Comparison between O Serotyping Method and Multiplex Real-Time PCR To Identify DiarrheagenicEscherichia coliin Taiwan

Abstract

To compare the diarrheagenic Escherichia coli (DEC) identifications obtained between traditional O serotyping and modern virulence gene detection assays, we developed a multiplex real-time PCR assay by detecting six specific virulence genes for enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). Among 261 clinical diarrheal stool samples, a total of 137 suspected DEC (sDEC) isolates were identified by the use of commercially available antisera. The most prevalent serogroups were O1 (12/137; 8.7%), O25 (9/137; 6.5%), and O44 (9/137; 6.5%). The specific virulence genes for the 137 sDEC isolates were analyzed by the multiplex real-time PCR assay. Fifteen (10.9%) of 137 isolates were confirmed to be true DEC strains, indicating that the serotypic markers did not correlate with the specific virulence genes. ETEC (66.7%) was the most prevalent, followed by EIEC (20%) and EPEC (13.3%). No EHEC strains were identified in the specimens. Four novel serotypes were found in the study: two in EPEC strains (O111:H9 and O63:H6) and two in EIEC strains (O63:H9 and O169:H9). In conclusion, the real-time PCR assay considerably reduces the high false-positive rate from the use of serotyping alone, and thus, it is suggested that serogrouping-based methods are inadequate for the identification of DEC isolates, although they are useful for the identification of a limited number of serogroups. In addition, ETEC, EPEC, and EIEC strains were present in 5. 7% (15/261) of the diarrheal patients in northern Taiwan in 2006.Escherichia coli is a predominant member of the human intestinal flora. Some strains are rendered pathogenic by their ability to possess specific virulence factors, such as enterotoxin or adherent fimbriae, that are genetically encoded by plasmid, chromosome, and bacteriophage DNA (2, 14). These diarrheagenic E. coli (DEC) strains usually play important roles as the causes of endemic and epidemic human diseases, such as severe diarrhea, food poisoning, and similar outbreaks worldwide (9, 15). Infections with these kinds of pathogens have therefore been of increasing concern in the clinical diagnosis of diarrheal disease in recent years. These virulent organisms can be classified into five major categories on the basis of the nature of their infection and pathogenic mechanisms: they are enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) (14, 25). However, the current rates of infections by these important enteric pathogens are probably largely underestimated due to the limitations of existing clinical diagnostic methods to be able to distinguish them from normal nonpathogenic flora. In order to achieve the goal of epidemic prevention and control of DEC in Taiwan, we need a more reliable procedure to identify and categorize DEC isolates so that more reliable studies of DEC incidence rates can be conducted in the future.Phenotypic...

Foreign Travel Is a Major Risk Factor for Colonization with Escherichia coli Producing CTX-M-Type Extended-Spectrum β-Lactamases: a Prospective Study with Swedish Volunteers

“…Due to the frequent episodes of gastroenteritis among travelers who acquired ESBL-producing E. coli strains, six specific virulence genes associated with enteropathogenic, enterohemorrhagic, enterotoxigenic, enteroinvasive (33), and enteroaggregative E. coli (28) were explored. The methods were modified to fit the GeneAmp PCR 9700 system (Applied Biosystems, Inc., Foster City, CA).…”

Investigation of Pathogenic Escherichia coli Strains in Patients with Diarrhea

“…Patojenik E.coli suşlarının tanımlanmasında serolojik ve moleküler yöntemlerin karşılaş-tırıldığı çalışmalarda, diyarejenik izolatların tespitinde serolojik testlerin yanlış pozitif sonuç verdiği belirtilmektedir 9,11 . Örneğin Taiwan'da yapılan bir çalışmada, 261 E.coli kökeninin 137'si serogruplandırılmış, bunlardan sadece 15 (%10.9)'i PCR ile patojen köken olarak tespit edilmiştir 9 .…”

“…Örneğin Taiwan'da yapılan bir çalışmada, 261 E.coli kökeninin 137'si serogruplandırılmış, bunlardan sadece 15 (%10.9)'i PCR ile patojen köken olarak tespit edilmiştir 9 . Japonya'da yapılmış bir diğer çalışmada da, lam aglütinasyonu ile ishale neden olduğu tespit edilen E.coli izolatlarının %17.…”

“…Chen ve arkadaşları 15 ise yaptıkları çalışmada, diyarejenik E.coli'nin tanısında klasik PCR ile karşılaştırıldığında M-PCR'nin özgüllüğünün %100, duyarlılığının %99 olduğunu bildirmişlerdir. Flora bakterileri ile patojen E.coli'lerin ayrılmasındaki güç-lükler nedeniyle, PCR çalışmalarında en azından dört laktoz pozitif ve iki laktoz negatif koloninin çalışmaya alınması veya en az 10 koloni içeren bir sürüntünün petri kutusundan seçilmesinin patojen bakterilerin yakalanma olasılığını artıracağı söylenebilir 9,14 . İshal yapan E.coli'lerin tanımlanmasında daha önce yapılmış çalışmalar incelendiğinde, bu konuda halen bir standardın olmadığı görülmektedir.…”

“…Rutin testlerle Salmonella, Shigella, rotavirus ve adenovirus pozitifliği saptanan örnekler çalışma dışı bırakıldı. Dışkı örneklerinden izole edilen E.coli suşları konvansiyonel yöntemlerle tanımlandı; tanımlamada güçlük çekilen kolonilerde VITEK-2 cihazı ve VITEK-2 GN tanımlama kartı (BioMérieux, Fransa) kullanıldı 9 . Serogruplandırma için izolatlar nutrient agar ve beyin-kalp infüzyon buyyonu (Brainheart infusion broth) besiyerlerinde üretildi.…”