on CFA agar differed considerably. Mannose-sensitive haemagglutinating (MSHA) patterns were more homogeneous than mannose-resistant haemagglutinating (MRHA) patterns. Forty-seven percent of the non-enterotoxigenic MRHA⁺ strains were haemolytic while only 6 % of the remaining strains were (X² correction=34.01; p<0.001). CFA/I was only detected in the four enterotoxigenic MRHA⁺ strains which were positive only with human and calf erythrocytes when grown on CFA agar. CFA/II was detected in three of a total of six enterotoxigenic strains which were MRHA⁺ only with calf erythrocytes when grown on CFA agar. K88 and K99 antigens were not detected. All strains in which bacteria with fimbriae or fibrils were observed showed haemagglutinating activity. Thus, 18 (66.7 %) of the 27 haemagglutinating strains grown on CFA agar showed fimbriae or fibrils while none of the 19 non-haemagglutinating strains did (X² correction=18.10; p<0.001).     

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.     

Escherichia coli pili as possible mediators of attachment to human urinary tract epithelial cells.

Presence of pili of fimbriae on Escherichia coli bacteria isolated from the urine of patients with urinary tract infection was related to the ability of the bacteria to attach to human uroepithelial cells. Piliated E. coli strains agglutinated guinea pig erythrocytes. D-Mannose and alpha-methyl-D-mannopyranoside inhibited this agglutination with all but one of the 12 strains tested. D-Mannose, D-galactose, alpha-methyl-D-mannopyranoside, and L-fucose did not afect attachment of piliated strains to uroepithelial cells. Heating as well as washing of piliated strains caused a parallel decrease of piliation and adhesive ability. Growth in glucose-enriched medium increased capsule formation but decreased piliation and adhesion. Capsulated strains retained their adhesive ability provided that pili extended outside the capsule.     

Serotypes of attachment pili of enterotoxigenic Escherichia coli isolated from humans.

Pili from enterotoxigenic Escherichia coli isolated from humans have been partially purified, and antisera have been prepared. These pili were initially attached to erythrocytes and then removed by thermal elution for purification. Three distinct antigenic types of pili have been identified. Antisera against these three pili types reacted with 60 of 106 (56%) enterotoxigenic E. coli isolated from humans but not with nontoxigenic, normal human fecal isolates of E. coli nor with enterotoxigenic E. coli strains isolated from animals. There was no correlation between pili serogroup and any of the following toxin production (heat labile, heat stable, or both), O antigenic type, geographical source of isolation, or mannose-resistant hemagglutination patterns of various erythrocyte types.     

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.     

Regulation of production of type 1 pili among urinary tract isolates of Escherichia coli.

The piliation and hemagglutination properties of 54 consecutive Escherichia coli isolates from women with recurrent urinary tract infections were studied. Mannose-sensitive hemagglutination (MSHA) of guinea pig erythrocytes, characteristic of type 1-piliated bacteria, was produced by 75% of the isolates, 32% produced mannose-insensitive hemagglutination, and 14% produced no hemagglutination reaction. The production of type 1 pili was examined in those strains that produced MSHA only. Studies with antiserum prepared against purified pili suggested that at least three subtypes of type 1 hemagglutinins were represented among the isolates. All of the type 1-piliated isolates produced MSHA after serial subculture in static broth. After growth on agar, selected type 1-piliated isolates were subdivided into two groups. Many strains apparently suppressed piliation during growth on agar (regulated variants); all colonies became MSHA negative and were composed of nonpiliated cells as shown by electron microscopy. The loss of the MSHA phenotype often occurred after a single overnight passage on agar, and any remaining hemagglutinin was gradually lost with one to three additional passages. Seven strains, however, retained a significant hemagglutination titer after multiple subcultures on agar, and they produced colonies consisting of a mixed population of piliated and nonpiliated cells. These strains were apparently able to oscillate between states of pilus expression and nonexpression during growth on agar (random phase variants). When nonpiliated cells isolated from the mixed, random variant population were plated on agar, they gave rise to hemagglutination-positive colonies that consisted of both piliated and nonpiliated cells. The distinction between random variants and regulated variants was also observed in shaking broth cultures inoculated with nonpiliated cells. The random variants produced MSHA-positive cultures composed of piliated and nonpiliated cells, whereas the regulated strains remained nonpiliated. The results indicate that type 1 pili are a predominant adhesin of uropathogenic E. coli and that during growth on agar only about one-fourth of the type 1-piliated isolates regulate pilus expression by random phase variation.     

Production and characterization of monoclonal antibodies to a pilus colonization factor (colonization factor antigen III) of human enterotoxigenic Escherichia coli

Three monoclonal antibodies (MAbs) to a pilus colonization factor (colonization factor antigen III [CFA/III]) of human enterotoxigenic Escherichia coli (ETEC) were developed and characterized. All of the MAbs isolated belonged to the immunoglobulin G2a subclass. The specificity of these MAbs for CFA/III pili was demonstrated by the immunogold-labeling technique. The presence of more than one epitope in CFA/III pili was suggested. One of the three MAbs appears to recognize a polymeric conformational epitope(s) of CFA/III. CFA/III antigenicity distinct from that of other pilus colonization factors of ETEC was demonstrated by both a bacterial agglutination test and a sandwich enzyme-linked immunosorbent assay using the MAbs. Of the 100 strains of ETEC isolated from persons with traveler's diarrhea, 8% were found to carry CFA/III pili. Two enzyme-linked immunosorbent assay systems which could detect as little as several or 50 ng of CFA/III per ml were developed.     

Virulence factors of bacteraemic Escherichia coli with particular reference to production of cytotoxic necrotising factor (CNF) by P-fimbriate strains

Thirty-seven strains of Escherichia coli isolated from bacteraemia and 40 faecal strains isolated from healthy individuals were O serogrouped and investigated for the production of colicins, haemolysin (Hly), cytotoxic necrotising factor (CNF), lethal activity for mice, the expression of P fimbriae, mannose-resistant (MRHA) and mannose-sensitive (MSHA) haemagglutination, and relative cell surface hydrophobicity. Virulence factors significantly associated with bacteraemic strains were: serogroups O2, O4, O6, O7, O8 and O75 (54% versus 10%, p less than 0.001), production of Hly (32% versus 8%, p less than 0.02) and CNF (38% versus 10%, p less than 0.01), expression of P fimbriae (27% versus 5%, p less than 0.02), MRHA types III, IVa and IVb (51% versus 8%, p less than 0.001), and possession of a moderate cell surface hydrophobic charge (35% versus 13%, p less than 0.05). Virulence factors were strongly associated with strains expressing defined MRHA types. Thus, all strains belonging to MRHA types III and IVa were toxigenic, whereas only 11% of strains belonging to MRHA types IVb, V or VI were toxigenic (p less than 0.001). Virulence factors were concentrated in strains belonging to O serogroups usually found in E. coli that cause extra-intestinal infections, especially in strains of O4 and O6 groups. The most interesting result of this study was that all 12 P-fimbriate strains expressed the MRHA type IVa and 11 of them synthesised CNF.     

Identification of plasmid-encoded mannose-resistant hemagglutinin and HEp-2 and HeLa cell adherence factors of two diarrheagenic Escherichia coli strains belonging to an enteropathogenic serogroup.

Two Escherichia coli strains (B/M 369 and C-35) belonging to enteropathogenic serogroup O86 were isolated from patients with infantile diarrhea and studied with respect to their cellular adherence properties. Both strains exhibited adherence (Ad+) to HEp-2 and HeLa cell monolayers in vitro and expressed mannose-resistant hemagglutinating (MRHA+) activity towards human, chicken, and sheep (but not mouse, rabbit, or guinea pig) erythrocytes. Cellular adherence properties of both strains could be substantially reduced by pronase treatment and by heat treatment (100 degrees C for 5 min) of bacteria. Electron microscopic examination failed to reveal fimbria- or pilus-like structures on the bacterial cell surface. Conjugation experiments conducted with these strains suggested that both MRHA and HEp-2 and HeLa cell adherence factors were encoded by the same plasmid, with a size of 55 to 57 megadaltons (MDa). Further biochemical studies indicated that the cellular adherence factors were associated with cell surface structures of bacteria that were proteinaceous in nature. An antiserum, rendered specific for the 57-MDa plasmid (pRP201) products of B/M 369 by adsorption, reacted with both MRHA+ Ad+ strains, B/M 369 and C-35, but not with their 57- or 55-MDa plasmidless MRHA- Ad- transconjugants or with other MRHA- Ad- E. coli strains. Immunological studies showed that the absorbed antiserum recognized two proteins with subunit molecular sizes of 18 and 14.5 kDa that were present on the cell surfaces of both strains. Furthermore, the absorbed antiserum at subagglutinating dilutions did inhibit, although only partially, the MRHA and HEp-2 and HeLa cell adherence activities of both E. coli strains. All these results would indicate that some of the E. coli strains belonging to enteropathogenic serogroups express their adherence potential through factors that were hitherto unrecognized.