Molecular Profiling and Phenotype Analysis of Escherichia coli O26:H11 and O26:NM: Secular and Geographic Consistency of Enterohemorrhagic a
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微生物临床杂志 2005年第8期
Institute for Hygiene and the National Consulting Laboratory on Hemolytic Uremic Syndrome, University Hospital Münster, Robert Koch Str. 41, 48149 Münster, Germany
Departments of Pediatrics and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri
ABSTRACT
Fifty-eight enterohemorrhagic Escherichia coli O26:H11 or O26:NM (nonmotile) strains and 44 atypical enteropathogenic E. coli O26:H11 or O26:NM strains isolated from patients in 11 countries during 52 years share a common pool of non-stx virulence genes, fitness loci, and genotypic and phenotypic diagnostic markers. These findings indicate close relatedness between these pathotypes and provide a basis for their clinical laboratory diagnosis.
TEXT
Escherichia coli strains expressing somatic (O) antigen 26 and flagellar (H) antigen 11 or no H antigen (i.e., they are nonmotile) (NM) are classified as either atypical enteropathogenic E. coli (aEPEC) (29) or enterohemorrhagic E. coli (EHEC) (18). aEPEC O26 strains, first identified as causes of infantile diarrhea in the 1940s (20), have caused pediatric diarrhea worldwide (11, 24, 29). Such strains contain eae encoding intimin (11), but in contrast to typical EPEC (20, 29), aEPEC O26 strains lack the EPEC adherence factor (EAF) plasmid (11, 24) encoding bundle-forming pili (20). They also lack genes encoding Shiga toxins (Stx), which are the major virulence factors of EHEC O26 (20). Since being recognized as pathogens >20 years ago (16, 17), EHEC O26 strains have emerged as the most common non-O157 EHEC serogroup causing hemolytic uremic syndrome (HUS) and diarrhea in Europe (5, 7, 28) and have been associated with human diseases worldwide (12, 14).
How related are EHEC O26 and aEPEC O26 Analysis of housekeeping genes (22) and comparative indexing of the core genomes (1) demonstrate that EHEC and aEPEC O26 share a variety of chromosomal regions. However, rosters of putative virulence characteristics of EHEC and aEPEC O26 have not been compared. Moreover, despite the importance of EHEC and aEPEC O26 as human pathogens, diagnostic procedures that identify both groups have not been established. To gain insight into their relatedness and to improve laboratory diagnosis of these organisms, we compared putative virulence genes of EHEC and aEPEC O26 isolates from patients from 11 countries, collected during more than 50 years, and sought genotypes and phenotypes that can be exploited to detect both of these pathogenic groups in clinical laboratories.
Bacterial strains. Fifty-eight EHEC and 44 aEPEC O26 strains were isolated between 1952 and 2003 from patients with HUS (25) (n = 31) or diarrhea (n = 71) in 11 countries (Austria [n = 2], Czech Republic [n = 22], Denmark [n = 3], France [n = 2], Germany [n = 59], Italy [n = 2], Norway [n = 1], the United Kingdom [n = 2], Australia [n = 1], Peru [n = 1], and the United States [n = 7]). Isolates from the same country showed no apparent geographical or temporal linkage. Sixty-two strains belonged to the serotype O26:H11, and 40 were of the serotype O26:NM. Restriction fragment length polymorphism analysis of the fliC gene demonstrated that all belong to the H11 clone complex.
Genotypic and phenotypic analyses. The strains were analyzed for putative virulence genes and diagnostic markers (Table 1) by PCRs as described previously (3, 4, 6, 7, 8, 9, 13, 21, 23, 26, 30). Long polar fimbriae of EHEC O26 (LPFO26) (27) were identified with primers lpfAO26-1 (5'-CCGCTTGTGTTGTGTCTCC-3') and lpfAO26-2 (5'-AGCAGATTTACCAGTATTCA-3') derived from a sequence of the lpfA gene of E. coli O26:H11 strain 843/02 (27) (GenBank accession number AB161111); the PCR was performed in 30 cycles of denaturing (94°C, 30 s), annealing (50°C, 1 min), and extension (72°C, 1 min), followed by a final extension (72°C, 5 min). Molecular analysis of the high-pathogenicity island (HPI) was accomplished as previously described (15). The enterohemolytic phenotype was sought on enterohemolysin agar (2) (Sifin, Berlin, Germany). Tellurite resistance was determined based on an organism's ability to grow on cefixime-tellurite (CT)-sorbitol MacConkey agar (SMAC) (Oxoid, Hampshire, United Kingdom) and on CT-rhamnose MacConkey agar (CT-RMAC) (Sifin) (12). Urease activity was examined in urea degradation broth (Heipha, Heidelberg, Germany) after a 24-h incubation (37°C).
Stool analysis for E. coli O26. Two-hundred-microliter aliquots of enrichment cultures (7) from 636 stools from patients with HUS (n = 177) or diarrhea (n = 459) were inoculated on SMAC, CT-SMAC, and enterohemolysin agar between January 2002 and December 2003. The overnight cultures were PCR screened for stx1, stx2, eae, and rfbO157 (7). Seventy-eight rfbO157-negative stools that were stx positive/eae positive or stx negative/eae positive were further investigated for E. coli O26 (described below).
Distribution of putative virulence and fitness genes among EHEC and aEPEC O26:H11/NM strains. The presence of stx genes in 58 EHEC strains and their absence in the 44 aEPEC strains was the only statistically significant difference (Table 1). All other genes investigated that encode various toxins and adhesins were either present with comparable frequencies among EHEC and aEPEC O26:H11/NM strains or absent from all strains of each pathotype (Table 1). The presence of irp-2 and fyuA, components of the HPI which encodes an iron uptake system and has been proposed to be a "fitness island" (15), in each of the EHEC and aEPEC O26 strains (Table 1) prompted us to investigate whether a complete HPI is present in such strains. Two EHEC strains and two aEPEC strains were subjected to 14 additional PCRs which target the other HPI genes or link consecutive genes (15). This analysis demonstrated that each of the EHEC and aEPEC O26 strains contained a complete HPI which was structurally conserved; this further supports the close relatedness between these two pathotypes. Most EHEC and aEPEC O26 strains had genes encoding tellurite resistance (terZ, terA, terB, terC, terD, terE, and terF) (26) and urease production (ureD, ureA, ureB, ureC, ureE, ureF, and ureG) (9) (Table 1). The finding of the widespread distribution of the ure genes within both E. coli O26 pathotypes is particularly important in light of a recent work (19) which demonstrated that the presence of the ureC gene distinguishes EHEC of the major serogroups, including O26, from diarrheagenic E. coli of other pathotypes, including EPEC; based on this, ureC was recommended as a target to screen for EHEC (19). In contrast, our data demonstrate that ureC is also a suitable target to seek aEPEC O26. However, in accordance with the observation by Nakano et al. (19), urease activity was expressed by only a minority of ure+ EHEC and aEPEC O26 strains (Table 2), indicating that the urease phenotype is not a suitable diagnostic marker for these strains.
Diagnostically useful phenotypes of EHEC and aEPEC O26:H11/NM strains. Each of 47 EHEC O26:H11/NM strains and 33 aEPEC O26:H11/NM strains that harbored the EHEC hlyA gene demonstrated an enterohemolytic phenotype (Table 2). Moreover, the presence of ter genes was perfectly correlated with tellurite resistance of EHEC and aEPEC O26 strains (Table 2). On CT-SMAC, all strains fermented sorbitol. In contrast, on CT-RMAC, 50 (94.3%) of 53 EHEC O26 strains and 30 (83.3%) of 36 aEPEC O26 strains generated rhamnose-nonfermenting, colorless colonies, allowing their differentiation from rhamnose-fermenting (reference 12 and this study), normal intestinal flora. Most EHEC and aEPEC O26 strains displayed both enterohemolytic and tellurite resistance phenotypes (Table 2). Only four of the 102 E. coli O26 strains investigated displayed neither of these two phenotypes (Table 2), because they lacked the encoding genes and could not be, therefore, identified on either enterohemolysin agar or tellurite-containing media.
Isolation of EHEC and aEPEC O26 from stools. To validate the utility of the enterohemolytic and tellurite resistance phenotypes for isolating EHEC and aEPEC O26 from stools, 78 fecal samples that were stx positive/eae positive or stx negative/eae positive by PCR screening were subjected to colony blot hybridization with stx1, stx2, and eae digoxigenin-labeled probes (7) (used as a gold standard). In parallel, they were cultured on enterohemolysin and CT-SMACs. Resulting enterohemolytic and sorbitol-fermenting colonies, and hybridizing colonies from master plates, were screened by slide agglutination with O26 antiserum. By use of colony blot hybridization, 14 EHEC O26 strains and 5 aEPEC O26 strains were isolated (Table 3). Enterohemolysin agar culture identified 13 of the 14 EHEC strains and 4 of the 5 aEPEC strains detected by colony blot hybridization (sensitivities of 92.9% and 80.0%, respectively) (Table 3). Culture on CT-SMAC identified 10 of the 14 EHEC strains and 3 of the 5 aEPEC strains identified by colony blot hybridization (sensitivities of 71.4% and 60.0%, respectively) (Table 3). The two strains that were not detected on enterohemolysin agar lacked the EHEC hlyA gene. In contrast, the six strains which were missed on CT-SMAC contained complete ter operons. The failure to identify these strains probably resulted from low numbers of these organisms in the respective stools, as indicated by colony blot hybridization, combined with the absence of a distinct sorbitol fermentation phenotype. CT-SMAC is a superior medium for the isolation of non-sorbitol-fermenting EHEC O157:H7 (7, 20); it was also successfully used to isolate EHEC O26 from acid-pretreated bovine feces (10). However, it does not distinguish sorbitol-fermenting E. coli O26 from other intestinal coliforms that are usually, but not always, reduced in density on CT-SMAC, because some of them also contain ter genes (26). This might explain why the sensitivity of CT-SMAC for the isolation of E. coli O26 in our study was lower than that of enterohemolysin agar, which allows a direct phenotypic differentiation of these pathogens from normal intestinal flora. However, culture on CT-SMAC identified the two EHEC hlyA-negative strains that were not detected on enterohemolysin agar. Conversely, all six strains that were missed on CT-SMAC were detected on enterohemolysin agar. Taken together, the results of this 2-year prospective study demonstrated that the combined culture on enterohemolysin agar and CT-SMAC is a simple and sensitive procedure to isolate both EHEC and aEPEC O26 from eae-positive stool samples.
Our data demonstrate that EHEC and aEPEC O26:H11/NM strains are largely conserved, without evidence of geographic or secular variation. These two pathotypes share a common pool of fitness and virulence genes, with stx genes being the only difference identified. Some of the easily detectable phenotypes shared by EHEC and aEPEC O26 strains, such as an enterohemolytic phenotype and growth on tellurite-containing media, can be exploited for the isolation of these pathogens from stool samples.
ACKNOWLEDGMENTS
This study was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) Project Network of Competence Pathogenomics Alliance "Functional Genomic research on enterohaemorrhagic Escherichia coli" (BD no. 119523), by a grant from BMBF Verbundprojekt, Forschungsnetzwerk "Emerging foodborne pathogens in Germany" (no. 01KI 9903), and by National Institutes of Health grant R01 AI47499.
We thank M. Hülsmann and N. Brandt for excellent technical assistance.
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Departments of Pediatrics and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri
ABSTRACT
Fifty-eight enterohemorrhagic Escherichia coli O26:H11 or O26:NM (nonmotile) strains and 44 atypical enteropathogenic E. coli O26:H11 or O26:NM strains isolated from patients in 11 countries during 52 years share a common pool of non-stx virulence genes, fitness loci, and genotypic and phenotypic diagnostic markers. These findings indicate close relatedness between these pathotypes and provide a basis for their clinical laboratory diagnosis.
TEXT
Escherichia coli strains expressing somatic (O) antigen 26 and flagellar (H) antigen 11 or no H antigen (i.e., they are nonmotile) (NM) are classified as either atypical enteropathogenic E. coli (aEPEC) (29) or enterohemorrhagic E. coli (EHEC) (18). aEPEC O26 strains, first identified as causes of infantile diarrhea in the 1940s (20), have caused pediatric diarrhea worldwide (11, 24, 29). Such strains contain eae encoding intimin (11), but in contrast to typical EPEC (20, 29), aEPEC O26 strains lack the EPEC adherence factor (EAF) plasmid (11, 24) encoding bundle-forming pili (20). They also lack genes encoding Shiga toxins (Stx), which are the major virulence factors of EHEC O26 (20). Since being recognized as pathogens >20 years ago (16, 17), EHEC O26 strains have emerged as the most common non-O157 EHEC serogroup causing hemolytic uremic syndrome (HUS) and diarrhea in Europe (5, 7, 28) and have been associated with human diseases worldwide (12, 14).
How related are EHEC O26 and aEPEC O26 Analysis of housekeeping genes (22) and comparative indexing of the core genomes (1) demonstrate that EHEC and aEPEC O26 share a variety of chromosomal regions. However, rosters of putative virulence characteristics of EHEC and aEPEC O26 have not been compared. Moreover, despite the importance of EHEC and aEPEC O26 as human pathogens, diagnostic procedures that identify both groups have not been established. To gain insight into their relatedness and to improve laboratory diagnosis of these organisms, we compared putative virulence genes of EHEC and aEPEC O26 isolates from patients from 11 countries, collected during more than 50 years, and sought genotypes and phenotypes that can be exploited to detect both of these pathogenic groups in clinical laboratories.
Bacterial strains. Fifty-eight EHEC and 44 aEPEC O26 strains were isolated between 1952 and 2003 from patients with HUS (25) (n = 31) or diarrhea (n = 71) in 11 countries (Austria [n = 2], Czech Republic [n = 22], Denmark [n = 3], France [n = 2], Germany [n = 59], Italy [n = 2], Norway [n = 1], the United Kingdom [n = 2], Australia [n = 1], Peru [n = 1], and the United States [n = 7]). Isolates from the same country showed no apparent geographical or temporal linkage. Sixty-two strains belonged to the serotype O26:H11, and 40 were of the serotype O26:NM. Restriction fragment length polymorphism analysis of the fliC gene demonstrated that all belong to the H11 clone complex.
Genotypic and phenotypic analyses. The strains were analyzed for putative virulence genes and diagnostic markers (Table 1) by PCRs as described previously (3, 4, 6, 7, 8, 9, 13, 21, 23, 26, 30). Long polar fimbriae of EHEC O26 (LPFO26) (27) were identified with primers lpfAO26-1 (5'-CCGCTTGTGTTGTGTCTCC-3') and lpfAO26-2 (5'-AGCAGATTTACCAGTATTCA-3') derived from a sequence of the lpfA gene of E. coli O26:H11 strain 843/02 (27) (GenBank accession number AB161111); the PCR was performed in 30 cycles of denaturing (94°C, 30 s), annealing (50°C, 1 min), and extension (72°C, 1 min), followed by a final extension (72°C, 5 min). Molecular analysis of the high-pathogenicity island (HPI) was accomplished as previously described (15). The enterohemolytic phenotype was sought on enterohemolysin agar (2) (Sifin, Berlin, Germany). Tellurite resistance was determined based on an organism's ability to grow on cefixime-tellurite (CT)-sorbitol MacConkey agar (SMAC) (Oxoid, Hampshire, United Kingdom) and on CT-rhamnose MacConkey agar (CT-RMAC) (Sifin) (12). Urease activity was examined in urea degradation broth (Heipha, Heidelberg, Germany) after a 24-h incubation (37°C).
Stool analysis for E. coli O26. Two-hundred-microliter aliquots of enrichment cultures (7) from 636 stools from patients with HUS (n = 177) or diarrhea (n = 459) were inoculated on SMAC, CT-SMAC, and enterohemolysin agar between January 2002 and December 2003. The overnight cultures were PCR screened for stx1, stx2, eae, and rfbO157 (7). Seventy-eight rfbO157-negative stools that were stx positive/eae positive or stx negative/eae positive were further investigated for E. coli O26 (described below).
Distribution of putative virulence and fitness genes among EHEC and aEPEC O26:H11/NM strains. The presence of stx genes in 58 EHEC strains and their absence in the 44 aEPEC strains was the only statistically significant difference (Table 1). All other genes investigated that encode various toxins and adhesins were either present with comparable frequencies among EHEC and aEPEC O26:H11/NM strains or absent from all strains of each pathotype (Table 1). The presence of irp-2 and fyuA, components of the HPI which encodes an iron uptake system and has been proposed to be a "fitness island" (15), in each of the EHEC and aEPEC O26 strains (Table 1) prompted us to investigate whether a complete HPI is present in such strains. Two EHEC strains and two aEPEC strains were subjected to 14 additional PCRs which target the other HPI genes or link consecutive genes (15). This analysis demonstrated that each of the EHEC and aEPEC O26 strains contained a complete HPI which was structurally conserved; this further supports the close relatedness between these two pathotypes. Most EHEC and aEPEC O26 strains had genes encoding tellurite resistance (terZ, terA, terB, terC, terD, terE, and terF) (26) and urease production (ureD, ureA, ureB, ureC, ureE, ureF, and ureG) (9) (Table 1). The finding of the widespread distribution of the ure genes within both E. coli O26 pathotypes is particularly important in light of a recent work (19) which demonstrated that the presence of the ureC gene distinguishes EHEC of the major serogroups, including O26, from diarrheagenic E. coli of other pathotypes, including EPEC; based on this, ureC was recommended as a target to screen for EHEC (19). In contrast, our data demonstrate that ureC is also a suitable target to seek aEPEC O26. However, in accordance with the observation by Nakano et al. (19), urease activity was expressed by only a minority of ure+ EHEC and aEPEC O26 strains (Table 2), indicating that the urease phenotype is not a suitable diagnostic marker for these strains.
Diagnostically useful phenotypes of EHEC and aEPEC O26:H11/NM strains. Each of 47 EHEC O26:H11/NM strains and 33 aEPEC O26:H11/NM strains that harbored the EHEC hlyA gene demonstrated an enterohemolytic phenotype (Table 2). Moreover, the presence of ter genes was perfectly correlated with tellurite resistance of EHEC and aEPEC O26 strains (Table 2). On CT-SMAC, all strains fermented sorbitol. In contrast, on CT-RMAC, 50 (94.3%) of 53 EHEC O26 strains and 30 (83.3%) of 36 aEPEC O26 strains generated rhamnose-nonfermenting, colorless colonies, allowing their differentiation from rhamnose-fermenting (reference 12 and this study), normal intestinal flora. Most EHEC and aEPEC O26 strains displayed both enterohemolytic and tellurite resistance phenotypes (Table 2). Only four of the 102 E. coli O26 strains investigated displayed neither of these two phenotypes (Table 2), because they lacked the encoding genes and could not be, therefore, identified on either enterohemolysin agar or tellurite-containing media.
Isolation of EHEC and aEPEC O26 from stools. To validate the utility of the enterohemolytic and tellurite resistance phenotypes for isolating EHEC and aEPEC O26 from stools, 78 fecal samples that were stx positive/eae positive or stx negative/eae positive by PCR screening were subjected to colony blot hybridization with stx1, stx2, and eae digoxigenin-labeled probes (7) (used as a gold standard). In parallel, they were cultured on enterohemolysin and CT-SMACs. Resulting enterohemolytic and sorbitol-fermenting colonies, and hybridizing colonies from master plates, were screened by slide agglutination with O26 antiserum. By use of colony blot hybridization, 14 EHEC O26 strains and 5 aEPEC O26 strains were isolated (Table 3). Enterohemolysin agar culture identified 13 of the 14 EHEC strains and 4 of the 5 aEPEC strains detected by colony blot hybridization (sensitivities of 92.9% and 80.0%, respectively) (Table 3). Culture on CT-SMAC identified 10 of the 14 EHEC strains and 3 of the 5 aEPEC strains identified by colony blot hybridization (sensitivities of 71.4% and 60.0%, respectively) (Table 3). The two strains that were not detected on enterohemolysin agar lacked the EHEC hlyA gene. In contrast, the six strains which were missed on CT-SMAC contained complete ter operons. The failure to identify these strains probably resulted from low numbers of these organisms in the respective stools, as indicated by colony blot hybridization, combined with the absence of a distinct sorbitol fermentation phenotype. CT-SMAC is a superior medium for the isolation of non-sorbitol-fermenting EHEC O157:H7 (7, 20); it was also successfully used to isolate EHEC O26 from acid-pretreated bovine feces (10). However, it does not distinguish sorbitol-fermenting E. coli O26 from other intestinal coliforms that are usually, but not always, reduced in density on CT-SMAC, because some of them also contain ter genes (26). This might explain why the sensitivity of CT-SMAC for the isolation of E. coli O26 in our study was lower than that of enterohemolysin agar, which allows a direct phenotypic differentiation of these pathogens from normal intestinal flora. However, culture on CT-SMAC identified the two EHEC hlyA-negative strains that were not detected on enterohemolysin agar. Conversely, all six strains that were missed on CT-SMAC were detected on enterohemolysin agar. Taken together, the results of this 2-year prospective study demonstrated that the combined culture on enterohemolysin agar and CT-SMAC is a simple and sensitive procedure to isolate both EHEC and aEPEC O26 from eae-positive stool samples.
Our data demonstrate that EHEC and aEPEC O26:H11/NM strains are largely conserved, without evidence of geographic or secular variation. These two pathotypes share a common pool of fitness and virulence genes, with stx genes being the only difference identified. Some of the easily detectable phenotypes shared by EHEC and aEPEC O26 strains, such as an enterohemolytic phenotype and growth on tellurite-containing media, can be exploited for the isolation of these pathogens from stool samples.
ACKNOWLEDGMENTS
This study was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) Project Network of Competence Pathogenomics Alliance "Functional Genomic research on enterohaemorrhagic Escherichia coli" (BD no. 119523), by a grant from BMBF Verbundprojekt, Forschungsnetzwerk "Emerging foodborne pathogens in Germany" (no. 01KI 9903), and by National Institutes of Health grant R01 AI47499.
We thank M. Hülsmann and N. Brandt for excellent technical assistance.
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