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 São 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.

A bacterial type III secretion system inhibits actin polymerization to prevent pore formation in host cell membranes.

The bacterial pathogen Yersinia pseudotuberculosis uses type III secretion machinery to translocate Yop effector proteins through host cell plasma membranes. A current model suggests that a type III translocation channel is inserted into the plasma membrane, and if Yops are not present to fill the channel, the channel will form a pore. We examined the possibility that Yops act within the host cell to prevent pore formation. Yop- mutants of Y.pseudotuberculosis were assayed for pore-forming activity in HeLa cells. A YopE- mutant exhibited high levels of pore-forming activity. The GTPase-downregulating function of YopE was required to prevent pore formation. YopE+ bacteria had increased pore-forming activity when HeLa cells expressed activated Rho GTPases. Pore formation by YopE- bacteria required actin polymerization. F-actin was concentrated at sites of contact between HeLa cells and YopE- bacteria. The data suggest that localized actin polymerization, triggered by the type III machinery, results in pore formation in cells infected with YopE- bacteria. Thus, translocated YopE inhibits actin polymerization to prevent membane damage to cells infected with wild-type bacteria.

Identification of a cluster of strains bearing a new adhesin among genetically diverse enterotoxigenic Escherichia coli isolates of serogroup O20.

About one-third of the enterotoxigenic Escherichia coli isolates lack any of the known colonization factors. Among this group, those of serogroup O20 are the most frequently isolated in Argentina. By combining analysis of adhesion to Caco-2 cells, random amplified polymorphic DNA, and pulsed-field gel electrophoresis techniques, we were able to identify three sets of closely related strains with different binding properties. Further analysis of the most prevalent group revealed that all these isolates expressed the recently described adhesin CS22.

CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15.

Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H- strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.

Prospective cohort study of enterotoxigenic Escherichia coli infections in Argentinean children.

In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.

Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly amplified polymorphic DNA analysis.

The genetic relatedness among 29 enterotoxigenic Escherichia coli strains of serotype O6:H16 was investigated by randomly amplified polymorphic DNA (RAPD) analysis. The strains were isolated in different parts of the world, displayed CS1-CS3 or CS2-CS3 profiles, and expressed heat-labile toxin (LT) and heat-stable toxin; a single strain expressed only LT. Ten RAPD types were distinguished and showed significant similarity, having on average 82% of the amplified bands in common. These results indicated that, irrespective of the different geographical origin or virulence factors, these strains belonged to a widespread clonal group.

Colonization factors of enterotoxigenic Escherichia coli isolated from children in north India.

Colonization factor antigens (CFAs) mediate attachment of enterotoxigenic Escherichia coli (ETEC) to the intestinal mucosa and induce protective immunity against ETEC diarrhea. ETEC strains (n = 111) isolated from North Indian children from 1985 to 1989 were examined for CFAs and putative colonization factors (PCFs). CFA/IV was the most common factor (26%), followed by coli surface antigen 17 (CS17) (19%), CFA/I (14%), PCFO166 (7%), and CFA/II (5%), while 24% of the isolates were negative for CFAs and PCFs. Among the strains producing heat-stable and heat-labile toxin (ST+LT+ strains), the STaI gene was strongly associated with the absence of known CFAs and PCFs, making the STaI+LT+ isolates an interesting target for the identification of previously undescribed factors. Repetitive sequence--based polymerase chain reaction revealed that the CS17+ strains, although clonally related, represented endemically circulating strains with a diversity greater than that of the CFA/I+ strains, which showed a substantial clonal clustering.

Binding of enterotoxigenic Escherichia coli expressing different colonization factors to tissue-cultured Caco-2 cells and to isolated human enterocytes.

The binding of ETEC strains expressing different colonization factors to the human enterocyte-like cell line Caco-2 and to isolated human enterocytes were determined. Strains expressing CFA/I, CS2, CS4+CS6, CS5+CS6, CS7, CFA/III+CS6 and PCFO166 adhered well to both types of cells whereas bacteria producing CS1, CS6 only, or CS17 did not adhere to either Caco-2 cells or to jejunal human enterocytes, suggesting that similar binding structures are present in both cell types. However, in some instances, binding of bacteria to the two types of cells differed, e.g. bacteria expressing CS3 or PCFO9 bound well to human enterocytes but not to Caco-2 cells. Conversely, bacteria producing PCFO20 or PCFO159 only adhered to Caco-2 cells and not to jejunal enterocytes. With few exceptions, this inability to bind to human enterocytes was the same for cells from all parts of the small intestine. This study contradicts previous reports suggesting that the binding structures of Caco-2 cells are identical to those of human enterocytes.

The structural gene encoding human enterotoxigenic Escherichia coli PCFO20 is homologous to that for porcine 987P.

Putative colonization factor PCFO20 was recently identified in an enterotoxigenic Escherichia coli (ETEC) strain of serogroup O20 isolated from a child with diarrhea in Argentina. The gene encoding the structural subunit of PCFO20 fimbriae, fotA, was cloned from strain ARG-2 in the expression phage vector lambda ZAP Express. One positive clone, pGV29, that carried a 3.3-kb fragment was identified on the basis of fimbrillin production by using a monospecific rabbit anti-PCFO20 serum. Nucleotide sequencing of a 1.3-kb Sau3A-ClaI fragment of the subclone pGV292 containing the region coding for PCFO20 fimbrillin revealed two open reading frames of which one was complete. A western blot (immunoblot) showed that the cloned protein, FotA, migrated like the PCFO20 fimbrial subunit protein did. Fimbriae were not detected on the surface of E. coli host bacteria containing pGV292 or pGV29, suggesting that the genes needed for assembly of PCFO20 fimbriae are lacking in both clones. The fotA gene encodes a 20,574-Da prefimbrillin protein which contains a 21-amino-acid signal sequence; the mature protein has a size of 18.1 kDa. The subunit protein FotA was found to be more homologous to the subunit of porcine 987P than to any fimbrial subunit produced by human ETEC. Alignments of the amino acid sequences of the two proteins indicate that they are partly identical, with an overall similarity of 82%. FotA fimbrillin was shown to be transported and assembled by the fimbria assembly machinery in porcine ETEC strain 987. PCFO20 and 987P may have evolved from a common ancestral gene. They are immunologically related but have affinity for different host cell receptors, since PCFO20-producing bacteria do not bind to neonatal piglet enterocytes.

Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli.

Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus). Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV. ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences. lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone. lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease. We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins. A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC.

A new fimbrial putative colonization factor, PCFO20, in human enterotoxigenic Escherichia coli.

The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Several colonization factor antigens (CFAs) and putative colonization factors (PCFs) have been described for ETEC. However, there are still many ETEC strains isolated from patients with diarrhea which do not possess any of these antigens. To identify CFAs in ETEC lacking the above-mentioned antigens, we exploited the ability of ETEC to adhere to tissue-cultured cells from an enterocyte-like cell line, Caco-2. An ETEC strain producing heat-labile toxin and heat-stable toxin of serotype O20:K27:H- (ARG-2) that was isolated from a child with diarrhea in Argentina and bound to Caco-2 cells was studied in further detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of this strain revealed a band of 25 kDa when bacteria were grown at 37 degrees C that was missing when the same strain was cultured at 20 degrees C. Furthermore, electron microscopy examination revealed the presence of fimbriae on the surfaces of cells of this strain when cells were grown at 37 degrees C but not at 20 degrees C. Rabbit antiserum raised against purified fimbriae reacted with the 25-kDa protein in immunoblotting and bound specifically to the fimbriae, as shown by immunoelectron microscopy. The presence of fimbriae, adhesion to Caco-2 cells, and the 25-kDa band seen in the SDS-PAGE were all simultaneously lost by single-insertion mutations. The N-terminal amino acid sequence of the protein subunit of the fimbriae showed no relation with those of the known colonization factors of ETEC. Furthermore, the fimbriae of the ARG-2 strain did not cross-react immunologically with any of the previously described adhesive factors in human ETEC when specific antisera against colonization factor antigens and putative colonization factors were used. Moreover, a specific antiserum raised against the fimbriae in ARG-2 did not react with ETEC carrying known colonization factors. We propose to name these new fimbriae PCFO20.

Characterization of monoclonal antibodies against putative colonization factors of enterotoxigenic Escherichia coli and their use in an epidemiological study.

Monoclonal antibodies (MAbs) against five putative colonization factors (PCFs), i.e., colonization factor antigen (CFA)/III, coli surface antigen (CS)7 and CS17, PCFO159, and PCFO166 of enterotoxigenic Escherichia coli (ETEC), were produced. Hybridomas (one each) producing specific antibodies against the respective PCFs were selected. All the MAbs reacted with the corresponding fimbriae but not with CFA/I, CFA/II, or CFA/IV or the heterologous PCFs in bacterial agglutination and enzyme-linked immunosorbent assays (ELISAs). In immunoelectron microscopy these MAbs bound along the fimbriae, and they also reacted with the corresponding subunits in immunoblots. The five MAbs were used to evaluate the prevalence of CFA/III, CS7, CS17, PCFO159, and PCFO166 in ETEC strains isolated from children with diarrhea in Argentina. One hundred five ETEC isolates negative for CFA/I, CFA/II, and CFA/IV were tested in slide agglutination or in a dot blot test for spontaneously agglutinating strains; positive results were confirmed by inhibition ELISAs. It was found that 27% of the CFA-negative ETEC strains carried one of the PCFs. The sensitivity of slide agglutination with these MAbs was similar to that with specific polyclonal antisera; however, the specificity was higher. PCFO166 was found in 9.5% of the strains tested, mainly in ETEC of serogroup O78 producing heat-stable toxin alone. CS17 and CS7 were identified in 6.7 and 5.7%, respectively, of strains producing heat-labile toxin only, most of which belonged to serogroup O114. PCFO159 was found in 3.8% of the isolates tested, whereas CFA/III was detected in only one ETEC strain.

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.

Relationship between enterotoxigenic Escherichia coli and diarrhea among children in Buenos Aires.

The incidence of enterotoxigenic Escherichia coli (ETEC) has been studied in 85 children with acute diarrhea in patients in the Hospital de Niños Pedro de Elizalde, Buenos Aires, and in 38 healthy children. All of them were up to four years old and none had received antibiotic treatment within 7 days before sampling. ETEC was recovered in 9 out of 85 (10.6%) children with diarrhea. From these positive cases, 6 were associated with heat-stable (ST), 1 with heat-labile (LT) and 2 with both LT and ST enterotoxins. Only one case (2.6%) of LT-producing ETEC was detected in the control group. In 5 out of 9 ETEC diarrhea cases (55.5%) the isolated strains expressed human colonization factor antigens (CFA); four of them were CFA/I and one CFA/II. The characteristics of the CFA, biotype, serotype and antibiotic sensitivity pattern were studied in 23 E. coli isolates from 10 ETEC positive children. Of the 12 ST only strains, 5 (41.7%) expressed CFA/I and 2 (16.7%) CFA/II (CS2 + CS3). One out of 2 LT/ST strains expressed CFA/I. CFAs were not detected in the ETEC-LT nor in the toxin negative E. coli strains. From the ETEC isolated, 82.4% were resistant to 4 or more antibiotics, whereas only 50% of simultaneously isolated toxin-negative E. coli presented this sensitivity pattern. The different ETEC strains belonged to several different serotypes, some of them rarely observed in other countries. None of these serotypes correlated either with the toxin profile or with the sugar fermentation pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between enterotoxigenic Escherichia coli and diarrhea among children in Buenos Aires.

The incidence of enterotoxigenic Escherichia coli (ETEC) has been studied in 85 children with acute diarrhea in patients in the Hospital de Niños Pedro de Elizalde, Buenos Aires, and in 38 healthy children. All of them were up to four years old and none had received antibiotic treatment within 7 days before sampling. ETEC was recovered in 9 out of 85 (10.6

) children with diarrhea. From these positive cases, 6 were associated with heat-stable (ST), 1 with heat-labile (LT) and 2 with both LT and ST enterotoxins. Only one case (2.6

) of LT-producing ETEC was detected in the control group. In 5 out of 9 ETEC diarrhea cases (55.5

) the isolated strains expressed human colonization factor antigens (CFA); four of them were CFA/I and one CFA/II. The characteristics of the CFA, biotype, serotype and antibiotic sensitivity pattern were studied in 23 E. coli isolates from 10 ETEC positive children. Of the 12 ST only strains, 5 (41.7

) expressed CFA/I and 2 (16.7

) CFA/II (CS2 + CS3). One out of 2 LT/ST strains expressed CFA/I. CFAs were not detected in the ETEC-LT nor in the toxin negative E. coli strains. From the ETEC isolated, 82.4

were resistant to 4 or more antibiotics, whereas only 50

of simultaneously isolated toxin-negative E. coli presented this sensitivity pattern. The different ETEC strains belonged to several different serotypes, some of them rarely observed in other countries. None of these serotypes correlated either with the toxin profile or with the sugar fermentation pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between enterotoxigenic Escherichia coli and diarrhea among children in Buenos Aires.

The incidence of enterotoxigenic Escherichia coli (ETEC) has been studied in 85 children with acute diarrhea in patients in the Hospital de Niños Pedro de Elizalde, Buenos Aires, and in 38 healthy children. All of them were up to four years old and none had received antibiotic treatment within 7 days before sampling. ETEC was recovered in 9 out of 85 (10.6

) children with diarrhea. From these positive cases, 6 were associated with heat-stable (ST), 1 with heat-labile (LT) and 2 with both LT and ST enterotoxins. Only one case (2.6

) of LT-producing ETEC was detected in the control group. In 5 out of 9 ETEC diarrhea cases (55.5

) the isolated strains expressed human colonization factor antigens (CFA); four of them were CFA/I and one CFA/II. The characteristics of the CFA, biotype, serotype and antibiotic sensitivity pattern were studied in 23 E. coli isolates from 10 ETEC positive children. Of the 12 ST only strains, 5 (41.7

) expressed CFA/I and 2 (16.7

) CFA/II (CS2 + CS3). One out of 2 LT/ST strains expressed CFA/I. CFAs were not detected in the ETEC-LT nor in the toxin negative E. coli strains. From the ETEC isolated, 82.4

were resistant to 4 or more antibiotics, whereas only 50

of simultaneously isolated toxin-negative E. coli presented this sensitivity pattern. The different ETEC strains belonged to several different serotypes, some of them rarely observed in other countries. None of these serotypes correlated either with the toxin profile or with the sugar fermentation pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

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.