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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 204-210
 

Spectrum of diarrhoeagenic Escherichia coli in paediatric population suffering from diarrhoea and as commensals in healthy children


1 Department of Microbiology, UCMS, Guru Teg Bahadur Hospital, New Delhi, India
2 Department of Paediatrics, UCMS, Guru Teg Bahadur Hospital, New Delhi, India

Date of Web Publication5-Jul-2017

Correspondence Address:
Shukla Das
Department of Microbiology, UCMS, Guru Teg Bahadur Hospital, Dilshad Garden, New Delhi - 110095
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_16_21

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 ~ Abstract 

Background: Diarrhoeagenic Escherichia coli (DEC) is associated with early death of children in developing countries and are being identified now as an important evolving pathogen. The objective of this study was to perform multiplex polymerase chain reaction (PCR) for simultaneous detection of six categories of DEC in two sets of PCR reactions using 11 virulent genes. Materials and Methods: During 1-year study period, forty isolates each from outpatient, inpatient and healthy groups were collected from children. E. coli was identified using conventional biochemical methods. DNA extraction was done using kit, and the extracted DNA was used as a template for multiplex PCR. Results: Virulent genes of DEC were detected in 106 (88.33%) samples. Overall, elt and est were detected in 8.33% and 30.83% of specimens; typical, atypical enteropathogenic E. coli and bfp were detected in 13.33%, 29.16% and 19.16% specimens; eagg was detected in 39.16% and east in 13.33% specimens and stx and hyla were isolated in 1.66% specimens each. While diffusely adherent E. coli and enteroinvasive E. coli genes were not isolated. Conclusion: Multiplex PCR is a rapid method for the simultaneous detection of 11 virulent genes of DEC at a time and it will provide a platform in understanding the diarrheal diseases in a more improved manner.


Keywords: Diarrhoeagenic Escherichia coli, multiplex polymerase chain reaction, pathogenesis, virulent genes


How to cite this article:
Singh T, Das S, Ramachandran V G, Dar SA, Snehaa K, Saha R, Shah D. Spectrum of diarrhoeagenic Escherichia coli in paediatric population suffering from diarrhoea and as commensals in healthy children. Indian J Med Microbiol 2017;35:204-10

How to cite this URL:
Singh T, Das S, Ramachandran V G, Dar SA, Snehaa K, Saha R, Shah D. Spectrum of diarrhoeagenic Escherichia coli in paediatric population suffering from diarrhoea and as commensals in healthy children. Indian J Med Microbiol [serial online] 2017 [cited 2017 Sep 26];35:204-10. Available from: http://www.ijmm.org/text.asp?2017/35/2/204/209574



 ~ Introduction Top


In India, diarrhoea still remains the most common cause of death among children under five. Every year, an estimated 2.5 billion death occurs due to diarrhoea; among them, 30%–40% is contributed only to diarrhoeagenic  Escherichia More Details coli (DEC).[1],[2]

In India, very few studies have documented the prevalence of all DEC types in paediatric population. On the basis of discrete epidemiological and clinical features, virulence determinants and association with certain serotypes, the pathogenesis of E. coli is well known and has been categorised as: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC).[3] Cytolethal distending toxin- producing E. coli are a distinct E. coli pathotype, implicated in diarrhoea. ETEC produces one or more enterotoxins that are heat-labile LT (LT-1 and LT-2) or heat-stable ST (STa and STb);[4] EPEC harbours a pathogenicity island that encodes a series of proteins involved in the attaching and effacement lesions of the intestinal microvilli of the host cell and the presence of the large EPEC adherence factor (EAF) plasmid, on which the cluster of genes encoding bundle-forming pili (bfp) is present.[5] Based on these, EPEC strains are classified as typical when they possess the EAF plasmid, whereas atypical EPEC strains do not possess the EAF plasmid;[6] EHEC is characterised by the production of two potent cytotoxins, Shiga-like toxins and enterohaemolysin.[7] EAEC, first discovered by studies of adherence to HEp-2 cells, displays a pattern of adherence characterised by self-agglutination that is designated as aggregative adherence. EIEC and DAEC categories are less prevalent. The profile of DEC in healthy children as carriage is not very clear. The existence of DEC in healthy gut can promote the horizontal transfer of virulence genes to the other non-pathogenic E. coli.

Conventional methods are time consuming and in vitro assays may not always be accurate. Polymerase chain reaction (PCR) provides a rapid, sensitive and cost-effective method in the detection of enteric pathogen in developing countries. This study was designed to determine the prevalence of DEC by multiplex PCR in children under 5 years of age suffering from diarrhoea and their 'presence' as commensal in non-diarrhoeal stool samples of healthy children.


 ~ Materials and Methods Top


Clinical specimens

The study population comprised three groups. Group 1 included forty children (not receiving any antibiotics) with diarrhoea for <72 h duration attending the outpatient department. The non-diarrhoeal group included forty children hospitalised and receiving antibiotic (oral or intravenous) for 72 h or more for reasons other than diarrhoea as Group 2. Group 3 represented the healthy controls of forty children below 5 years of age not suffering from diarrhea or any other disease.

Fresh stool samples were collected and inoculated on media as per standard laboratory methods, and E. coli was identified based on biochemical reactions.[8]

DNA extraction

Isolated lactose-fermenting colonies on MacConkey agar confi rmed as E. coli were selected for DNA extraction using a commercial kit (Real Biotech Corporation, Taiwan).

Primer selection

Primers were selected from previous published literature as shown in [Table 1].[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19] The presence of the following genes depicted the various DEC: elt and est for ETEC, eagg and east for EAEC, atypical EPEC with eae and eae + bfp (eaf) for typical EPEC; stx and hyla for EHEC; ipah for EIEC and daaE for DAEC. GAPDH was used as amplification internal quality control.
Table 1: Primer sequences, annealing temperatures and size of amplified products from selected genes of diarrheagenic Escherichia coli

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Polymerase chain reaction conditions

0.2 ml tubes were used for each set of multiplex PCR assay. Each tube contain a total volume of 25 μl including 2.5 μl buffer (10X), 1 μl dNTPs (200 μM), MgCL2 1 μl (1.5 mM), 1 μL of each primer, forward and reverse (10 μM), 5 μl of the extracted DNA and nuclease-free water to make up the volume. All PCR reagents were purchased from Genei, Bengaluru, and amplification was performed on a thermocycler (Eppendorf).[17]

An initial denaturation was performed at 94°C for 10 min which was followed by 35 amplification cycles of 40 s at 94°C and 30 s at 50°C and 55°C and 50 s at 72°C, and final extension of 7 min at 72°C. The amplified PCR products were analysed by electrophoresis on 1% agarose gel, stained with ethidium bromide at 125 V and 15 mA current in an 18-slot apparatus for 30 min. Molecular marker of 100 bp was used to determine the size of the amplicons.[20] Uniplex PCR (Fermentas, India) was also performed in DEC isolates that showed the presence of multiple genes, for the confirmation of mixed infection. All the isolates were also screened for their phylogenetic groups using multiplex PCR as described by Clermont et al., 2013.[21]

Standard bacterial control strains were obtained from the National Institute of Cholera and Enteric Diseases (Kolkata, India). Positive controls for PCR were E. coli ATCC 35401 (ETEC est+/elt+), E. coli ATCC 43887 (EPEC eaf+/bfpa+/eaeA+), E. coli ATCC 35150 (EHEC stx1+/hyla+/eaeA+) and E. coli ATCC 43893 (EIEC ipah+). Non-pathogenic E. coli ATCC 1175 was used as a negative control.

Isolates producing virulent genes were subjected to sequencing using the same set of primers. Purification of the PCR products of virulent genes and DNA sequencing was performed commercially (Yaazh Xenomics, Chennai). To increase the accuracy of the results, sequencing was performed with both forward and reverse primers.

Statistical analysis

Statistical analysis was done using Statistical Package for Social Sciences package (version 20.0, IBM, USA). Chi-square test and Fisher's exact test were used to determine the statistical significance of data. P < 0.05 was considered statistically significant. To identify the preponderance of DEC, multivariable logistic regression analysis was also done.


 ~ Results Top


One hundred and twenty stool specimens were collected and analysed from children during a period of 1 year from July 2012 to July 2013. Multiplex PCR detected target genes of DEC in 106 (88.33%) diarrhoeal stool samples as shown in [Figure 1] and [Figure 2]. The distribution of DEC amongst various age groups in children is depicted in [Table 2].
Figure 1: Multiplex PCR for virulent genes of diarrheagenic E. coli (DEC). Agarose gel electrophoresis of virulent genes polymerase chain reaction (PCR) amplification products of EAEC, EPEC, ETEC and EHEC genes; with GAPDH as internal control (170bp) run on 1.5 % agarose gel. (L-R) lane1: 100bp ladder, lane 2: eagg (630 bp), lane 3: east (111 bp), lane 4: Enteroaggregative E. coli (eagg and east), lane 5: eae (482 bp), lane 6: eaf (397 bp), lane 7: bfpa (326bp), lane 8: Enteropathogenic E. coli (eae, eaf and bfpA), lane 9: 100bp ladder, lane 10: east (111 bp), lane 11: elt (218 bp), lane 12: est (190 bp), lane 13: Enterotoxigenic E. coli (east, elt and est), lane 14: stx (308 bp), lane 15: hyla (165 bp), lane 16: Enterohemorrhagic E. coli (stx and hyla) and lane 17: 100bp ladder; respectively.

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Figure 2: Uniplex PCR for virulent genes of diarrheagenic E. coli (DEC). Agarose gel electrophoresis of virulent genes polymerase chain reaction (PCR) amplification products on 1.5 % agarose gel. (L-R) lane1: 100bp ladder, lane 2: east (111bp), lane 3: hyla (165bp), lane 4: est (190bp), lane 5: elt (218bp), lane 6: stx (308bp), lane 7: bfpa (326bp), lane 8: eaf (397bp), lane 9: eae (482bp), lane 10: eagg (630bp), lane 11: eaf (397 bp), lane 12: hyla (165bp) and lane 13: 100bp ladder; respectively.

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Table 2: Age- and sex-wise distribution of diarrheagenic Escherichia coli

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The most frequent category of DEC detected was EPEC followed by EAEC, ETEC and EHEC, as shown in [Table 3]. In ETEC, virulent gene est was detected in 47.5%, 42.5% and 2.5% whereas elt was observed in 17.5%, 7.5% and 0% in Groups 1, 2 and 3, respectively. In EAEC, eagg (55%, 30% and 32.5%) was more frequently detected than east (10%, 25% and 5%) gene in Groups 1, 2 and 3. Typical EPEC was detected in 22.5%, 15% and 2.5% samples and atypical EPEC was detected in 12.5%, 37.5% and 37.5% samples in the Groups 1, 2 and 3, respectively. Atypical EPEC was more common in healthy children as compared to diarrhoeal cases. EHEC was also differentiated into typical (stx + hyla + eae) and atypical (stx with or without eae), however typical EHEC variety was not detected in any sample as compared to atypical EHEC observed in 2.5% of isolates in children representing Group 2. Our results showed the preponderance of phylogenetic Group B2 with 44 isolates (36.66%) followed by Groups B1, A, F, D, E and C with 26 isolates (21.66%), 19 isolates (15.83%), 7 isolates (5.83%), 6 isolates (5%), 4 isolates (3.33%) and 3 isolates (2.5%), respectively. About 11 isolates (9.16%) remain unidentified as they were negative for all the genes in quadruplex PCR.
Table 3: Distribution of enterotoxigenic Escherichia coli, enteroaggregative Escherichia coli, enteropathogenic Escherichia coli and enterohaemorrhagic Escherichia coli in three groups according to the type of virulence genes

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A multivariable logistic regression model was performed to find the predominance of different DEC types in children >1 year in three groups. It was observed that EAEC was significant in all the groups. Coefficient of odds ratio was 8.535 and 5.013 times higher in children >1 year of age in Groups 1 and 2, respectively, while it was 0.121 times lower in control group. EPEC did not register a significant value in any group but its coefficient of odds ratio was 0.856 times lower in Group 1 and 1.798 and 1.15 times higher in Groups 2 and 3. Coefficient of odds ratio of EHEC was 4.938 times higher in Group 1 and 0.528 times lower in Group 2. EHEC was associated significantly in Group 1 with odds ratio 0.57 times lower in children >1 year of age in comparison to Group 2 where its chance of occurrence was 1.496 times higher [Table 4].
Table 4: Multivariable logistic regression models exploring significant risk of predominant diarrhoeagenic Escherichia coli infection in age <1 year (reference category) versus >1 year in three groups separately

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The most common mixed infections observed in our study were EAEC, EPEC and ETEC in 36/120 (30%); EAEC and EPEC in 17/120 (14.16%) and EPEC and ETEC in 5/120 (4.16%) isolates. Coexistence of all the four categories of DEC together was present in 2/40 isolates (5%) in Group 1 and 1/40 (2.5%) in Group 2. In healthy controls, the coexistence of EAEC and EPEC occurred in 11/40 isolates (27.5%) [Table 5].
Table 5: Coinfection of diarrhoeagenic Escherichia coli among three groups

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Analysis of nucleotide sequences of DEC was done by performing nucleotide BLAST which was obtained by the chromatograms and predicted amino acid sequences of the above genes. MEGA 6.06 software (MEGA 6.06 Pennsylvania State University) was used for phylogenetic tree production as shown in [Figure 3].
Figure 3: Evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbour-Joining method. The evolutionary distances were computed using the maximum composite likelihood method[41] and are in the units of the number of base substitutions per site. The analysis involved 34 nucleotide sequences. Codon positions included were 1st +2nd +3rd + non-coding. All positions containing gaps and missing data were eliminated. There were a total of four positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

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 ~ Discussion Top


DEC still remains the main cause of childhood diarrhoea in developing countries, found most common in children <5 years of age. In our study, DEC was detected in 88.33% of isolates. EPEC being the predominant pathotype at 72.5% (13.33% typical and 29.16% atypical EPEC) was followed by EAEC 63.33%, ETEC 45.83% and EHEC 9.16% (2.5% atypical EHEC, none of the typical EHEC in any group).[21] EIEC and DAEC were not detected in any of the study groups, indicating that they are less prevalent in this area and perhaps in India as well.[22],[23]

Among diarrhoeal group, percentage of EPEC isolation was the highest (80%), followed by EAEC (77.5%), ETEC (75%) and EHEC (12.5%); however, the overall distribution of DEC was higher than those reported elsewhere.[13],[24],[25],[26] Molecular analysis has shown that both chromosomal and plasmid-encoded virulence determinants are involved in EPEC pathogenesis.[27],[28] Similar to other studies, typical EPEC, though known to exist in gut as carriers, was significantly associated with diarrhoea (P < 0.05) compared to atypical EPEC. Earlier studies had predicted that atypical EPEC may have an innate property of longer persistence in intestine; hence, their existence as colonizers in healthy children is not unusual. Atypical strains have eae gene only and they also possess parts of the plasmid but do not express bundle-forming pili for adherence.[27],[29] EAEC was the other important etiological agent associated with diarrhoea in our study. Its characteristic unique patterns of stacked bricks' adherence to Hep-2 cells is associated with the presence of large plasmids and it has been known to cause persistent diarrhoea in children in developing countries.[18] ETEC has been associated with watery diarrhoea in children and 20%–40% of traveller's diarrhoea.[24] EHEC can induce severe attaching and effacing lesions, but percentage isolation of atypical EHEC was not significant in this study.[21] Based on several molecular studies, it is evident that EHEC having eae, stx and ehxA genes (typical Shiga toxin-producing E. coli [STEC]) is considered epidemiologically important as strains exhibiting this virulence gene profile were associated with severe diarrhoea/haemolytic-uraemic syndrome outbreaks worldwide. In contrast, EHEC harbouring only the stx (atypical STEC) seems less virulent and it is associated with sporadic infection.

In hospitalised children not presenting with diarrhoea, the isolation of DEC was evident in similar order to that of diarrhoeal children with EPEC (80%), followed by EAEC (75%), ETEC (60%) and EHEC (10%).[24] The presence of pathogenic DEC in Group 2 in children under 5 years of age admitted to the hospital receiving antibiotics may be existing as an associated pathogen or their presence as mere commensal in the gut could not be established. Their role as pathogens causing acute infection, however, could not be ascertained as the children did not present with diarrhoeal symptoms. It is worth noting that the usage of antibiotics in this population may eventually develop a selective pressure and provide a suitable niche for the proliferation and survival of DEC in the gut.

In healthy controls, the existence of EPEC (57.5%) and EAEC (37.5%) was remarkably higher than ETEC (2.5%) and EHEC (5%) pathogens as observed in other studies.[30],[31] Presence of higher percentage of EPEC that comprised 37.5% of atypical EPEC in healthy children cannot be ignored which is in agreement with studies elsewhere.[32],[33] Atypical EPEC is known to cause prolonged diarrhoea, and apart from the host factors, they may carry other virulence factors responsible for their pathogenicity.[34]

Our results are in agreement with other studies reported.[35] Commensal isolates belong to Groups A and B1 and virulent E. coli belongs to Groups B2 and D as described by other studies also.[36] About 9.16% of E. coli isolates remained unclassified; it may be due to their existence in rare phylogroups or due to occurrence of more than one phylogroups.[21]

Occurrence of coinfection with one or more DEC was also found in our study. This can be explained by the plasticity of E. coli genome that has the potential of undergoing continuous rearrangements. Horizontal gene transfer by mobile genetic elements such as transposons and integrons plays a major role in genome flexibility.[37],[38]

Multivariable logistic regression model predicted the risk of incidence of different DEC types. We selected children <1 year as reference category. EAEC was found significantly in all the groups. The possibility of existence of EAEC infection was 8.535 and 5.013 times higher than other DEC types in children aged more than 1 year in diarrhoeal and non-diarrhoeal groups. The production of excess mucus in EAEC results in the formation of a heavy biofilm leading to enhanced pathogenicity of the organism and perhaps leading to persistent colonisation and diarrhoea. The enhanced adherence in the gut is more favourable in malnourished children deficit in immune response.[39] Hence, its ability to cause infection in healthy children was found reduced by 0.121 times which was lower than that of EPEC (1.150). The pathogenesis of ETEC was 4.938 times higher in children above 1 year of age in Group 1 as compared to Group 2 where its pathogenesis was reduced (0.528 times). The antigenic heterogeneity conferred by the presence of multiple fimbrial antigens may be responsible for its varied role as an etiological agent in different geographical areas and ethnic groups.[39] However, the frequency of occurrence of EHEC was reduced by 0.57 times in diarrhoeal group but simultaneously its presence was found to be increased by 1.496 times in non-diarrhoeal group.

Genotypic demonstration of a virulence plasmid is not synonymous with its expression; nonetheless, the presence of a pathogen with appropriate virulence factors in a 'symptomatic' patient would imply a cause and effect relationship. Thus, the detection of several different virulence plasmids would help in a well-tuned epidemiological dissection of public health burden. The prevalence of different categories of DEC in symptomatic patients, with different clinical severity, and in healthy controls may reflect the socio-sanitary ambience of the epidemiological setting and the potential harmful impact of usage of empirical antibiotics in paediatric population; if this is true, control measures have to be taken for specific situations.

DNA sequence analysis showed high identity levels ranging from 96% to 100% to the gene bank sequence database, confirming specificity of the primers.[40],[41],[42] No mutations were detected in these genes, when we compared our sequences with already existing sequence in NCBI database. It may be believed that the same copy of each virulent gene may have been transferred to other E. coli isolates in an environment made conducive by the excessive use of antibiotics in the hospital.


 ~ Conclusion Top


DEC was recovered at higher rate from healthy children and children without diarrhoea, demonstrating extensive and fast spread of these pathogens in community. Multiplex PCR being time saving can be used for simultaneous detection of pathogenic genes. The occurrence of atypical EPEC in healthy children is of a great concern and cannot be neglected.

Acknowledgement

We like to thank all children (and their parents) who participated in the research. We also acknowledge the Council of Scientific and Industrial Research, Library Avenue, Pusa, New Delhi - 110 012, India, for financial support.

Financial support and sponsorship

This study was supported by Council of Scientific and Industrial Research (CSIR), New Delhi.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04