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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 30  |  Issue : 3  |  Page : 279-284
 

Detection of diarrheagenic Escherichia coli by multiplex PCR


1 Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore IC, India
2 Department of Microbiology, Manipal University, Manipal, India

Date of Submission20-Feb-2012
Date of Acceptance15-Jul-2012
Date of Web Publication8-Aug-2012

Correspondence Address:
A Hegde
Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore IC
India
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DOI: 10.4103/0255-0857.99485

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 ~ Abstract 

Background: Diarrheagenic E.coli (DEC) are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus) forEnteropathogenic E.coli (EPEC), hlyA for Enterohemorrhagic E.coli (EHEC), elt and stla for Enterotoxigenic E.coli (ETEC), CVD432 for Enteroaggregative E.coli (EAEC) and ial for Enteroinvasive E.coli (EIEC). Results: In 200 children with diarrhea 52 (26%) DEC infections were found. Among 100 controls 8 (8%) DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%)and controls (5,5%), followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5%) and 3 (1.5%) of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.


Keywords: Diarrhea, E. coli, Multiplex PCR, Virulence


How to cite this article:
Hegde A, Ballal M, Shenoy S. Detection of diarrheagenic Escherichia coli by multiplex PCR. Indian J Med Microbiol 2012;30:279-84

How to cite this URL:
Hegde A, Ballal M, Shenoy S. Detection of diarrheagenic Escherichia coli by multiplex PCR. Indian J Med Microbiol [serial online] 2012 [cited 2014 Dec 22];30:279-84. Available from: http://www.ijmm.org/text.asp?2012/30/3/279/99485



 ~ Introduction Top


Diarrhea can be caused by a wide range of viruses, bacteria and parasites. Among the bacterial pathogens E coli is an important etiologic agent of childhood diarrhea and represents a major public health problem in developing countries. [ 1] Diarrheagenic strains of E.coli can be divided into five main categories on the basis of distinct epidemiological and clinical features and specific virulence determinants. [2] The five categories of diarrheagenic E.coli (DEC) are Enteropathogenic E.coli (EPEC), Enterohemorrhagic E.coli (EHEC), Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC) and Enteroinvasive E.coli (EIEC). Identification of DEC strains requires that these organisms be differentiated from nonpathogenic members of the normal flora. It is not sufficient to identify a strain as diarrheagenic based on O serotyping, because it does not correlate, in most cases, with the presence of virulence factors. [2] Thus, for identification of diarrheagenic E. coli strains, factors that determine the virulence of these organisms should be identified. With the advent of PCR, it has become possible to detect pathogenic genes in bacterial isolates, allowing the rapid diagnosis of diarrheagenic E. coli. PCR methods using single primer sets have been reported elsewhere [3],[4] but screening of bacterial isolates requires a large number of individual PCRs if single primer sets are used in separate reactions. To reduce the number of tests needed for diagnosis of diarrheagenic E. coli, several multiplex PCR systems have been reported previously. [5],[6] However, usually more than one multiplex PCR is required for identification of a diarrheagenic E. coli strain. Pass et al. [7] reported a multiplex PCR to detect 11 virulence genes, but it has not been fully evaluated against a large panel of isolates. This study attempted to use the two multiplex PCR assays for identification and studying the prevalence of DEC in young children and infants with acute diarrhea.


 ~ Materials and Methods Top


Clinical specimens

During the period from April 2009and August 2011, 300 stool samples from children who were from 0-60 months of age were investigated in Mangalore, Karnataka, India to determine the prevalence of diarrheagenic  Escherichia More Details coli as the etiological agents of diarrhea. This included 200 children with and 100 children without diarrhea. Cases and controls were selected from patients attending the outpatient clinic of three different hospitals after receiving permission from institutional ethical committee.The sample size was determined with 85% confidence level and 80% power according to similar studies conducted earlier. [1],[6] Children were enrolled in the study if they had diarrhea characterized by the occurrence of three or more, loose watery stool or at least one bloody loose stool in a 24 hour period. Control subjects were healthy children with no history of diarrhea for at least one month. Children whose diarrhea could be attributed to classic pathogens like  Salmonella More Details or Shigella or gross infestation with parasites were excluded from the study. Neither patients nor controls had been treated with antibiotics in the week preceding sampling. The stool samples were cultured on Mac Conkey agar (HiMedia Laboratories Pvt. Ltd., Mumbai, India). As a rule about 5 lactose fermenting colonies presumed to be E.coli by colony morphology, were selected and submitted to biochemical tests. Biochemically confirmed E.coli isolates were stored at - 80°C in Trypticase soy broth supplemented with 20% glycerol for further procedures.

Bacterial strains

The reference strains were E.coliATCC® 43887 positive for eaeA and bfpA genes of EPEC; E.coli ATCC® 35401 positive fo relt and stla genes of ETEC; E.coli ATCC® 43893 positive for ial gene of EIEC. Local isolates of E.coli positive for CVD432 gene to detect EAEC and hly gene to detect EHEC were used for standardization of the multiplex PCR assays. E.coli isolated from children with and without diarrhea was subjected to the multiplex PCR assays described below to detect the presence of DEC.

Multiplex PCR assays

The multiplex PCR assays were standardized for the detection of five types of DEC.The reference strains were cultured on Mac Conkey agar. A sweep of about five E.coli like colonies was used for PCR. The DNA was isolated from colonies by suspending the colonies in 50μl of deionized water. The suspension was boiled for 10 min at 95°C and centrifuged at 10,000 × g for 10 min. The supernatant was then used as the DNA template. The DNA templates were subjected to multiplex PCR with specific primers [Table 1], for the detection of the following virulence markers: eaeA for the structural gene of intimin of EPEC and EHEC, bfpA for the structural gene of the bundle forming pilus of EPEC, hlyA for the plasmid encoded enterohemolysin of EHEC, elt and stla for the enterotoxins of ETEC, ial for the invasion associated locus of the invasion plasmid found in EIEC, CVD432 for the nucleotide sequence of the EcoR1-Pst DNA fragment of EAEC.The primers were selected on the basis of similar studies done earlier. [2],[8],[9] All primers were obtained from Sigma-Aldrich Co., St. Louis, MO, USA. The specificity of each primer was confirmed by monoplex PCR. The multiplex PCR assays were tested with several PCR cycling protocols.
Table 1: PCR Primers used in the multiplex PCR assays for the detection of virulence genes of Diarrheagenic E.coli

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The minimum criteria for determination of DEC were defined as follows: the presence of bfpA and eae for typical EPEC (but the presence of only eaeA for atypical EPEC), the presence of elt and/or stla for ETEC, the presence of ial for EIEC, the presence of hlyA for EHEC (the additional presence of eaeA confirms the detection of a typical EHEC isolate) and the presence of CVD432 for EAEC. A maximum of four pairs of primers could be used in a single multiplex PCR, therefore two multiplex PCRs were standardized for the identification of all DEC.

Multiplex PCR 1 was standardized for the detection of ETEC, EAEC and EHEC

Multiplex PCR 2 was standardized for the detection of EPEC and EIEC

Multiplex PCR assay 1

The reaction mixture containing optimized protocol was carried out with a 50 μl mixture containing 10mMTris-HCL (pH 8.3), 50mM KCL, 2.0 mM MgCl 2, a 2mM concentration of each deoxynucleoside triphosphate (Fermentas Inc, Maryland, USA) , 2 U 0f Hot start Taq DNA polymerase (Fermentas Inc., Maryland, USA), 5 μl of the DNA template. 0.5 μM of each of the primers (Sigma-Aldrich Co., St. Louis, MO, USA) i.e. elt and stlA for ETEC isolates, CVD432 for EAEC isolates and hlyA and eaeA for EHEC isolates. The cycling conditions in Eppendorf's Mastercycler pro (Eppendorf India Limited Chennai, India)were as follows: 95°C for 1 min for one cycle followed by 35 cycles of 94°C for 1 min, 55°C for I min, 72°C for 1min and72°C for 5 min.

Multiplex PCR assay 2

The protocol used was as explained above for Multiplex PCR 1except that the primers used were eaeA and bfpA for EPEC isolates, and ial for EIEC isolates. The thermocycling conditions were as follows: 95°C for 1 min for one cycle followed by 35 cycles of 94°C for 1 min, 57°C for I min, 72°C for 1min and 72°C for 5 min.

The PCR products (10 μl) were analyzed by gel electrophoresis with 2.0% (W/V) agarose gels (HiMedia Laboratories Pvt. Ltd., Mumbai, India) in Trisborate- buffer (Fermentas Inc., Maryland, USA). The DNA bands were visualized and photographed under UV light after staining the gel with ethidium bromide (Fermentas Inc., Maryland, USA).

Sensitivity of multiplex PCR assays

To determine the detection limit of the multiplex PCR assays stool samples negative for diarrheagenic E.coli were spiked with a phosphate buffered saline suspension of reference strains of EAEC and EIEC in serial 10 fold dilutions to give 10° to 10 8 CFU/ml. Each serial dilution of the spiked stool sample was spread onto a Mac Conkey agar and incubated at 37°C. Multiplex PCR was performed with each dilution of spiked stool sample. The sensitivity of the assay was defined as the lowest concentration of DEC that yielded positive results for each dilution. The sensitivity of detection was 10 3 CFU per assay for all target genes. The PCR assays were also used to detect DEC directly from fecal samples spiked with different concentrations of the reference strains of EIEC and EAEC. The reference strains were suspended in phosphate buffered saline at different concentrations. The diluted suspensions were used to spike 180 mg of stool specimen. The DNA was isolated from the spiked stool specimens by using QIAamp stool mini Kit (QIAGEN India Pvt. Ltd, New Delhi, India) and subjected to multiplex PCR assays. The detection limit by the multiplex PCR assays for both the reference strains was 10 8 CFU.

Specificity of multiplex PCR assays

The specificity of the two multiplex PCR assays was determined by using standard reference and ATCC strains of DEC. The strains were subjected to both multiplex PCRs, and the results were compared with those obtained by monoplex PCR assays. Both multiplex PCR assays showed 100% specificity in identifying the reference strains as shown in [Figure 1]. Non-specific bands were not visualized.
Figure 1: Multiplex PCR of reference strains and clinical samples. Lane 1: DNA molecular size marker (100bp ladder), Lane 2: Typical EPEC (eaeA amplicon size 229 and bfpA amplicon size 450), Lane 3: ETEC (elt amplicon size 322 and Stla amplicon size 170, Lane 4: EAEC (CVD432 amplicon size 630bp), Lane 5: EHEC (hlyA amplicon size 534bp), Lane 6: EIEC (ial amplicon size 320), Lane 7 and 8: clinical isolates of EAEC (CVD432) and atypical EPEC (eae)

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Detection and identification of DEC strains from stool samples

The Multiplex PCR assays were then used to detect DEC in the stool samples collected from children with and without diarrhea. Template DNA from the stool samples was extracted as described above. The Multiplex PCR assays were also used for detecting DEC directly from the stool samples which were positive for DEC. DNA was extracted from stool samples using QIAamp DNA Stool Mini Kit (QIAGEN India Pvt. Ltd, New Delhi, India)and subjected to multiplex PCR for detecting the presence of DEC.

Detection of two DEC strains in a spiked stool sample

Stool samples spiked with each pair of the two different categories of DEC i.e. EAEC and EPEC were tested by the multiplex PCR. The multiplex PCR could correctly detect the two DEC strains in a spiked stool sample.

Statistical analysis

The chi-square test and Fisher exact test was used to determine the statistical significance of the data. A P value of <0.05 was considered significant.


 ~ Results Top


Multiplex PCR for reference strains

In this study in order to detect five different categories of DEC simultaneously, a mixture of seven primer pairs were used in two different multiplex PCR assays. Both multiplex PCR assays showed 100% specificity in identifying the reference strains. Nonspecific bands were not visualized. [Figure 1] and [Figure 2] show the PCR products of the two multiplex PCR assays derived from pure cultures of reference strains of EAEC, EIEC, EPEC, EHEC, ETEC and clinical isolates.
Figure 2: Multiplex PCR of reference strain of EPEC and clinical samples. Lane 1: DNA molecular size marker (100bp ladder), Lane 2: ATCC 43887 Typical EPEC (eaeA amplicon size 229 and bfpA amplicon size 450), Lane 3: Negative control ATCC E.coli 25922, Lane 4 and Lane 9: Clinical isolates of EAEC (CVD432 amplicon size 630bp), Lane 5and Lane 11: Negative clinical samples, Lane 6 and Lane 7: Atypical isolates of EPEC (eaeA amplicon size 229), Lane 8: Clinical isolate of EIEC (ial amplicon size 320), Lane 10: Clinical isolate of ETEC (elt amplicon size 322)

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Sensitivity of multiplex PCR assay

Sensitivity of the multiplex PCR assays was determined from the number of DEC cells (in CFU/ml) spiked into each milliliter of stool sample that could be detected by these assays. The limit of detection of DEC was approximately 10 3 CFU/ml of the stool suspension. [Figure 3] shows the limit of detection of EIEC by multiplex PCR assay 1.
Figure 3: Detection limit of EIEC (ial amplicon size 320) from spiked stool samples. Lane 1: DNA molecular size marker (100bp ladder), Lane 2: Non-spiked stool sample, Lane 3 to 7: dilutions from 102 to 107

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Detection and Identification of DEC Strains From Stool Sample

A total of 52 (26%) DEC was isolated from 200 stool samples from children with diarrhea. DEC was detected in 16 (16%) out of 100 stool samples from children without diarrhea [Table 2]. The prevalence of DEC in both groups was significantly different (P <0.05). The prevalence was greatest for EAEC both among children with and without diarrhea.The PCR assays detected 26 (13%) EAEC isolates, 16 (8%) EPEC isolates, 7 (3.5%) ETEC isolates and 3 (1.5%) EIEC isolates in children with diarrhea. The prevalence of EAEC and EPEC in the children without diarrhea were 5 (5%) and 3 (3%) isolates respectively. There was a significant difference (P < 0.05) in the prevalence of EAEC between children with and without diarrhea. ETEC and EHEC were not isolated from the stool samples of children without diarrhea. EHEC was not isolated from any of the children tested [Table 2]. Of the ETEC strains isolated 5 (71.4%) produced heat labile toxin only and 2 (28.6%) produced heat labile and heat stable toxin. 12 of the 16 EPEC isolates from children with diarrhea and all EPEC isolates from children without diarrhea were atypical EPEC (positive for attaching and effacing gene eaeA only). The four remaining isolates of EPEC from children with diarrhea were typical EPEC isolates (positive for attaching and effacing gene eaeA and bundle forming pili bfpA). The Multiplex PCR assays were also used for detecting DEC directly from the stool samples which were positive for DEC. DNA was extracted from stool samples using QIAamp DNA Stool Mini Kit and subjected to multiplex PCR for detecting the presence of DEC. The multiplex PCR assays were found to be effective for direct detection of DEC in stool samples. The assays correctly identified 100% of the DEC strains directly in stool samples.
Table 2: Diarrheagenic E.coli among children with and without diarrhea

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 ~ Discussion Top


PCR is a highly sensitive and specific molecular biology technique for the detection of target DNA in various clinical specimens. Diarrheagenic E.coli is classified into five main categories according to the presence of different virulence genes. In stool samples it can help to differentiate diarrheagenic E.coli strains from those of the normal flora. DEC strains are usually characterized by phenotypic assays in most laboratories, but it is not possible to identify all the five pathotypes of DEC by these methods. [8] DEC can be identified by molecular methods based on the presence of different chromosomal and/or plasmid encoded virulence genes that are absent in commensal E.coli. The genes that code for virulence factors of the different DEC have been extensively studied and characterized. [9] Numerous PCR methods have been developed to identify the virulence genes of DEC. [2],[10],[11] In this study we were able to detect DEC from pure cultures and spiked stool samples using control strains by the multiplex PCR assays. The limit of detection of DEC by the multiplex PCR assays was approximately 10 3 CFU/ml.

The prevalence and other epidemiological features of these pathogens as causative agents of diarrhea vary from region to region. This variation is also seen between and within countries in the same geographical area. [11],[12] Four categories of DEC were detected in children with diarrhea in this study. We found that EAEC was the most common DEC prevalent in children with diarrhea. This is in agreement with a study conducted earlier by CMC, Vellore to detect DEC in children. [13] EAEC has been known to cause persistent diarrhea in children in developing countriesand is an emerging pathogen associated with diarrhea. It has been identified in travelers and children in the developing world and in human immunodeficiency virus infected patients. [14],[15] EAEC has also been reported as the predominant DEC in children in studies conducted in Vietnam and USA. [10],[14]

EPEC are classified into two types. Type I or typical EPEC which are positive for both eae gene and the bfp gene and Type II or atypical EPEC which are positive for eae gene only. BfpA is the structural gene encoding BFP (the bundle forming pilus). These fimbriae are formed only under certain cultural conditions. Typical EPEC is well recognized as a cause of gastroenteritis in infants. EPEC usually causes infection in infants younger than 2 years of age. The isolation of fewer typical EPEC strains confirms a recent trend that has been observed in many other countries. [16],[17] It is now considered as an emerging pathogen. Several recent studies have reported an increase in prevalence of atypical EPEC strains. [12] Similarly in our study too most of the EPEC were of atypical type. The prevalence of ETEC (3.6% in the diarrhea group) was lower in our study than in someprevious studies. [18],[19] Most of the earlier studies have used primers for ST gene only in the multiplex PCR assays. In one study conducted earlier the product sizes of Stla and Stlb primers is almost similar. [20] We subjected all our isolates to monoplex PCR assays using primers for both the subtypes. We detected only Stla gene in our isolates of ETEC by monoplex PCR assays, hence we included only detection of Stla gene in the multiplex PCR assay. In the present study we did not identify any EHEC strains. These results agree with the low prevalence of EHEC infection in developing countries. [9],[19],[20] The low frequencies of EIEC strains is also in agreement with other studies performed in different parts of the world. [10],[18],[20] There is not much data regarding previous studies using multiplex PCR assays for the detection of DEC from India. Our multiplex PCR assays have shown similar sensitivity and specificity to studies conducted earlier. [13],[21]

The limitations associated with traditional diagnostic techniques can be overcome by PCR which is a sensitive, specific and rapid method for diagnosis.In this study, we were able to save time and effort involved in testing for virulence factors, reducing the number of gene detection assays by use of two sensitive and specific multiplex PCR assays. These assays can also detect mixed infections involving two DEC strains. E. coli is considered as part of intestinal flora and no attempt is usually made for characterizing these strains further in routine diagnostic microbiology laboratory setting in a developing country. Accurate identification of DEC is important in understanding the disease spectrum and burden, tracing the sources of infection and routes of transmission. Such identification would also assist the clinician to dispense appropriate management. The rapid detection of DEC has important treatment implications. Treatment of diarrhea does not usually depend on the etiological diagnosis, but such diagnosis bears an implication on overall management of patients. Some DEC strains (e.g., ETEC) respond to antimicrobial agents, while others (e.g., STEC) do not and therefore antibiotics should be avoided. DEC strains are usually resistant to the commonly used antibiotics. Optimal treatment therefore depends on rapid detection of the specific pathogen. The overall time required to perform the assays is about 24 hours and the approximate cost per assay is about Rs 450. A clinical microbiology laboratory setting in a developing country could use these Multiplex PCR assays as a practical and rapid diagnostic tool for identification of DEC.

In conclusion, the two multiplex PCR assays were able to correctly determine the presence of corresponding DEC virulence genes in all the reference strains of DEC. They can save considerable time and effort involved in testing for various virulence factors of DEC. Although singly they cannot be used for the detection of all strains of DEC, the two multiplex PCR assays could be used for detection of DEC in routine diagnostic laboratories. This study helped to understand that DEC does contribute to the burden of diarrhea in children in the developing world.


 ~ Acknowledgments Top


This study received financial support from the Indian Council of Medical Research. Project No: 5/8-1(211)/D2006-ECD-II

 
 ~ References Top

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2.Aranda KR, Fagundes-Neto U, Scaletsky IC. Evaluation of Multiplex PCRs for the Diagnosis of Infection with Diarrheagenic Escherichia. J ClinMicrobiol2004; 42: 5849- 53.  Back to cited text no. 2
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]

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