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Cite this article: Gautam K, Pokhrel BM, Shrestha CD, Bhatta DR (2015) Prevalence of Diarrheagenic E.coli (DEC) Determined by Polymerase Chain Reaction (PCR) in Different Tertiary Care Hospitals in Nepal. JSM Microbiology 3(2): 1021.

*Corresponding authorKirtika Gautam, Ayurveda Campus, Institute of Medicine (IOM), Kirtipur, Nepal, Tel: 9841943521; Email:

Submitted: 07 April 2014

Accepted: 21 August 2015

Published: 24 August 2015

Copyright© 2015 Gautam et al.

OPEN ACCESS

Keywords•DEC•PCR•Diarrhea•EAEC•ETEC

Research Article

Prevalence of Diarrheagenic E.coli (DEC) Determined by Polymerase Chain Reaction (PCR) in Different Tertiary Care Hospitals in NepalKirtika Gautam¹*, Bharat Mani Pokhrel², CD shrestha³ and Dwij Raj Bhatta1

¹Department of Microbiology, Tribhuvan University, Nepal ²Department of Clinical Microbiology, Tribhuvan University, Nepal³Department of Microbiology, Kathmandu Medical College, Nepal

Abstract

Diarrheal diseases are the major cause of morbidity and mortality in resource limited countries including Nepal. E.coli strains are among the most important bacterial causes of childhood diarrhea. This study was undertaken to determine the prevalence of DEC in diarrheal stools specimen from patients with diarrhea using PCR.

Methods: Diarrheal Stool samples were cultured on Mac Conkey Sorbitol agar (MSA) at 37 degree Celsius for 24 hours to isolates diarrheagenic E.coli. Isolates were confirmed by biochemical tests. Antibiotic susceptibility testing was done by using Muller Hinton Agar according to CLSI guidelines. Further multiplex polymerase chain reaction assays were used to detect genes of five different types of DEC.

Results: A total of 120 strains of diarrheagenic E.coli (DEC) were isolated. These were analyzed by multiplex PCR assay. Molecular assay by PCR shows that in Nepal the incidence of enteroaggregative E.coli (EAEC) is the main prevalent DEC 74 (61.66%) followed by Enterotoxigenic E.coli (ETEC). It occurs in 46 (38.33%) of the total isolated cases. A total of 120 strains of diarrheagenic E.coli were isolated. Male (66.66%) had higher infection rate than female (33.33%). In this study it was seen that maximum incidence of diarrheagenic E.coli isolated in warmer season (50%) and least cases were seen in spring (8.33%). Likewise the incidence of disease was high in children under 5 years (70.83%) of age and elderly (22.5%).

Conclusion: The present study showed the prevalence of DEC in stool specimens from adults and children with acute diarrhoea using polymerase chain reaction (PCR).

INTRODUCTIONDiarrhea is defined by World Health Organization (WHO) as

having 3 or more loose or liquid stool per day or having more stools than a normal for that person [1]. Diarrhea can be caused by a wide range of viruses, bacteria, fungi and parasites. Among the bacterial pathogens, diarrheagenic E.coli is an important etiologic agent of childhood diarrhea and represents a major public health problem in developing countries including Nepal. The multiplex PCR is a rapid diagnostic tool for simultaneous detection of 5 categories of diarrheagenic E.coli.

Diarrheal diseases are the major cause of death in children

under 5 years of age in resource limited countries, resulting approximately 2.5 million deaths each year worldwide [2]. In recent years with the introduction of polymerase chain reaction in clinical laboratories, it has become possible to detect genes encoding virulence factors in bacterial isolates allowing rapid diagnosis of DEC strains [3]. Molecular identification and classification of DEC established by the presence or absence of one or more specific virulence genes, which are absent in the commensally E.coli [4].

Virulent factors they possess, virulent E.coli strains cause either non inflammatory diarrhea (watery diarrhea) or inflammatory diarrhea (dysentery with stools usually containing blood, mucus

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and leucocytes). Infection is common where sanitation is poor, both infants and susceptible travellers to developing countries are particularly at risk. The disease is most serious in infants and also in elderly people. E.Coli diarrheal disease is caused orally by ingestion of food or water contaminated with a pathogenic strain shed by an infected person.

Different categories of DEC

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 [5]. They are Enteropathogenic E.coli (EPEC), Enterohemorrhagic E.coli (EHEC), Enterotoxigenic E.coli (ETEC),Enteroaggregative E.coli (EAEC) and Enteroinvasive E.coli (EIEC).

MATERIALS AND METHODSThe study period consists of 5 years from November 2009 to

November 2014. During this period a total of 1250 stool samples from diarrheal patients of Kanti Children’s Hospital (KCH), Nepal Medical College (NMC) Jorpati and Kathmandu Medical College (KMC) Sinamangal Kathmandu were collected. All the samples were processed according to standard Microbiology techniques to isolate diarrheagenic E.coli. Samples were cultured at 37 degree Celsius for 24 hours. Next day, if there seen colourless Sorbitol Non Fermenting (SNF) colonies, it is identified as DEC. Further confirmation was done with the set of standard biochemical test. After identifying bacteria, sensitivity test was performed by modified Kirby Bauer disc diffusion method on Muller Hinton Agar (MHA) using antibiotics as per CLSI guidelines. All the culture media, biochemical media and antibiotics discs were from Hi-media. Identification of 5 strains of DEC was done by using 9 plex PCR assay.

Procedure

Isolate preparation: 24-48 hour colony growth from selective media was used.

DNA extraction: DNA from E.coli colony was extracted by using a 0.5% Triton boil method. 1ml of nuclease free water was added to 5micro litre of Triton x-100 to a micro centrifuge tube.

Test isolates: 1-2 colonies of each of the 5 E. coli morphotypes were add to the single tube. In this case, we should try to add an equal amount of each tube of Vortex 5 seconds.

Positive Controls: 2 colonies of each of the control strains were combined into a single tube Vortex 5 seconds.

a. Boiled or incubated in a heat block at 100C for 20 minutes.

b. It was centrifuge at 10,000 rpm for 10 minutes

c. A clear Supernant containing DNA genome was transfer to new tube and was use directly for PCR.

PCR The number of reactions was determine to set up per run. In a clean DNA free area, master mix was made and dispensed into the 96- well plates. Extracted DNA was then added. A negative control and positive control was included in each run.

- Thaw Qiagen multiplex MM, Q-solution, and primers in a DNA free room.

- Vortex Qiagen multiplex MM, Q-solution, and primers

- Master mixture was prepared in a 1.5ml micro centrifuge tube.

- 45micro litre of master mix was aliquot into each well.

- Lid was placed on the PCR plate and took that plate to another room for the DNA addition.

- Extracted DNA was vortex.

- 5 micro litre of DNA was added to sample or control wells

- Plate was sealed.

- PCR was run.

PCR cycling conditions

1 X 15 min 95 degree centigrade

40 X 30 sec 94 degrees centigrade

90 sec 58 degree centigrade

90 sec 72 degree centigrade

1 X 10 min 72 degree centigrade

1 X 4 degree centigrade

Amplification and sequencing of virulence gene

The amplified PCR products were withdraw from the thermal cycler and run on a 2% agarose gel in TAE buffer. The ethidium bromide stained gel bands were observed in a UV trans illuminator and photographed using Geldoc (ABI). A clear band was formed on agarose gel.

PCR primers used in the multiplex PCR assays for the detection of virulence genes of Diarrheagenic E. coli

ETEC (LT)

ETEC508F 5’-CACACGGAGCTCCTCAGTC-3’

ETEC508R 5’-CCCCCAGCCTAGCTTAGTTT-3’

ETEC (ST)

ETEC147F 5’-GCTAAACCAGTAGGTCTTCAAAA-3’

ETEC147R 5’-CCCGGTACAGCAGGATTACAACA-3’

EHEC (Stx1)

CHEC348F 5’-CAGTTAATGTGGTGGCGAAGG-3’

EHEC348R 5’-CACCAGACAATGTAACCGCTG-3’

EHEC (stx2)

ECEC584F 5’-ATCCTATTCCCGGGAGTTTACG-3’

EHEC584R 5’-GCGTCATCGTATACACAGGAGC-3’

EPEC (eae)

EPEC881F 5’-CCCGAATTCGGCACAAGCATAAGC-3’

EPEC881R 5’-CCGGATCCGTCTCGCCAGTTTCG-3’

EPEC (bfpA)

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EPEC300F 5’-GGAAGTCAAATTCATGGGGGTAT-3’

EPEC300R 5’-GGAATCAGACGCAGACTGGTAGT-3’

EIEC (ipaH)

EIEC423F 5’-TGGAAAAACTCAGTGCCTCT-3’

EIEC423R 5’-CCAGTCCGTAAATTCATTCT-3’

EAEC (aatA)

EAEC650F 5’-CTGGCGAAAGACTGTATCAT-3’

EAEC650R 5’-CAATGTATAGAAATCCGCTGTT-3’

EAEC (aaiC)

EAEC215F 5’-ATTGTCCTCAGGCATTTCAC-3’

EAEC215R 5’-ACGACACCCCTGATAAACAA-3’

A clear band was formed on an agarose gel at 508 BP, 2nd was formed on 650 BP regions confirming the LT and aatA virulent gene (Figure 1).

Safety

Specimens were handled, processed and disposed of using standard guidelines for bio-hazardous materials. Spills were immediately disinfected.

Quality Control

A positive control and a negative control were included in each run. The positive control is made up of a combination of characterized E.coli strains that includes every virulence gene tested. The negative control is E.coli ATCC 25922 or another E.coli strain negative for all virulence genes.

RESULTS AND DISCUSSIONThis study was undertaken to investigate the prevalence of

DEC in diarrheal patients by the use of PCR in adult and children with acute diarrhea in Nepal by PCR. Of the 1250 stool samples obtained from patients with diarrhea during the 5 years study period, DEC type was detected in 120 (8.57%) using the PCR method. Multiplex PCR systems as practical and rapid diagnostic tools for the routine identification of all human DEC (Table 1)

Table 2 Shows the distribution of diarrheagenic E.coli according to Gender. It shows that male were more prevalent then female. Male covers 66.66% & female were 33.33%. Similar to this study a study done in Egypt by Marwa et al found that male were 60% and female were 40% (Figure 2).

Table 3 Shows the seasonal variation in incidence of diarrheagenic E.coli. This study shows that the most prevalent of the disease is seen in warmer season i.e. from April to October. In contrast to this, a study in Norway found that diarrheagenic E.coli was isolated from children throughout the year but was found most frequently in the late summer/early autumn period [6].

Diarrheagenic E.coli which most frequently causes diarrhea in children less than 5 years of age, as has been reported from many studies in developing countries [6]. Elderly are also suffered by this because defences are frequently deficient or lacking in the infant and the elderly. In this study also children under 5 years of age and elderly peoples are more susceptible for

Table 1: Source wise distribution of diarrheagenic E.coli according to hospitals.

Samples processed from

(KCH)

Sample processed from

(NMC)

Sample processed from

(KMC)Total isolates

1050 200 150 120 (8.57%)No. of

isolates% of

isolatesNo. of

isolates% of

isolatesNo. of

isolates% of

isolates80 66.66 30 25.00 10 8.33

Table 2: Distribution of Diarrheagenic E.coli according to Gender in three different hospitals in Nepal.

Male Female Total

No. % No. %

80 66.66 40 33.33 120The above table shows the distribution of E.coli according to gender; It shows that out of 120 isolated cases, male shows 80 which constitutes 66.66% and female were 40, which constitutes 33.33%. (Table: 2).

Children under the age of five, 70.83%

Elderly people, 22.50%

Percentage distribution of diarrheagenic E.coli among infected group

Figure 2 Distribution of diarrheagenic E.coli (DEC) according to age.

80

40

male female

Distribution of diarrheagenic (DEC) E.coli according to sex

Figure 1 Distribution of diarrheagenic E. coli (DEC) according to age. Multiplex PCR of reference strains and clinical samples. Lane M: DNA molecular size marker (100bp ladder). Lane 2 and 12: ETEC (LT- amplicon size is 508 bp) and Lane 7 and 8: EAEC (aatA- amplicon size is 650 BP).

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diarrheagenic E.coli as in Table 4. An important example is the role of the immune system: Passive immune protection of infants by colossal antibody is important, breast feeding is especially relevant where crowding & poor economic condition prevail. Infection with these pathogens often excites an inflammatory cell response in the intestine as in frequently reflected in the diarrheal symptoms. In the present study showed that that were infected with diarrheagenic E.coli were all formulated fed. It has been suggested that breast feeding might have a protective action against diarrhea (Table 5). Factors in the milk such as specific secretary immunoglobulin a antibodies and receptor analogues as well as other innate and anti inflammatory properties might all contributes to decrease the infection.

The most prevalent DEC was EAEC accounting for 74 (61.66) cases followed by 46 cases of ETEC (38.33%). No EPEC, EIEC and EHEC strains were isolated from any of the examined stool samples. Diarrheal disease by diarrheagenic E.coli is endemic in many countries including Nepal. With poor sanitary condition but emerge sporadically as a serious public health threat. The role of EAEC and ETEC in developing countries is well known. However data from Nepal is limited. This is in agreement with a study conducted earlier by CMC Vellore to detect DEC in children [7]. EAEC has also been reported as the predominant DEC in children in a studies conducted in Vietnam and USA [8, 9]. In contrast, lower prevalence of EAEC have been reported from Tanzania (33%) and several other regions in India including Kolkata (9%), northern India (12.3%) and Manipal (22%) [10].In this study ETEC were detected in 46 (38.33%) of samples. In agreement to this study higher prevalence of ETEC was in some previous studies [11,12]. It is a ubiquitous pathogen, commonly transmitted via contaminated food and water and is major cause of traveler’s diarrhea and infantile diarrhea in developing countries (Figure 3). In this study those isolated strains produced only heat Labile Toxin (LT). To our knowledge, this is the first report from Nepal [13]. There is not much data regarding previous studies using multiplex PCR assays for the detection of DEC from Nepal.

Table 3: Seasonal variation in incidence of diarrheagenic E.coli.

Summer season (April-May-Jun)

Monsoon Season

(July-Aug.-Sep.)

Winter(Oct.-Nov.-

Dec.)

Spring(Jan.-Feb.-Mar.)

60 30 20 10In this study, it was seen that maximum incidence of diarrheagenic E.coli isolates in warmer season and least cases seen in spring season.

Table 4: Age wise Distribution of E.coli from different hospitals.

Age Group (years) KCH NMC KMC

0-9 30 15 20

10-19 3

20-29 2

30-39

40-49 1 2

50-59 5

60-69 14 8Above table shows the incidence was high in children below 5 years of age & elderly people.

Table 5: Incidence of each virulence gene in diarrheagenic E.Coli (DEC) isolates from diarrhoeal stool by PCR.Hospitals DEC Category Virulence gene NO %

KCH

EHEC (total

ETEC (total)

EPEC (total)

EIEC (total)

EAEC (total)

St x 1St x 2

LT onlySTeae

bfPAipaH

aaicaatA(only)

00

40

60

12.3

18.4

NMC

EHEC (total

ETEC (total)

EPEC (total)

EIEC (total)

EAEC (total)

St x 1St x 2

LT onlySTeae

bfPAipaH

aaicaatA(only)

2

8

4.0

16.0

KMC

EHEC (total

ETEC (total)

EPEC (Total)

EIEC (Total)

EAEC (Total)

St x 1St x 2

LT onlySTeae

bfPAipaH

aaicaatA(only)

4

6

16.0

24

Most frequently isolated DEC was EAEC 74 (61.66%). This was followed by 46 (38.33%) of ETEC.

Figure 3

CONCLUSIONThe overall result of the present study shows that in Nepal

LT and aatA virulent gene are responsible for causing diarrhea in case of diarrheagenic E.coli. The condition can be remarkably

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Gautam K, Pokhrel BM, Shrestha CD, Bhatta DR (2015) Prevalence of Diarrheagenic E.coli (DEC) Determined by Polymerase Chain Reaction (PCR) in Different Tertiary Care Hospitals in Nepal. JSM Microbiology 3(2): 1021.

Cite this article

improved by availing them with appropriate nutrition, safe drinking water and hygienic living conditions. Further studies are needed to evaluate the epidemiology and virulence properties of DEC. PCR assays could be used for detection of DEC in routine diagnostic laboratories.

ACKNOWLEDGEMENTWe are thankful to all the staffs of Microbiology laboratory of

Central Department of Microbiology, KCH, NMC and KMC as well as Walter Reed Afrim Research Unit Nepal (WARUN).We are also equally thankful to University Grant Commission (UGC) Nepal for providing fund to this project work

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