| Escherichia coli is an innocuous resident of the gastrointestinal tract and ubiquitous in the environment. The E. coli which can cause diarrhea is known as diarrheagenic E. coli (DEC). The diarrheagenic E. coli can be divided into six pathotypes:enterohemorrhagic E. coli (EHEC) (or Shiga toxin-producing E. coli [STEC]), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC). The children’s watery diarrhea caused by DAEC have been studied in both developed and developing countries, such as the United States, France, Bangladesh, Peru, Brazil and Ghana. And DAEC strains causing bloody diarrhea has been reported recently, which raised more concern on it.DAEC is identified by its characteristic adherence patterns on HEp-2 cells, Afa/Dr adhesins interact with decay-accelerating factor or carcinoembryonic antigen in the polarized epithelial cells, arouse the rearrangement of cytoskeletal proteins and the elongation of the microvilli. It can not to be distinguish from other commensal or diarrheagenic E. coli by culture or biochemical identification. Most DAEC strains harbor adhesins of the Afa/Dr family. In this study, a multiplex PCR system was establishedbased on four adhesion-associated genes afaB, afaC. afaD and ciaaE to identify DAEC.A total of 2876 fecal samples from diarrheal patients and health carriers was collected from 2011 to 2015 in Henan province, Zigong city of Sichuan province, Guizhou province, Anhui province and Longgang of Shenzhen city.151 DAEC strains were identified, giving a culture positive rate of 5.42%.6.26%. and 4.77% of fecal samples from the diarrheal patients and the healthy carriers were DAEC culture positive. DAEC strains were more common among diarrheal under five.Sixty-three different O:H serotypes were identified. O127:H18 (15,9.93%), O86:H18 (12,7.95%), O1:H6 (11,7.28%), O1:H15 (9,5.96%), O12:H5 (7,4.64%), O2:H30 (7, 4.64%), O15:H15 (5,3.31%) and O16:H5 (5,3.31%) were dominant. The remaining 55 serotypes contained strains less than 5 each. Fourty different O:H serotypes were identified in the 76 DAEC isolates from diarrheal patients and 36 were identified in the 75 DAEC isolates from healthy carriers.Afa/Dr family were detected using specific primers for the adhesin-encoded genes afaE. Of the 151 DAEC strains,61 were afaEA positive, which was the most frequent gene.34.21%(26/76) and 46.6%(35/75) were identified in the isolates from diarrheal patients and healthy carriers. afaE-X (19.74%), afaEA+afaE-5 (19.74%) and afaE-5 (13.16%) respectively in the 76 diarrheal isolates. afaE-X (17.33%), afaEA+afaE-5 (13.33%) and afaE-5 (2.67%) were positive in the 75 healthy isolates. Three strains were positive in the diarrheal isolates, and other afaEldaaE types were all less than seven strains.Thirteen virullent genes, i.e. aggA, agg3A, agg4A, aafA, sfaD, escV, nleE, pet, espP, shET2, subA, cdt-II, hlyA were all absent. Thirteen virullent genes including escJ (11/76), escN (24/76), nleB (10/76), irp2 (75/76),fyuA (66/76), sat (17/76), pic (3/76), cnfl (35/76), cnf2 (1/76), shETl (14/76), era (35/76), cdt-1 (15/76) and astA (61/76) were identified in the 76 diarrheal isolates and 11 virullent genes including escN (49/75), nleB (11/75), irp-2 (73/75), jyuA (74/75), sat (35/75), pic (8/75), shET1 (3/75), cdt-1 (19/75), astA (64/75), ent (45/75) and cnfl (35/75) in the 75 healthy isolates. Other four genes including IpfA, papC, fimH and hdaA were also identified,82.9%,68.4%,73.7%,44.7% were positive in the 76 diarrheal isolates and 82.7%,57.3%,57.3%,50.7% in the healthy isolates.The isolates were tested for susceptibility to a panel of 21 antimicrobials (ampicillin, amoxicillin-clavulanic acid, ampicillin-sulbactam, cefuroxime, cefotaxime, ceftriaxone, cefepime, cefoxitin, levofloxacin, norfloxacin, ciprofloxacin, gentamycin, kanamycin, streptomycin, imipenem, meropenem, trimethoprim-sulphamethoxazole. a ztreonam. chloramphenicol. tetracycline and nitrofurantoin) using the Kirby-Bauer disc-diffusion method. All isolates were sensitive to meropenem. Of the 151 isolates. 91.39%,75.5% and 73.51% were resistant to ampicillin. trimethoprim-sulphamethox azoic and tetracycline respectively; less than 50% were resistant to cefuroxime. cefotaxime, ceftriaxone, gentamycin and streptomycin; less than 10% were resistant to imipenem, nitrofurantoin, cefoxitin and kanamycin.100% and 77.33% were multiple-resistant strain in the 76 diarrheal isolates and in the 75 isolates.151 isolates were analysed by MLST and found to have 28 different sequence types (ST), of which 7 were novel types. ST38 (50,33.11%), ST10 (26,17.22%), ST131 (12, 7.95%), ST 1177 (10,6.62%) and ST648 (9,5.96%) were the most prevalent genotypes. Other 22 genotypes were all less than 5 strains. The seven novel STs were identified including ST5146, ST5147, ST5148, ST5150, ST5151, ST5152 and ST5153.This study developed a PCR system to identify DAEC and for the first time, DAEC strains were isolated from both diarrheal patients and healthy carriers fecal samples from 2011 to 2015 in China. The serotypes, virulence factors, antimicrobial resistance and sequence types were characterized. DAEC isolates were multidrug resistant. The serotypes, virulence gene profiles and STs were highly heterogenous. Serotypes O86:H18 and O127:H18 strains were isolated from China and other countries, but O1:H6 strains were popular only in our country. Thus emphasizing the necessary to detect and monitor the epidemiologic and molecular characteristics of DAEC in the diarrhea monitoring system, which will contribute to further understanding of DAEC in infectious diarrhea and how to deal with potential outbreaks caused by DAEC infection. |
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