Molecular Diagnosis of Diarrheagenic E. coli Infections Among the Pediatric Patients in Wasit Province, Iraq.
Diarrheal disease is still a global problem around the world specially in children under five years in developing countries (1). According to the World Health Organization (WHO), diarrheal diseases are the second leading cause of death (~760, 000 per year) in children (2, 3). The microbial causes of diarrhea are variety of bacterial, viral and parasite 4, Among these pathogens, diarrheagenic E. coli play a major role in causing diarrhea in children under 5 years (5, 6, 7).
When the microbial agent is bacteria, E. coli consider one of the major causes, specially to infantile diarrhea (8,9-10). Depending on specific virulence gene, clinical features, and serotypes diarrheagenic E.coli divided into 6 stains: Enteropathogenic E. coli, Enteroaggregative E. coli, Enteroinvasive E. coli, Enterotoxigenic E. coli, Shiga toxin-producing E. coli and Diffusely Adherence E. coli (11). Culture and biochemical test can't distinguished between commensal or pathogenic strains of E. coli in stool, therefor PCR used to detect the virulence genes in pathogenic strains (12), multiplex PCR provide detection to many Diarrheagenic E.coli strains virulence genes with high sensitivity, specificity (13).
The aim of thisstudy was detecting the distribution of diarrheagenic E. coli pathotypes among children with diarrhea in Wasit province, Iraq by multiplex PCR, and assessing the antimicrobial susceptibility profile of diarrheagenic E. coli, in order to contribute to the establishment of a more effective empirical antibiotic therapy for the disease.
MATERIALS AND METHODS
Collection of samples
During period from middle of September 2017 to middle of December 2017 a total of 210 stool samples were collected from children (males and females) with an ages under five years presented with diarrhea had been admitted at hospitals and attended at private clinics in Wasit province, Iraq. Otherwise, 40 stool samples were collected from healthy children considered as control. The stool samples transported on Carry Blair swabs and cultured on MacConkey agar, XLD, EMB, Blood agar, and CHROMagarSTEC and incubated aerobically at 37[degrees]C for 24 hours, the isolated bacteria was identified according to morphological, biochemical tests and API 20E kit.
Antibiotic susceptibility test: performed by Kirby-Bauer procedure on Muller Hinton agar 14 and results interpreted according to Clinical and Laboratory Standards Institute (15)
DNA extraction: was performed according to the procedure (Geneaid Genomic DNA extraction Kit).
Multiplex PCR technique: was used for amplifying the genes. The mixture reaction was performed in a total volume 50 [micro]l of PCR Mastermix Gold Multiplex 50x (DNA Template 4 [micro]l, Forward primer 1 [micro]l for each primer, Reverse primer 1 [micro]l for each primer, free water ddH2O 20[micro]l). PCR cycling program parameters used in this reaction for detection of (bfpB, eaeA, pic, aggR, astA, invE, ipaH, hly, stx1, stx2, elt, estla, estlb) genes as shown in table (1), the thermal cycling program(Initial denaturation 95[degrees]C for 5 min. 1cycle), (Denaturation 94[degrees]C for 30 sec 35 cycle), (Annealing 58[degrees]C for 30 sec. 35 cycle), (Extension 72[degrees]C for 1 min. 35cycle), (Final extension 72[degrees]C for 7min. 1 cycle) (Holding 4[degrees]C 1 cycle). The amplification products were electrophoresed through a 2% agarose gel and visualized with UV transilluminator after ethidium bromide staining. A 100 bp DNA ladder was used as a molecular size marker in gel. The statistical analysis of all the evidence was done using the system SPSS IBM version 20 software, Chi-squire test. P- value < 0.05 was considered statistically significant.
E. coliwere isolated in 100 (47.6%) of 210 collected samples followed by 78 (37.2%) of other gram negative bacteria (Salmonella, Klebsiella, Proteus, Pseudomonas) and 32 (15.2%) samples that were no growth. The results of primary diagnosis to these 100 E. coli isolates by selective and differential culture media were consistent with the microscopic and biochemical tests results.
Multiplex applied on theses 100 and 40 control samples and the results showed that DEC in were detected in 42/ 100 (42%) among diarrheal children compared with 0/100 (0%) among control children. The distribution of 42 DEC pathotype isolates were: EPEC was found in 19 (45.3%), EAEC in 17(40.5%), ETEC in 3 (7.1%), EIEC in 3 (7.1%) and 0 (0%) in STEC and controls.
From 19 isolates detected as EPEC which was watery diarrhea 10 (52.6%) isolates of them are atypical EPEC showed eaeA gene found without bfpB gene, and 9 (47.4%) were typical EPEC which showed eaeA gene together with bfpB gene. All 19 isolates in our study don't produce nether stx1 or stx2, in addition one of aEPEC showed astA gene.
Enteroaggregative E. coli 17 (40.5%) isolates came second after Enteropathogenic E.coli as causative agent of diarrhea among Diarrheagenic E. coli pathotypes in our study, aggR gene was appeared in all EAEC isolates detected in our study that mean all of them were typical EAEC. Enterotoxigenic E.coli account 3 isolates (7.1%) and Enteroinvasive E. coli was detected in 3 isolates (7.1%) that suggested these pathotype maybe play a lessimportant role in childhood diarrhoea in developingcountries. When age stratification was analysed high incidence of DEC E. coli recorded in first and second age group flowed by third and fourth age group, and there were no cases recorded in fifth age group.
The prevalence of Enteropathogenic E. coli infection infections was high in first and second years, also all Enteroaggregative E.coli infections were detected under 2 years, while Enteroinvasive E. coli were high between 2-3 years also cause infection in first age group, in time all Enterotoxigenic E.coli infections were all above 1 year as shown in figure (1).
E. coli pathotypes, in our study were identified and isolated successfully by using Multiplex PCR. PCR products visualized to measured product size results from amplification the primers in compared with (100 bp) ladder as shown in figures (2, 3, 4, 5, 6, 7, 8, 9).
Antibiotic susceptibility test results were showed in table (2): The highest level of resistance were to Amoxiclav (95.2%), Ampicillin (95.2%), Sulfa-Trimethoprim (92.9%) followed by Tetracycline (85.7%), Cephalthin (85.7%) Ceftriaxone (81%) Cefotaxime/clavulanicacid 71.4%. The maximum E. coli sensitivity was to Imipenem (100%) flowed by Amikacin (85.7%), Ciprofloxacin (78.6%) Chloramphenicol (73.8%).
The distribution of DEC in our study was 42 (20%) among 210 diarrheal cases. Our result concur to other study in Iraq reported by Hamada et al (12) in Kirkuk (36%) and globally with other studies in Iran Heidary et al (19) (28%), while our result contrast with other studies were showed less prevalence to Diarrheageic E. coli Konateet al (20) revealed (7.4%) in Burkina Faso, Salmaniet al (21) in Iran who showed (88%). These differences reflecting the difference in distribution of geographical areas, quality of sanitation.
Among all the Diarrheagenic E.coli pathotypes, Enteropathogenic E.coli (EPEC) were found to be the most common pathotypes for children with (45.3%), our result compatible with localstudy by Sakhi (22) who showed EPEC as most than other pathotypes (63%), and in contrast with Khalil 11 and Al-Dulaimi (23) where they show it came second after EAEC. Our finding was, however, similar to globally studies with Zhou et al (1), Thakur et al (7) and Chellapandietal (6) that also reported a high frequency of EPEC pathotypes associated with pediatric diarrhea.
EPEC are sub-grouped into typical (tEPEC, eae+ bfpA+) and atypical (aEPEC, eae+ bfpA-) strains that differ in several respects Naji and Nasser (24). From 19 isolates detected as EPEC which was watery diarrhea 10 (52.6%) isolates of them are atypical EPEC showed eaeA gene found without bfpB gene, and 9 (47.4%) were typical EPEC which showed eaeA gene together with bfpB gene.
All 19 isolates in our study don't produce nether stx1 or stx2, in addition one of aEPEC showed astA gene. Our study is close to study by Arif and Salih (23) in Sulaimani, Iraq, and global reports by Malvi (25) in India, that showed the distribution of atypical EPEC was higher than typical EPEC. Ochoa and Contreras (26) report that atypical EPEC (aEPEC) are more prevalent than typical-EPEC (tEPEC).
Enteroaggregative E.coli 17 (40.5%) isolates came second after Enteropathogenic E. coli as causative agent of diarrhea among Diarrheagenic E. coli pathotypes in our study, that agree with reports by Sakhi 22 in Dhi-Qar city, also Globally with Thakur et al (7). But EAEC considered the major cause of diarrhea between diarrheagenic E. coli pathotypes in local studies by Khalil (11) and Al-Dulami (23), also Globally, Rajendranet et al (27) in India, Ali et al 28 in Egypt, and Konateet et al (20) in Burkina Faso.
EPEC and EAEC were reported as the most common Diarrheagenic E. coli pathotypes Bueriset et al (29); Moyoet et al (30); Wang et al (31).
Enterotoxigenic E. coli account 3 isolates (7.1%) of Diarrheagenic cases, the detection of ETEC is in consonance with previous local findings by Hamada et al (12), and concur with global study by Raghavanet et al (32) in India. Our study differ from other reports by Chiyangiet et al (33) in Zambia which suggested high prevalence of ETEC 40%.
Enteroinvasive E. coli was detected in 3 isolates (7.1%) of Diarrheagenic isolates. Our study is nearly close with local study by Hamada et al (12) (10%), and globally with (0.5%) by Moshtagian (34), and (12.9%) by Konateet et al (20), and (3.7%) by Zhouet et al (1).
Vieira et al (35) also showed low rate of prevalence Enteroinvasive E. coli and suggested that this pathotype may be play a less important role in childhood diarrhea in developing countries. In this study, no Shiga toxin-producing E. coli were detected this result is similar to local reports of Sakhi (22) and Hamada et al (12). Globally, Ali et al 28 and Canzalez-Roman 36, also showed no isolates of STEC were detected in children with diarrhea. STEC appears to be more frequent in adults than children Okekeet et al (37). These difference between our results and other studies may be attributed to rout of infection, virulence factors, pathogen strains, difference in population selection, time of collection and size of samples.
Antibiotic susceptibility test
Resistance to Amoxiclave (95.2%) agreement with another study had been reported high resistance in studies done by Al-Hilali (38) 83.4 % in Al-Najaf. Our result; disagreed with previous study in Alkut by Shamkhi (39) who recorded low resistance to Amoxiclav with 7.1% AL-Shuwaikh et al (39) in Baghdad report 33.3%. Increased Amoxiclav resistance coincided with growing Amoxiclav consumption at the community level, similarly, the isolated Diarrheagenic E. coli pathotypes showed high resistance rates to Ampicillin and Sulfa-Trimethoprim Konateet et al (41), as mention in study by Goossenset et al (42); Llor and Bjerrum (43) about antibiotic resistant, there is high resistant in the most consumption antibiotic. Sulfa-Trimethoprim is widely used in developing countries to treat diarrhea because of their availability over the countries Nguyen et al (44). Attention should be given while prescribing Amoxiclav, Ceftriaxone, and Ampicillin to avoid increasing resistance pathotype by E. coli. Similar study conducted by Konate et al (41) in Burkina Faso revealed that 85% of E. coli isolates were resistance to Tetracycline.
Our study for Cefotaxime was agreement with previous local studies by, AL Hilali (38) 68%, Sakhi (22) 85.7%, Ugwa et al (45), also reported resistance to Ceftriaxone (91%) by E. coli isolates. Rajeshwari et al 46 reported similar finding for the high resistance of Ceftriaxone 75% and cefotaxime (77.5%) in Indian children with diarrhea, while disagreement Khalil (11) 4% and Hamada et al (12) 10%. Antibiotic susceptibility testing of isolates showed high resistance rate to Cephalothin (85.7%). The emergence of multidrug resistance especially in E. coli has become a critical public concern, which was designated as resistance to one agent in three or more antibiotic classes. Kamwati (47); Alizadi (48). Many factors responsible for an increase in rates of antimicrobial resistance include misuse/over use of antibiotic by healthcare professionals and general public Magiorakos et al (49); WHO (50); Konateet al (41), and inadequate surveillance systems and independence on reliable microbiological techniques which leads to inappropriate prescription of antibiotics Wellington et al (51).
Ciprofloxacin showed low resistant similar with local studies by Hamada et al 12 10 %, Khalil 11 8%. Globally Kamwati 47 4%, Canizalez-Roman et al 36 21%. Ciprofloxacin was one of the most active antimicrobial agent which currently recommended to treated diarrhea in children Ayat ollahi et al 52.
Amikacin showed low resistant agreement with 3.3% reported by Hamada et al (12) and Al-Hilali (38) 0%, and globally with Zhou et al (1) 7.4%.
Imipenem with no resistant agreement with 0% resistant from Al-Hilali (38) and Shamkhi (39). The Impenim was the most effective antibiotic against DEC followed by Amikacin, Ciprofloxacin and Chloramphenicol. Impinem has been highly effective against gram negative bacteria Mohammed et al (53), Alam et al (54). They should be used in life threatening multidrug resistance infections where there is no other alternative.
The statistical analysis to susceptibility test results in this study showed high resistance among Diarrheagenic E. coli isolates which were collected from hospitalized children than isolates collected from private pediatric clinics (without history of hospital admitted) as shown in table (3).
This result goes with report of Kamwati (47) who showed isolates from children who had been hospitalized were more resistance than those isolated from children not previously hospitalized, and he conclude that recent history of antimicrobial use and hospitalization is a serious predisposing factor to carriage of Multi Drug Resistant strains. Fox-Lewis (55) also mention that hospital-acquired Escherichia coli isolates were multidrug resistant than isolates were community-acquired. Multi drug resistance MDR may be acquired from other patients who have received antibiotics. Infections caused by Multi drug resistance gram negative bacteria are difficult to treat and so may cause more prolonged symptoms in the site of infection Hawkey et al (56).
Enteropathogenic E. coli and Enteroaggregative E. coli was the most common types of Diarrheagenic E. coli among children less than 2 years of age presented with diarrhea in Wasit province. Enterotoxigenic E. coli and Enteroinvasive E. coli were more common in children more than 2 years of age in Wasit province.
This study highlights the Using of multiplex PCR in identifying and successful isolation of Diarrheagenic E.coli from normal flora and can be used as a rapid and accurate method for the isolation of pathogenic strains of E. coli, this will greatly help pediatricians to decrease the use of antibiotic in treatment of diarrhea in children and decreasing the problem of increasing antibiotic resistance. The results of antibiotic sensitivity test revealed that the most active compound against Diarrheagenic E. coli isolates was Imipenem followed by Amikacin, Ciprofloxacin and Chloramphenicol.
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Zainab Kadhim Abdul-hussein, Rana Hussein Raheema and Ahmed Ibrahim Inssaf
Department of Medical Microbiology Faculty of Medicine, University of Wasit, Iraq.
* Correspondence: firstname.lastname@example.org
(Received: 13 October 2018; accepted: 29 November 2018)
Caption: Fig. 1. Prevalence diarrheagenic E. coli with age groups.
Caption: Fig. 2. Gel electrophoresis of amplified (eaeA, bfpB, aggR, astA, pic, hly, stx1, stx2 invE, ipaH, elt estIa, estIb) genes, the product size (482, 910, 400, 102, 1, 111, 688, 244, 324, 766, 437, 655, 157, 171 (bp) respectively), of E.coli strains using conventional PCR. Agarose 2%, and TBE (1X) at (75 V/cm for 90 min., stained with Ethydium bromide dye and visualized on a UV transilluminator. Lane(L): DNA ladder (100-3000 bp), Lanes: (1-12) stool samples.
Caption: Fig. 3. Gel electrophoresis of amplified (eaeA, bfpB, aggR, astA, pic, hly, stx1, stx2, invE, ipaH, elt, estIa, estIb) genes, the product size (482, 910, 400, 102, 1, 111, 688, 244, 324, 766, 437, 655, 157, 171 (bp) respectively), of E.coli strains using conventional PCR. Agarose 2%, and TBE(1X) at (75 V/cm for 90 min., stained with Ethydium bromide dye and visualized on a UV transilluminator. Lane(L): DNA ladder (100-3000bp) Lanes: (1-12) stool samples.
Caption: Fig. 4. Gel electrophoresis of amplified (eaeA, bfpB, aggR, astA, pic, hly, stxl, stx2, invE, ipaH, elt, estIa, estIb) genes, the product size (482, 910, 400, 102, 1, 111, 688, 244, 324, 766, 437, 655, 157, 171(bp) respectively), of E.coli strains using conventional PCR. Agarose 2%, and TBE (1X) at (75 V/cm for 90 min., stained with Ethidium bromide dye and visualized on a UV transilluminator. Lane (L): DNA ladder (100-3000bp), Lanes: (1-12) stool samples.
Caption: Fig. 5. Gel electrophoresis of amplified (eaeA, bfpB, aggR, astA, pic, hly, stxl, stx2, invE, ipaH, elt, estIa, estIb) genes, the product size (482, 910, 400, 102, 1, 111, 688, 244, 324, 766, 437, 655, 157, 171 (bp) respectively), of E.coli strains using conventional PCR. Agarose 2%, and TBE(1X) at (75 V/cm for 90 min., stained with Ethidium bromide dye and visualized on a UV transilluminator. Lane (L): DNA ladder (100-3000bp), Lanes: (1-12) stool sample.
Caption: Fig. 6. Gel electrophoresis of amplified (eaeA, bfpB, aggR, astA, pic, hly, stx1, stx2, invE, ipaH, elt, estIa, estIb) genes, the product size (482, 910, 400, 102, 1, 111, 688, 244, 324, 766, 437, 655, 157, 171 (bp) respectively), of E.coli strains using conventional PCR. Agarose 2%, and TBE (1X) at (75 V/cm for 90 min., stained with Ethidium bromide dye and visualized on a UV transilluminator. Lane (L): DNA ladder (100-3000 bp), Lanes: (1-12) stool samples.
Caption: Fig. 7. Gel electrophoresis of amplified (eaeA, bfpB, aggR, astA, pic, hly, stx1, stx2, invE, ipaH, elt, estIa, estIb) genes, the product size (482, 910, 400, 102, 1, 111, 688, 244, 324, 766, 437, 655, 157, 171 (bp) respectively), of E. coli strains using conventional PCR. Agarose 2%, and TBE (1X) at (75 V/cm for 90 min., stained with Ethidium bromide dye and visualized on a UV transilluminator. Lane (L): DNA ladder (100-3000bp), Lanes: (1-12) stool samples.
Caption: Fig. 8. Gel electrophoresis of amplified (eaeA, bfpB, aggR, astA, pic, hly, stxl, stx2, invE, ipaH, elt, estIa, estIb) genes, the product size (482, 910, 400, 102, 1, 111, 688, 244, 324, 766, 437, 655, 157, 171 (bp) respectively), of E. coli strains using conventional PCR. Agarose 2%, and TBE (1X) at (75 V/cm for 90 min., stained with Ethidium bromide dye and visualized on a UV transilluminator. Lane (L): DNA ladder (100-3000bp), Lanes: (1-12) stool samples.
Caption: Fig. 9. Gel electrophoresis of amplified (eaeA, bfpB, aggR, astA, pic, hly, stxl, stx2, invE, ipaH, elt, estIa, estIb) genes, the product size (482, 910, 400, 102, 1, 111, 688, 244, 324, 766, 437, 655, 157, 171 (bp) respectively), of E. coli strains using conventional PCR. Agarose 2%, and TBE (1X) at (75 V/cm for 90 min., stained with Ethidium bromide dye and visualized on a UV transilluminator. Lane (L): DNA ladder(100-3000 bp), Lanes: (1-12) stool samples.
Table 1. Primers used for multiplex PCR reaction E.coli Primers Primers (Sequence (5'-3')) strain EAEC aggR aggR-F: ACGCAGAGTTGCCTGATAAAG aggR-R: AATACAGAATCGTCAGCATCAGC astA astA-F: TGCCATCAACACAGTATATCCG astA-R: ACGGCTTTGTAGTCCTTCCAT pic pic-F: AGCCGTTTCCGCAGAAGCC pic-R: AAATGTCAGTGAACCGACGATTGG hly hly-F: TTCTGGGAAACAGTGACGCACATA hly-R: TCACCGATCTTCTCATCCCAATG 0.1 STEC stxl stx1A-F: CGATGTTACGGTTTGTTACTGTGACAGC stx1A-R: AATGCCACGCTTCCCAGAATTG stx2 stx2AF: GTTTTGACCATCTTCGTCTGATTATTGAG stx2A-R: AGCGTAAGGCTTCTGCTGTGAC invE invE-F: CGATAGATGGCGAGAAATTATATCCCG invE-R:CGATCAAGAATCCCTAACAGAAGAATCAC EIEC ipaH ipaH-F: GAAAACCCTCCTGGTCCATCAGG ipaH-R: GCCGGTCAGCCACCCTCTGAGAGTAC eaeA eae-F: TCAATGCAGTTCCGTTATCAGTT eae-R: GTAAAGTCCGTTACCCCAACCTG EPEC bfpB bfpB-F: GACACCTCATTGCTGAAGTCG bfpB-R: CCAGAACACCTCCGTTATGC elt elt-F: GAACAGGAGGTTTCTGCGTTAGGTG elt-R: CTTTCAATGGCTTTTTTTTGGGAGTC ETEC estIa estIa-F:CCT CTTTTAGYCAGACARCT GAATCASTTG estIa-R: CAGGCAGGATTACAACAAAGTT CACAG estIb estIb-F: TGTCTTTTTCACCTTTCGCTC estIb-R: CGGTACAAGCAGGATTACAACAC E.coli Primers Product References strain size (bp) EAEC aggR 400 astA 102 16 pic 1,111 hly 688 17 STEC stxl 244 stx2 324 16 invE 766 EIEC ipaH 437 18 eaeA 482 EPEC bfpB 910 elt 655 16 ETEC estIa 157 estIb 171 Table 2. Antibiotic susceptibility test Antibiotics CEC AMC CTR TE SXT AMP Sensitive 8 1 7 4 2 2 19% 2.4% 16.7% 9.5% 4.8% 4.8% Intermediate 4 1 1 2 1 0 9.5% 2.4% 2.4% 4.8% 2.4% 0% Resistance 30 40 34 36 39 40 71.4% 95.2% 81% 85.7% 92.9% 95.2% Chi squire 28 72.4 44.14 52 67 34.38 Antibiotics CTL C CIP IPM AK Sensitive 3 31 33 42 36 7.1% 73.8% 78.6% 100% 85.7% Intermediate 3 3 2 0 3 7.1% 7.1% 4.8% 0% 7.1 % Resistance 36 8 7 0 3 85.7% 19% 16.7% 0% 7.1% Chi squire 51.8 31.8 39.57 34.38 51.8 AK=Amikacin, AMC=Amoxiclav, AMP=Ampicillin, CTR=Ceftriaxone, CTL=Cephalothin, C= Chloramphenicol, CIP=Ciprofloxacin, CEC=Cefotaxime-Clavulanic, IPM=Imipenem TE=Tetracycline, STX=Sulfa- Trimethoprim
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Abdul-hussein, Zainab Kadhim; Raheema, Rana Hussein; Inssaf, Ahmed Ibrahim|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Dec 1, 2018|
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