Prevalence and Properties of Diarrheagenic Escherichia coli among Healthy Individuals in Osaka City, Japan

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1 Jpn. J. Infect. Dis., 62, , 2009 Epidemiological Report Prevalence and Properties of Diarrheagenic Escherichia coli among Healthy Individuals in Osaka City, Japan Sami Fujihara 1,2,3, Kentaro Arikawa 2, Tetsu Aota 2, Hiroshi Tanaka 2, Hiromi Nakamura 3, Takayuki Wada 3, Atsushi Hase 3, and Yoshikazu Nishikawa 2 * 1 Urban Living and Health Association, Osaka ; 2 Osaka City University Graduate School of Human Life Science, Osaka ; and 3 Osaka City Institute of Public Health and Environmental Sciences, Osaka , Japan (Received April 13, Accepted June 5, 2009) SUMMARY: The etiological roles of diarrheagenic Escherichia coli (DEC), including enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC) and EAST1EC---a strain of E. coli that possesses no diarrheagenic characteristics other than the EAggEC heat-stable toxin 1 (EAST gene---remain controversial. To clarify the prevalence of DEC among healthy individuals in Osaka City, Japan, and to compare the virulence properties of strains previously isolated from diarrheal patients, fecal specimens were examined for DEC. Isolation rates of Shiga toxin-producing E. coli, enterotoxigenic E. coli and EAggEC were significantly lower among healthy adults than sporadic adult patients. There were no differences in enteropathogenic E. coli (EPEC), DAEC and EAST1EC between patients and healthy carriers. Subtyping of the intimin gene (eae) of EPEC, and measuring the IL-8 inductivity of DAEC on epithelial cells could provide criteria to distinguish strains in diarrheal patients from those in healthy carriers. Proper criteria should be established in order to diagnose subtypes of DEC as causative agents. INTRODUCTION Escherichia coli is a normal inhabitant of the intestinal tract in humans and warm-blooded animals; however, certain strains cause enteric disease in their hosts and are referred to as diarrheagenic E. coli (DEC). Based on distinct epidemiological and clinical features, specific virulence determinants and other characteristic markers, such as enterotoxins and adherence phenotypes, DEC strains have been classified into the following six pathotypes (: enteropathogenic E. coli (EPEC); Shiga toxin-producing E. coli (STEC); enterotoxigenic E. coli (ETEC); enteroinvasive E. coli (EIEC); enteroaggregative E. coli (EAggEC); and diffusely adherent E. coli (DAEC). E. coli that has no diarrheagenic characteristics other than the EAggEC heat-stable toxin 1 (EAST gene is defined as EAST1EC, and is included in the possible seventh group of DEC in this investigation. Although EAST1 is reported to be an enterotoxin of EAggEC (2-4), it has not been well accepted as a virulence factor (5,6). However, we have focused our attention on EAST1EC because an outbreak due to EAST1EC O166:H15 occurred in Osaka City in 1996 (7,8). EPEC possessing both the bundle-forming pilus gene (bfpa) and intimin gene (eae) for E. coli-attaching and -effacing is a well-recognized pathogen in developing countries as class I EPEC (9) or typical EPEC (10). However, atypical EPEC organisms possessing eae alone have been reported to be more prevalent in both developing and developed countries (1, and animals can be reservoirs of atypical EPEC, in contrast to typical EPEC, in which humans are the sole reservoir (12). To accumulate precise information on the prevalence of DEC, we previously reported isolation rates of DEC among *Corresponding author: Mailing address: Graduate School of Human Life Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka , Japan. Tel & Fax: , nisikawa@life.osaka-cu.ac.jp sporadic diarrheal patients in Osaka City (13). We then suggested that DAEC comprises heterogeneous groups of organisms with variable virulence and that the subgroup of DAEC possessing afimbrial adhesin (Afa) genes and/or inducing high levels of interleukin-8 (IL-8) secretion in epithelial cells plays a role in causing sporadic diarrheal illnesses, particularly in pediatric fields (14-16). However, the enteropathogenicity and etiological roles of these atypical EPEC (17,18), EAggEC (19,20), DAEC (18,2 and EAST1EC strains remain somewhat controversial. Clinical microbiologists often find it difficult to assess the etiological significance of these isolates, particularly when the organisms are isolated from sporadic patients. It is helpful for clinical laboratories to know the prevalence of DEC among healthy people beforehand. In this study, a total of 2,230 fecal specimens from healthy adults were examined for DEC. The isolation rates and virulence properties were discussed in comparison with those among diarrheal patients in our previous study (13). Salmonella was examined as a representative enteric pathogen for comparison with DEC. MATERIALS AND METHODS Specimens: Between August 2006 and November 2007, a total of 2,230 stool specimens from healthy adult individuals, including food handlers, were examined. Bacterial examination: Fecal specimens were examined for the presence of E. coli, Salmonella and Shigella with desoxycholate hydrogen sulfide lactose agar (Nissui Pharmaceutical, Tokyo, Japan). More than two suspicious colonies were tested in TSI (Nissui) and LIM media (Nissui), and were identified based on biochemical tests. E. coli and Salmonella were serotyped with antisera (Denka Seiken Co. Ltd., Tokyo, Japan). Bacterial strains: DEC strains isolated from diarrheal patients throughout our previous study (13) were used to 318

2 compare subtypes of the eae of EPEC and IL-8 induction due to DAEC. Detection of enterovirulent genes and production of enterotoxin: Our newly developed real-time PCR was used to examine the presence of 10 enterovirulence genes (eae, stx1, stx2, elt, est for STh, est for STp, virb, aggr, asta and afab) in each isolate (22). Briefly, bacterial cells were recovered from a 0.5-ml pure culture of each strain grown for 18 h at 37 C, and genomic DNA was extracted with a Genomic DNA Purification kit (Gentra Systems, Minneapolis, Minn., USA) according to the manufacturer s protocols. Duplex or triplex primer-probe reactions were performed in QuantiTect Multiplex PCR solution (QIAGEN, Hilden, Germany). Reaction mixtures (48 l) were dispensed into 96-well, thin-wall PCR plates (Applied Biosystems, Foster City, Calif., USA), and 2 l of genomic DNA solution was added as a template. Plates were covered with optically clear sealing film and briefly centrifuged. PCR was performed with the ABI PRISM 7000 Sequence Detection System under the following cycle conditions: denaturation at 95 C for 15 min, followed by 40 cycles of 95 C for 1 min and 60 C for 1 min. Shiga toxin (Stx) production was examined using Duopath Verotoxins (Merck Ltd., Tokyo, Japan). Subtyping of enterovirulent genes: According to the report of Blanco et al. (23), the eae genotype was identified using 26 types of intimin type-specific PCR primers complementary to the heterogeneous 3 -end of the genes. DAEC strains were assigned to subgroups according to the scheme described by Le Bouguénec et al. (24). Strains that amplified the primer set for afac were identified as afapositive, and these strains were assigned to Afa/Dr + and Afa/ Dr groups on the basis of positive or negative reactions to primers for afabc. The Afa/Dr group also belongs to the Afa group, but the Dr antigen was not recognized. Organisms that were Afa/Dr + but did not react with primers for any afa subtypes (afae1, 2, 3, 5 and daae) were classified as afaex; bacteria possessed genes amplified with primers for Afa, but the gene was not identified as part of a particular subgroup. Tissue culture adhesion tests and IL-8 assay: Bacterial strains that showed positive reactions on PCR for eae, aggr or afab were further examined by adhesion tests, as described previously (25). Monolayers of HEp-2 cells grown on cover slips (diameter 13 mm) in 24-well plates were prepared in the absence of antibiotics. Two-day-old monolayers of HEp- 2 cells were used for the tests. Bacterial strains were grown statically overnight at 37 C in 1% buffered peptone water (Oxoid, Basingstoke, England). Before the test, monolayers were washed once with Dulbecco s PBS. One milliliter of Basal Eagle s medium containing D-mannose (1% w/v) without antibiotics or sera was added to each well. Overnight bacterial culture (20 l) was inoculated into each well, and plates were incubated at 37 C for 3 h. Monolayers were washed three times with PBS, and 1 ml of medium was added to each well. After a further 3-h incubation period, monolayers were washed thoroughly three times with PBS, fixed with absolute methanol, and stained with 10% (v/v) Giemsa. For an enzyme-linked immunosorbent assay of IL-8, adhesion tests were performed as described above, but the epithelial cells were not washed at 3 h, in contrast to the ordinary adhesion tests. A sample of 100 l of the culture medium was taken after 6-h incubation, and a commercial kit IL-8, Human, Assay (GE Healthcare UK Ltd., Buckinghamshire, UK) was used according to the manufacturer s instructions. Pulsed-field gel electrophoresis (PFGE): PFGE was performed by the method of Pei et al. (26). Statistics: Differences between isolation rates were analyzed by performing chi-square tests with Yates continuity correction or Fisher s exact probability test. The Kruskal- Wallis test was used to analyze seasonal variations of the isolation rates. The chi-square statistic for an M N contingency table was used to compare the overall distribution of intimin subtypes between strains isolated from healthy carriers and those from patients. RESULTS Prevalence of DEC in healthy individuals: A total of 2,230 specimens from healthy individuals were examined between August 2006 and November DEC strains were isolated from 109 stool samples (Table. EPEC, EAST1EC, and DAEC were prevalent and did not show significant differences in isolation rates from those among diarrheal patients in our previous study. By contrast, no STEC, ETEC, or EIEC strains were isolated from healthy individuals in this study, and there were significantly fewer EAggEC organisms. The cumulative isolation rates of EPEC, DAEC and EAST1EC among healthy individuals in this study were assessed by season. Isolation rates of EPEC showed significant seasonal variation (Table 2; Kruskal-Wallis, P < Characteristics of DEC in healthy individuals: Varieties of DEC serogroups were isolated through this surveillance, as shown in Table 3. Possession of eae, which plays an important role in intimate adhesion to intestinal epithelial cells and in producing attaching and effacing lesions, was thought to be the primary criterion for defining isolates as EPEC in this investigation. Consequently, 18 strains for which sero- Table 1. Prevalence of each subpopulation of diarrheagenic E. coli Healthy adult Diarrheal adult patient 2) Total no. of specimens 2,230 (%) 126 (%) Age (Mean ± SD, Median) 38.5 ± 15.7, ± 14.7, 32 EPEC 32 (1.4) 0 STEC 0* 1 (0.8) ETEC 0*** 4 (3.2) EIEC 0 0 EAggEC 5 (0.2)*** 4 (3.2) Afa(+)DAEC 19 (0.9) 2 (1.6) EAST1EC 53 (2.4) 6 (4.8) Subtotal 109 (4.9) 17 (13.5) Salmonella spp. 1 (0.04)*** 10 (7.8) : P values were determined by chi-square tests with Yates continuity correction. * and *** mean significantly fewer among healthy individuals compared to patients at P < 0.05 and 0.001, respectively. 2) : The data of adult patients were extracted from our previous study (13). Table 2. Cumulative number of strains of each subgroup of diarrheagenic E. coli isolated during this study by season Spring Summer Autumn Winter No. of specimens 330 (%) 768 (%) 242 (%) 890 (%) EPEC 1 (0.3) 21 (2.7) 5 (2. 5 (0.6) EAST1EC 7 (2. 26 (3.4) 5 (2. 15 (1.7) DAEC 0 4 (0.5) 4 (1.7) 11 (1.2) : Seasonal change in the isolation rates of EPEC was statistically significant (Kruskal-Wallis, P <

3 Table 3. O antigen serotype and number of strains isolated throughout this study STEC EPEC EAggEC DAEC EAST1EC stx1/stx2 eae aggr/asta afab asta O103 ( O15 ( O78 (4) O1 (2) O1 (2) O27 ( O126 ( O25 (3) O8 (2) O55 ( O74 ( O15 (2) O74 (3) O86a (3) O18 (5) O103 ( O151 ( O20 ( O124 ( UT (9) 2) O25 (5) O127a ( O28ac ( O128 ( O74 (2) O145 ( O86a ( O166 ( O121 ( O167 ( O166 ( O168 ( O169 ( UT (18) 3) UT (29) Subtotal ( (32) (5) (19) (53) : Numbers in parentheses indicate number of strains. UT; could not be serotyped with commercial antisera. 2) : One strain possessed both afab and asta. 3) : One strain possessed both eae and asta. Table 4. Subtypes of eae and serotypes of EPEC isolated from healthy carriers and patients in Osaka, Japan Isolates from Isolates from Type of eae healthy carriers patients 2) O antigen serogroup (n = 32) (n = 23) UT 3) ) 1 2 6** 26, 119, 153, 167, 168, UT / 2B , UT / / 2O , 145, UT 1 4 8* 15, 55, 74, 157, UT / 2 8* 3 27, 74, 111, 124, 153, UT , UT R/ , UT , 119, UT R/ UT B a B UT 4 1 UT Total 5) : P values were determined by Fisher s exact probability test. * and ** mean prevalence at P = and <0.05, respectively. 2) : Strains of patients were all from non-adults (13) since no EPEC was isolated from adult patients. 3) : Untypable. 4) : EPEC indicated by bold underlined O antigen number were reported to possess the eae subtypes (23,27-29). 5) : Six strains from healthy carriers and one from a patient showed positive reactions with two sets of primers simultaneously. types could not be determined with the set of commercially available antisera were also included as EPEC, along with organisms of well-established serotypes such as O55 and O127. Only one strain possessing eae of type 1 and an untypable O antigen showed positive reactions with PCR primers for bfpa. A total of 32 strains were identified as EPEC (32/2,230, 1.4%), although 9 of these did not show localized adhesion in adhesion tests. Fig. 1. PFGE patterns of enteroaggregative E. coli O78 strains. Lanes M, Marker; 1, 5 and 9, strains SK34; 2, 6 and 10, strain SK37; 3, 7 and 11, strain SK50; 4, 8 and 12, strain SK60. DNA on lanes 1-4, 5-8 and 9-12 was digested by XbaI, BlnI and SfiI, respectively. Subtyping (23,27-29) of the eae assigned the strains isolated from carriers and from patients into 14 and 9 groups, respectively (Table 4): strains of patients were all from nonadults (13) since no EPEC was isolated from adult patients. Five EPEC strains did not produce amplicons with the typing primers of Blanco et al. (23) used in this study, and were designated as UT (untypable). No statistically significant difference was recognized in the overall distribution of intimin subtypes between healthy carriers and patients (chi-square M N method, P = 0.23). However, both subtypes of 1 and 1 were prevalent (Fisher s test, P = and 0.050, respectively) and were present among 61% of EPEC strains from patients, while among strains isolated from healthy carriers, / 2 was the most prevalent (24%; Fisher s test, P = 0.050), followed by R/ 2B (12%), 1 (12%), / / 2O (9%) and 1 (9%). Six EPEC strains from healthy carriers showed positive reactions with two sets of typing primers: one O15 strain was 1- and 1-positive; one O74 was 1- and / / 2O-positive; one O168 was 1- and R/ 2-positive; one O128 and another O-untypable strain were 1- and -positive; and one O-untypable strain was / 2- and R/ 2-positive. One O-untypable strain isolated from a patient was 1- and -positive. Nineteen strains of DAEC were isolated (19/2,230, 0.9%); three strains each of O86a and O25, two strains of O1, and one strain each of O74 and O151 were assigned into DAEC with another nine O-untypable strains because of their positive reactions with primers for afab in our real-time PCR system (Table 5). Three strains were not reconfirmed as being afa-positive when afac primers were used according to the scheme of Le Bouguénec et al. (24). These strains were assigned as putative new afa-positive strains because of their clear diffuse adhesion on HEp-2 cells. Two strains did not show clear diffuse adhesion on HEp-2 cells, despite the positive results in PCR. Another two strains were Afa/Dr. Afa/ Dr + DAEC strains isolated from healthy carriers induced less IL-8 secretion (<200 pg/ml) than strains isolated from diarrheal patients. In all these isolates except for two, afae was not assigned into subtypes by the typing primers of Le Bouguénec et al. (24) and the gene was named afaex temporarily. One O126 strain and four O78 strains of EAggEC were 320

4 Table 5. Serotypes, motility and afa subtypes of DAEC isolated in Osaka, Japan, and their induction of IL-8 secretion in Caco-2 cells Strain Origin Serogroup afa subtype Adhesion Motility IL-8 induction in Caco-2 cells 164 Carrier O86a AfaEX DA + 41 ± Carrier UT AfaEX DA + 19 ± Carrier O74 AfaEX DA + 18 ± Carrier UT AfaEX ± Carrier O1 AfaEX DA + 21 ± Carrier O1 New-Afa DA + 82 ± Carrier UT New-Afa DA ± Carrier UT Afa/Dr DA + 19 ± Carrier UT AfaEX DA ± Carrier O86a AfaEX DA ± 59 ± Carrier O25 AfaEX DA ± 11 ± Carrier O25 Afa/Dr DA + 43 ± Carrier O151 AfaEX ± Carrier UT AfaEX DA 15 ± Carrier UT AfaEX DA 16 ± Carrier UT New-Afa DA + 31 ± Carrier O25 AfaEX DA ± 15 ± Carrier UT AfaE2 DA + NE 1472 Carrier O86a AfaE3 DA + 41 ± 3 V546 Patient UT EAggEC AA ± 2 V561 Patient UT AfaE1 DA ± 20 V64 Patient O1 AfaEX DA ± 10 V679 Patient UT AfaE3 DA 16 ± 0 DH5 - ± 43 ± 1-19 ± 2 DA, diffuse adhesion; AA, aggregative adhesion. isolated (5/2,230, 0.2%). The O78 strains were isolated from samples from individuals working at the same location. Although infection in this group was strongly suggested, their PFGE patterns showed differences (Fig.. Infection was not suspected in the remaining case. A total of 61 strains showed positive reactions on real-time PCR for asta. Three DAEC and all five EAggEC cases possessed asta with virotype-specific genes. However, the remainder (53 strains of 13 serogroups) possessed no other virulence genes except asta (Table 3), and these were identified as EAST1EC (53/2,230, 2.4%). The serogroup O169 and O166 strains did not include O169:H41 and O166:H15, which have caused outbreaks in the past (8,30,3. Salmonella: Salmonella Manhattan was isolated from only one healthy individual throughout this study, although Salmonella was detected in 10 stools (7.8%) of diarrheal adult patients in a previous investigation (13). Isolation rates of Salmonella were significantly higher in the patient group than among healthy individuals (Table. DISCUSSION When compared with Salmonella, DEC is isolated unexpectedly often from healthy individuals. Pathogens are typically isolated from patients more frequently than from healthy individuals. The present isolation rates were thus compared with our previous investigation of patients in Osaka City (13), although these investigations were performed separately at different periods. STEC, ETEC and EAggEC organisms were significantly less common among healthy individuals than in diarrheal adult patients (Table. EAggEC is considered to be a DEC, based on accumulated epidemiological data from case-control studies and outbreaks due to this organism (6,32-37). Clinicians regard EAggEC as a causative agent, in addition to the well-recognized STEC and ETEC, when isolated from sporadic diarrheal adult patients. By contrast, isolation rates of EPEC, DAEC and EAST1EC did not show significant differences between healthy individuals and diarrheal patients (Table. Germani et al. reported that afa-possessing DAEC strains isolated from 2- to 6-year-old children were epidemiologically important (38). We found that the subgroup of DAEC possessing Afa genes and/or inducing high levels of IL-8 was significantly prevalent among patients age 1 to 4 years (16). Thus, DAEC can be principally recognized as a pediatric enteric pathogen, similarly to EPEC. There were few fecal specimens from children in this study, as healthy children do not typically undergo fecal examination, in contrast to food handlers, and we were unable to obtain a sufficient number of specimens to determine whether DAEC and EPEC are significantly more prevalent among patients than healthy children. The present study suggests that clinical microbiologists may not be able to regard these categories of DEC as causative agents, even if the organisms were isolated from sporadic adult diarrheal patients, although the prevalence of EPEC in adults may be a source of infection to children. We previously reported that diffusive adhesiveness itself is unlikely to be sufficient for afa-positive DAEC to induce IL-8, and that additional stimulation by flagella may be required to cause high levels of chemokine induction (15). All afa-positive motile strains isolated from patients induced IL-8 to a comparable degree as EAggEC, while non-motile DAEC strains did not. However, the DAEC strains isolated from healthy individuals in this study induced IL-8 secretion to a lesser degree than strains from patients, despite their good motility (Table 5). Pathogenic mechanisms other than diffuse adhesion and motility thus appear to be necessary for high IL-8 induction. The ability to induce IL-8 secretion from 321

5 intestinal epithelial cells may thus be a criterion to assess the diarrheagenicity of DAEC. Although EPEC has been recognized as a DEC (17,18), we were unable to find any significant differences in isolation rates between healthy carriers and patients. As described the excellent review by Chen and Frankel (39), intimin, the product of eae, has various subtypes, and there appears to be a direct correlation between intimin type and specific EPEC clones. Cell-binding activity is localized to the C-terminal 280 amino acids (Int280), and Int280 has two immunoglobulinlike domains and a C-type lectin domain, which possibly binds to 1-integrins. As the C-terminal region that binds to receptors on epithelial cells is highly divergent and likely contributes to tissue tropism, we surmised that intimin type may discriminate EPEC strains isolated from patients from those of healthy individuals. The types of 1 and 1 were very prevalent among strains isolated from patients, as Jenkins et al. reported (40). The presence of these eae types is an important criterion to predict the diarrheagenicity of atypical EPEC strains isolated from sporadic patients at clinical laboratories. Reports suggesting the diarrheagenicity of EAST1 genepossessing E. coli have gradually increased (41-46). EAST1EC O166:H15 was detected as the sole etiological agent among adult patients in outbreaks in Japan (7,8). Although we believe that EAST1EC may cause diarrheal illness in adults based on this outbreak data, the higher isolation rate (4.8%) in patients did not reach the level of statistical significance when compared to that in healthy individuals (2.4%). As with ETEC, some organisms possessing genes for enterotoxins may not be pathogenic in humans. Only a subset of EAST1EC that possesses a set of genes for particular colonization factors with the EAST1 gene is diarrheagenic in humans. The varieties of sequences of asta and/or numbers of gene copies harbored in bacterial strains are also supposed to regulate their diarrheagenicity (47,48). EAST1EC isolates from healthy carriers and diarrheal patients should be scrutinized from this molecular viewpoint in future studies. The cumulative isolation rates of EPEC, DAEC and EAST1EC among healthy individuals during this study were assessed by season (Table 2). EPEC and EAST1EC showed an increased isolation rate in summer among healthy individuals, which is similar to the trend we observed in diarrheal specimens (13). On the other hand, DAEC is likely to be prevalent in winter (16); we also observed this phenomenon in diarrheal specimens. These findings suggest that DAEC is not foodborne. As discussed above, clinicians should resist regarding DAEC as a foodborne pathogen in adult diarrheal patients at present. 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