RESUMO
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.
Assuntos
Técnicas de Tipagem Bacteriana , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/genética , Proteínas de Fímbrias , Variação Genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado , Enterotoxinas/metabolismo , Escherichia coli/patogenicidade , Proteínas de Escherichia coli , Humanos , Fenótipo , Sorotipagem , VirulênciaRESUMO
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.
Assuntos
Adesinas de Escherichia coli/genética , Proteínas de Escherichia coli , Escherichia coli/classificação , Escherichia coli/genética , Argentina , Toxinas Bacterianas/genética , Criança , Análise por Conglomerados , Diarreia/microbiologia , Eletroforese em Gel de Campo Pulsado , Enterotoxinas/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Variação Genética , Humanos , Fenótipo , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sorotipagem , Especificidade da Espécie , Células Tumorais CultivadasRESUMO
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.
Assuntos
Adesinas Bacterianas/genética , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Enterotoxinas , Proteínas de Escherichia coli , Escherichia coli/patogenicidade , Proteínas de Fímbrias , Adesinas Bacterianas/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Células CACO-2 , Diarreia/microbiologia , Escherichia coli/classificação , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Humanos , Dados de Sequência Molecular , Mutação , Antígenos O , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Fímbrias , Proteínas de Bactérias/análise , Pré-Escolar , Estudos de Coortes , Diarreia/etiologia , Enterotoxinas/análise , Escherichia coli/classificação , Escherichia coli/patogenicidade , Feminino , Humanos , Lactente , Recém-Nascido , Intestinos/microbiologia , Masculino , Estudos Prospectivos , SorotipagemRESUMO
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.
Assuntos
Toxinas Bacterianas/biossíntese , Enterotoxinas/biossíntese , Proteínas de Escherichia coli , Escherichia coli/classificação , Escherichia coli/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Técnicas de Tipagem Bacteriana , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Humanos , Filogenia , Sorotipagem , VirulênciaRESUMO
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.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Diarreia/microbiologia , Infecções por Escherichia coli/epidemiologia , Escherichia coli/imunologia , Argentina/epidemiologia , Criança , Infecções por Escherichia coli/diagnóstico , Fímbrias Bacterianas/imunologia , Humanos , Imuno-Histoquímica , Microscopia ImunoeletrônicaRESUMO
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)
Assuntos
Diarreia Infantil/microbiologia , Infecções por Escherichia coli/complicações , Proteínas de Fímbrias , Argentina , Proteínas de Bactérias/análise , Pré-Escolar , Resistência Microbiana a Medicamentos , Enterotoxinas/biossíntese , Escherichia coli/efeitos dos fármacos , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Humanos , Lactente , Recém-NascidoRESUMO
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)
RESUMO
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)
RESUMO
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.