MOLECULAR DETECTION OF VIRULENCE DETERMINANTS OF DIARRHOEAGENIC

ESCHERICHIA COLI PATHOTYPES ISOLATED FROM YOUNG CHILDREN IN KATSINA

STATE
BY

ANAS UMAR
MSC/MCB/19/0364

BEING A DISSERTATION
SUBMITTED TO THE

DEPARTMENT OF MICROBIOLOGY,
FACULTY OF NATURAL AND APPLIED SCIENCES
UMARU MUSA YAR’ADUA UNIVERSITY KATSINA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE
OF MASTER OF SCIENCE IN MICROBIOLOGY

SUPERVISED BY
DR. BASHIR ABDULKADIR
PROF. ABDULHADI S. KUMURYA

1
FEBRUARY, 2023
 Introduction

• Background of the study

Diarrheal disease is a major health problem, and it is considered
an important cause of morbidity and mortality, especially in
infants and children under five years old(Mare et al., 2021),
with about 525,000 young children dying and 1.7 billion cases
of diarrhoea diagnosed worldwide annually (WHO, 2017).

Diarrhoea can be caused by a wide range of microbial agents

including viruses, bacteria, and parasites. Such diarrhoeal
agents can be acquired from fecally contaminated food, water,
fingers, etc., through the fecal–oral route (Saha et al., 2022).

Among the bacterial agents, Escherichia coli is one of the most

common causes of diarrhoea (Saka et al., 2019).
2
 Statement of the Research Problem
 Despite the fact that Escherichia coli is a commensal bacterium found
as normal fecal flora of a variety of animals including man, not all the
strains are harmless for some of them have evolved specific virulence
mechanisms that enable them cause debilitating and sometimes fatal
diseases in humans and animals (Jafari et al., 2012).
 Diarrhoea has been reported to kill 2,195 children younger than five
years per day, which is more than the combined deaths from malaria,
acquired immunodeficiency syndrome (AIDS), and measles (Atimati
and Eki-Udoko, 2022).

 Diarrhoea is transmitted via fecal-oral route and has been linked to a

lack of safe water, as well as poor sanitation and personal hygiene.

 As at the end of 2015, Nigeria still ranked second among the top 15
countries with high child mortality due to diarrhoea and pneumonia
(Adesoji and Liadi, 2020). 3
 JUSTIFICATION OF THE RESEARCH
 The occurrence of diarrhoeagenic E. coli (DEC) in
children is rarely investigated in Katsina State and even
the very few studies that isolated E. coli in diarrheic
children did not characterize the E. coli to strains level

 and also did not investigate the genetic background,

including the presence of virulence factors (VFs), which
are not routinely evaluated, and hence the proportion of
diarrhoea attributable to DEC remains undefined.

 This study therefore, was conducted to investigate the

occurrence and frequency of diarrhoeagenic E. coli
pathotypes as a cause of infectious diarrhoea in children
below five years of age in Katsina State, 4
 Materials and Methods
 Study design
 Ethical Approval
 Study Population
 Inclusion and Exclusion Criteria
 Sample Collection and Transportation
 A total of165 stool samples, 135 from diarrheic subjects and
30 from non-diarrheic subjects (Adesoji and Liadi, 2020).
 Laboratory Procedures
 Isolation and Identification of E. coli (Adesoji and Liadi,
2020). Antibiotic Susceptibility Testing
 Kirby-Bauer Disc Diffusion Technique

5
 Materials and Methods Cont’d.
 Tested antibiotics (Oxoid, UK): amoxicillin clavulanate (30μg),
ampicillin (10μg), ceftriaxone (30μg), ciprofloxacin (5μg), tobramycin
(10μg) and tetracycline (30μg), respectively.

 Molecular Detection of Virulence Determinants of Diarrhoeagenic E.

coli
 DNA Extraction: DNA was extracted from the bacterial culture using
the AccuPower® genomic DNA extraction kit (Bioneer Corporation)
following the manufacturer’s instruction as previously described by
Alverin, 2022.
 Statistical Analysis: Chi square analysis and percentage prevalence.

 A p-value of less than or equal to 0.05 was considered statistically

significant.
6
 Results
Table 1: Distribution of Socio-demographic and Clinical Characteristics of Diarrheic
  and Non-diarrheic underFacility
Five Children in Katsina StateControl
MMGHD(N= TYMCHK(N=3 GHMN(N=1 GHML(N=25) KPHC(N=34) n=135 n=30
  29) n(%) 7) n(%) 0) n(%) n(%) n(%) Total n(%) Control n(%)
Sociodemography
Gender: Female 17(58.62) 16(43.24) 6(60.00) 16(64.00) 18(52.94) 73(54.07) 16(53.33)
Male 12(41.38) 21(56.76) 4(40.00) 9(36.00) 16(47.06) 62(45.93) 14(46.67)
Age group(year)              
0-1 12(41.38) 10(27.03) 5(50.00) 9(36.00) 16(47.06) 52(38.52) 10(33.33)
2-3 9(31.03) 16(43.24) 3(30.00) 10(40.00) 12(35.29) 50(37.04) 8(26.67)
4<5 8(27.59) 11(29.73) 2(20.00) 6(24.00) 6(17.65) 33(24.44) 12(40.00)
FP-Breast milk            
only 9(31.03) 11(29.73) 7(70.00) 9(36.00) 12(35.29) 48(35.56) 9(30.00)
Formula only 4(13.79) 8(21.62) 1(10.00) 9(36.00) 5(14.71) 27(20.00) 10(33.33)
Mixed 16(55.17) 18(48.65) 2(20.00) 7(28.00) 17(50.00) 60(44.44) 11(36.67)
Clinical Characteristics
OD-<24hrs 3(10.34) 6(16.22) 1(10.00) 2(8.00) 5(14.71) 17(12.59) -
1-2dys 7(24.14) 10(27.03) 3(30.00) 4(16.00) 9(26.47) 33(24.44) -
3-4dys 11(37.93) 12(32.43) 2(20.00) 10(40.00) 13(38.24) 48(35.56) ­-
>4dys 8(27.59) 9(24.32) 4(40.00) 9(36.00) 7(20.59) 37(27.41) -
               
NS-Loose 12(41.38) 20(54.05) 4(40.00) 20(80.00) 21(61.76) 77(57.04) -
Watery 17(58.62) 17(45.95) 6(60.00) 5(20.00) 13(38.24) 58(42.96) -
               
CS-Vomiting 8(27.59) 13(35.14) 4(40.00) 8(32.00) 14(41.18) 47(34.81) -
Fever 17(58.62) 13(35.14) 3(30.00) 11(44.00) 13(38.24) 57(42.22) -
Dehydration 4(13.79) 11(29.73) 3(30.00) 6(24.00) 7(20.59) 31(22.96) -
Key: MMGHD= Mallam Mande General Hospital Dutsin-Ma, TYMCHK= Turai Yar’adua Maternity and Children Hospital Katsina, GHMN= General Hospital
7
Mani, GHML= General Hospital Malumfashi, KPHC= Karfi Primary Health Centre, d=diarrheic, N= total number examined, FP= feeding pattern, OD= onset of
diarrhoea, NS= nature of stool, CS= clinical symptoms.
Table 2: Distribution of E. coli among the study Population
based on Sampled Facilities
  Number Number positive Number negative
examined n(%) n(%) n(%)

Facilities      

MGHD 29 (17.58) 15(17.44) 14 (17.72)

TYMCHK 37 (22.42) 22 (25.58) 15 (18.99)
GHMN 10 (6.06) 7 (8.14) 3 (3.79)
GHML 25 (15.15) 19 (22.09) 6 (7.59)
KPHC 34 (20.61) 23 (26.74) 11 (13.92)
Total 165 (100.0) 86 (100.0) 79 (100.0)
Key: MMGHD= Mallam Mande General Hospital Dutsin-Ma, TYMCHK= Turai Yar’adua
Maternity and Children Hospital Katsina, GHMN= General Hospital Mani, GHML= General
Hospital Malumfashi, KPHC= Karfi Primary Health Centre, n= number, %= per cent.
8
 Control Subjets
17

Total E. coli
Diarrheic Sub- 86
jects
69

Figure 1: Distribution of E. coli among Diarrheic and Non-diarrheic

under five Children in Katsina State.
9
Table 3: Distribution of E. coli among under Five Years Children in Katsina State
According to Gender

Gender No. Positive for E. coli % 0f Occurrence

Male 35 40.69

Female 51 59.31

Total 86 100.0

Key: No. = number, %= per cent, E. coli= Escherichia coli

10
 Table 4: Distribution of E. coli among under Five Years Children in
Katsina State According to Age
Age groups (years) No. Positive for E. coli % 0f
Occurrence
0-1 34 39.00

2-3 31 36.05

<5 21 24.42

Total 86 100.0

Key: No. = number, %= per cent, E. coli= Escherichia coli

11
 Results Cont’d.
Molecular Detection of Virulence Genes from E.coli Isolates 

Figure 2: Agarose Gel Electrophoresis of Virulence Genes Detected from E. coli Isolates
Legend: L=ladder, N= negative control, P= positive control, bp= base pairs, Lane 1= ipaH,12
positive for EIEC, Lane 2= aggR, positive for EAEC, Lanes 3 and 5= est and elt, positive for
 Table 4: Distribution of Virulence Genes Detected in Isolates of
E. coli from Diarrhoeic and Non-diarrhoeic under Five Children
in Katsina State
Virulence Genes No. Detected Percentage of Occurrence
(%)
eae 6 13.95
aggR 25 58.14
est 3 6.98
elt 4 9.30
ipaH 5 11.63
Total 43 100.0

n-number, %-per cent

13
 Results Cont’d.
Table 5: Distribution of DEC Pathotypes based on Detected Virulence Genes
Virulence Genes

DEC eae est elt aggR stx1 stx2 ipaH Total

Pathotype DECn(%)
EPEC 6 - - - - - - 6(15.00)

ETEC - 3 4 - - - - 4(10.00)

EHEC - - - - - - - 0(0)

EAEC - - - 25 - - - 25(62.50)

EIEC - - - - - - 5 5(12.50)

Total 6 3 4 25 0 0 5 40(100.0)

Key: EPEC=Enteropathogenic E. coli, EIEC= Enteroinvasive E. coli, EAEC=Enteroaggregative

E. coli Enterotoxigenic E. coli, %= per cent, DEC= Diarrhoeagenic Escherichia coli,14-=
negative
Table 6: Distribution of DEC Pathotypes among under Five Years
Children in Katsina State According to Gender
Pathotypes
χ2 Test
  EPEC EAEC ETEC EIEC Total DEC P-value
n(%)
Diarrheic           0.037*
Subjects
Gender
Male 0(0) 10(14.49) 1(1.45) 1(1.45) 12(17.39)  
Female 5(7.25) 12(17.39) 2(2.90) 2(2.90) 21(30.43)  
Totald 5(7.25) 22(31.38) 3(4.35) 3(4.35) 33(47.83)  
Control Subjects           0.021*
Gender
Male 1(1.45) 1(5.88) 0(0) 0(0) 2(11.74)  
Female 0(0) 2(11.74) 1(5.88) 2(11.74) 5(7.25)  
Totalc 1(1.45 3(17.65) 1(5.88) 2(11.74) 7(41.18)  
Grand Total 6(15.00) 25(62.50) 4(10.00) 5(12.50) 40(100.0)  

Key: EPEC=Enteropathogenic E. coli, EIEC= Enteroinvasive E. coli, EAEC=Enteroaggregative E.

coli Enterotoxigenic E. coli, %= per cent, DEC= Diarrhoeagenic Escherichia coli, χ2 =Chi square,
15
Table 7: Distribution of DEC Pathotypes among under Five Years
Diarrheic and Non-diarrheic Children in Katsina State According to Age
Pathotypes χ2 Test
  EPEC n(%) EAEC n(%) ETEC n(%) EIEC n(%) Total DEC P-Value

n(%)
Diarrheic           0.045*
Age group
(years)
0-1 3(4.35 11(15.94) 1(1.45) 1(1.45) 16(23.19)  
2-3 2(6.45) 9(13.04 2(6.45) 2(6.45) 15(21.74)  
<5 0(0) 2(6.45) 0(0) 0(0) 2(6.45)  
Totald 5(7.25) 22(31.88) 3(4.35) 3(4.35) 33(47.83)  
Control            
Age group
(years)
0-1 0(0) 1(5.88) 1(1.45) 2(11.74) 4(23.53) 0.753
2-3 1(5.88) 2(11.74) 0(0) 0(0) 3(17.65)  
<5 0(0) 0(0) 0(0) 0(0) 0(0)  
Totalc 1(5.88) 3(17.65) 1(1.45) 2(11.74) 7(41.18)  
Grand Total 6(15.00) 25(62.50) 4(10.00) 5(12.5) 40(100.0)  
Key: EPEC=Enteropathogenic E. coli, EIEC= Enteroinvasive E. coli, EAEC=Enteroaggregative E. coli
Enterotoxigenic E. coli, %= per cent, DEC= Diarrhoeagenic Escherichia coli, χ2 =Chi square,
16 *=
statistically significant.
 Table 8: Antibiotic Susceptibility Profile of DEC Pathotypes Isolated
from under Five Children in Katsina State
Antibiotic Disc content S n(%) I n(%) R n(%) Total

(µg)

Ciprofloxacin 5 25(62.50) 9(22.50) 6(15.00) 40

Augmentin 30 28(70.00) 5(12.50) 7(17.50) 40

Tobramycin 10 23(57.50) 10(25.00) 7(17.50) 40

Ampicillin 10 6(15.00) 1(2.50) 33(82.50) 40

Ceftriaxone 30 19(47.50) 11(27.50) 10(25.00) 40

Tetracycline 30 10(25.00) 9(22.50) 21(52.50) 40

Key: S= Sensitive, I= Intermediate, R= Resistant, µg= microgram, n=

number
17
 25
24

20

17
15

10

5
4
2
1
0
Total MDR EAEC EIEC EPEC ETEC

Figure 3: Multidrug Resistance among DEC Pathotypes

Key: MDR= Multidrug Resistance, EPEC=Enteropathogenic E. coli, EIEC= Enteroinvasive E. coli,
18
EAEC=Enteroaggregative E. coli Enterotoxigenic E. coli
 Discussion
 Diarrhoea is one of the leading causes of morbidity and
remains the second leading cause of death in children
younger than 5 years globally, particularly in developing
countries. (Mandeel et al., 2022).
 Diarrhoeagenic Escherichia coli (DEC) are one of the major
causes of diarrhoea in Nigeria (Onanuga et al., 2014).
 In this study, the prevalence of DEC pathotypes was
significantly higher among the children with diarrhoea
(47.83%) than those without diarrhoea (41.18%).
 Similar results have also been reported from other parts of
Nigeria, for example, from Kano State , FCT Abuja, Ile Ife,
Osun State and, of course, from other countries across the
world, including Switzerland, Iraq, Burkina Faso( Saka et al.,
2019, Onanuga et al., 2014, Pabst et al., 2003, Mandeel 19et
 Discussion Cont’d.
 The results of the current study indicated that EAEC (31.88%) is the
most frequently detected diarrhoeagenic E. coli pathotype among
under five diarrheic children in Katsina State and 17.65% among non-
diarrheic group followed by EPEC (7.25%) and (5.88%), respectively.

 This correlates with the findings of Saka et al. (2019) and that of
Onanuga et al. (2016).
 In the present study, highest resistance was observed in ampicillin
antibiotic (82.50%). This corresponds with high resistance rate (85%)
of diarrhoeagenic E. coli to ampicillin reported by Ochoa et al. (2009)
in Peru and 82.90% reported by Saleh et al. (2021).
 Highest susceptibility to Augmentin (70.0%%) by the isolates was
observed in this study followed by Ciprofloxacin (62.50%). This is
however contrary to 100% Augmentin resistance rate reported by
Ugwu et al. (2017).
20
 Conclusion
The findings of this study revealed that
enteroaggregative E. coli (EAEC) and
enteropathogenic E. coli (EPEC) are the most
circulating DEC pathotypes among under five years
children in Katsina State.

The DEC pathotypes had highest resistance to

ampicillin antibiotic (82.50%).

Augmentin antibiotic was found to be the most

potent drug (70.0%) against the DEC strains. EAEC
was the most MDR DEC pathotype. 21
Selected References
Atimati, A. O., & Eki-Udoko, F. E. (2022). Diarrhoea prevalence,
characteristics and outcome among children admitted into the
emergency ward of a tertiary hospital in Southern Nigeria. Annals
of Clinical and Biomedical Research, 3(2). https://
doi.org/10.4081/acbr.2022.218
Onanuga, A., Igbeneghu, O. and Lamikanra, A. (2014). A Study of the
Prevalence of Diarrhoeagenic Escherichia coliin Children from
Gwagwalada, Federal Capital Territory, Nigeria. Pan African
Medical Journal, 17:146.
Saka, H.K., Dabo, N.T., Muhammad, B,,García-Soto S., Ugarte-Ruiz, M.
and Alvarez, J.(2019) Diarrheagenic Escherichia coli Pathotypes
From Children Younger Than 5 Years in Kano State, Nigeria.
Front. Public Health 7:348. doi: 10.3389/fpubh.2019.00348
WHO (2018). Outbreaks of E. coli in Europe. Accessed on 12th August,
2022. 22
Thanks for Listening

23