Prevalent phenotypic and genotypic profile of Enterotoxigenic Escherichia coli among
Iranian children
Shahram Nazarian, Seyed Latif Mousavi Gargari*,Iraj Rasooli, Masoome Alerasol ,Samane
Bagheri,Shakiba Darvish Alipoor
Department of Biology, Shahed University, Tehran-Qom Express Way, Opposite Imam
Khomeini's Shrine, Tehran-3319118651, Iran
Category of paper: Original articles
Running title: Phenotypic and genotypic ETEC profile in Iran
*
Corresponding author: Department of Biology, Shahed University, Tehran-Qom Express
Way, Opposite Imam Khomeini's shrine, Tehran-3319118651, Iran
Tel,: +98(21)51212600; Fax: +98(21)51212601 E-mail address: slmousavi@shahed.ac.ir
1
Abstract
Background: Enterotoxigenic Escherichia coli (ETEC) are the most common causes of diarrhea
among children. ETEC strains express colonization factors (CFs) mediating adherence to the
small intestinal epithelium and entrotoxin inducing diarrhea. The present work was designed to
characterize the phenotypes and genotypes of ETEC associated with diarrhea among Iranian
children.
Methods:Stool samples from 422 affected children were collected for microbiological testing.
Multiplex PCR technique was employed for the detection of the entrotoxin and CFs genes of
ETEC strains. Phenotypic detection of the toxins and CFs was used by immunoassay
techniques.The strains were analyzed for serogroups, antimicrobial resistance and entrotoxin
activity.
Results: Most of ETEC isolates were positive for heat labile and heat stable toxins. CFA/I, CS3,
CS2 and CS5 were detected from some of the clinical isolates. 33.3% of the isolates did not
express CFs. The majority of ETEC isolates were identified as O127 and O128 serotypes. 57%
of the strains were resistant to more than one antimicrobial agent. Enterotoxin activity was
confirmed by Chinese hamster ovary cell assay, Rabbit ileal loop and adenylate cyclase
activation tests.
Conclusion:Phenotypic and genotypic characterization of regional ETEC could help evaluate the
pathogenicity of ETEC in order to efficiently reduce the burden of illness brought about by
ETEC. This may lead to development of effective prophylactic measures.
Keywords
Enterotoxigenic Escherichia coli ;Diarrhea; Virulence gene ; Multiplex PCR
2
Introduction
Diarrheagenic E.coli strains are known to cause gastrointestinal illness in humans.1 ETEC is an
important causative agent of diarrheal disease
associated with morbidity and mortality in
children up to 5 years of age.2 ETEC strains have two major virulence determinants: the heatlabile toxin (LT) and heat-stable toxin (ST) and the colonization factors (CFs).3 To cause
diseases, ETEC must adhere to the epithelium of the small intestine by means of CFs. Secretory
diarrhea is then produced due to the effects of the enterotoxins. Several types of colonization
factor antigens,coli surface antigens (CS) and putative colonization factors(PCFs) have been
identified of which the most common ones are: CFA/I, CS1 to CS6 and CS21.
3-5
Since ETEC
strains have diverse toxins or CFs, therefore it is important to characterize various clinical ETEC
isolates with respect to their virulence profiles.
Due to increasing antibiotic resistance among ETEC strains, the knowledge of antimicrobial
resistance patterns in different countries is important when prescribing antibiotics and can serve
as a guide for implementing public health interventions against diarrheal disease. 4,6 ETEC
phenotype variability across regions, populations, complicates these efforts over time.3 Gupta et
al. highlighted a need for more information on the distribution of ETEC phenotypes.7 Iran is one
of those world regions where ETEC has not been deeply characterized. Hence the present work
was designed to characterize the phenotypes and genotypes of enterotoxigenic Escherichia coli
associated with diarrhea among Iranian children less than five years of age. With respect to
migration of people from surrounding countries to Iran and spread of ETEC infection through
these routes, the findings are expected to characterize the bacteria transmitted from neighboring
countries with low-Human Development Index.
3
Methods
Bacterial strains
The specimens were obtained from children under 5 years of age. The study was conducted in
Iranian communities between April 2009 to October 2011. After initial screening, 422 stool
samples were investigated for the presence of ETEC in samples from central, south and north
regions of IRAN.
Genotypic detection of virulence factors
ETEC strains were identified using primers for LT, STp and STh designed to amplify toxin
encoding genes (Table 1). Once strains were confirmed by PCR as ETEC, they were tested
sequentially to detect genes encoding CFAs with primers listed in Table 1. Reverse transcriptase
(RT) was used to analyze CF mRNA expression under culture conditions at 37oC. RT-PCR was
conducted using gene specific first strand cDNA from total RNA.
Serotyping
The O serotypes of E.coli strains were determined by agglutination tests in a commercially
available serotype kit (Bahar-afshan, Iran) according to the manufacturer’s instruction.
Antimicrobial resistance phenotyping
The commercial grade antimicrobial disks (ampicillin, nalidixic acid, chloramphenicol,
tetracycline, ciprofloxacin, gentamicin, cefoxitin, cefalothin, trimethoprim/sulfamethoxazole) .
ETEC strains were analyzed for their antimicrobial susceptibilities by disk diffusion method
according to The National Committee for Clinical Laboratory Standards breakpoints. 8
Phenotypic detection of toxins and colonization factors
4
ST production of the isolates was determined by E. COLIST-EIA kits (Denka Seiken, Tokyo,
Japan). Ganglioside-GM1-ELISA to detect LT was performed. Microtiter wells were coated with
GM1. 100 µl from each of the E. coli strains in LB broth was added to wells and incubated
overnight at 37°C. The released LT bound to GM1 was detected with rabbit anti cholera toxin
antiserum and goat anti rabbit immunoglobulin conjugated to horseradish peroxidase (Dako,
Roskilde, Denmark) using H2O2 and OPD (O-phenylenediamine dihydrochloride) as substrates
(Sigma Aldrich). 9Only isolates confirmed to be ETEC by ELISA were further tested for the
expression CFs by an immunodot blot assay. 9
Rabbit ileal loop test
New Zealand withes rabbits (1.5 kg) were kept in a starvation condition for 48 h. After general
anesthesia, 500 µl aliquots from E.coli strains cultivated in Brain Heart Infusion (BHI) broth
containing 107 cfu/ml were injected into each 6 loop in mid portion of the ileum. The animals
were sacrificed 18 h later, and the ratio of fluid accumulation against loop length (g/cm) was
calculated as an index of enterotoxigenicity.10 Research was conducted in compliance with the
Animal Welfare Act and regulations related to experiments involving animals.
Adenylate cyclase activation
The ability of the toxin to stimulate adenylate cyclase was determined by incubating CHO cells
with the toxin and measuring the amount of cyclic AMP (cAMP). CHO cells were seeded in 24well sterile culture plates at a density of 5 × 104 cells/well and were grown overnight to
approximately 80% confluence. Bacteria-free BHI culture supernatant was added and the plates
were incubated in a CO2 incubator. After a wash with PBS, the cells were lysed with 0.1 M HCl
and the lysate was centrifuged.
11
The supernatant was collected and tested for intracellular
cAMP levels using a cAMP immunoassay kit (Abnova).
5
ETEC binding assay
ETEC strains were subjected to a Caco-2 adhesion test with the method described by Taniguchi
et al. The number of ETEC cells adhering to each Caco-2 cell was counted and the adhesion
index was determined by examining 100 Caco-2 cells.12
Statistical analyses
Binding of the bacteria to the cells was expressed as the mean percentage of cells with bound
bacteria ± standard deviation. Results were obtained from three different experiments.
Results
Clinical data
A total of 422 samples were collected and screened for presence of E.coli strains.
295 of the fecal samples (69.9%) were positive for E.coli. Twenty one (4.97%) of samples was
detected as ETEC positive. ETEC affected patients had an average age of 28.23 months, with
42.85 % (9 Nos.) under 24 months old (Table 2). The most common symptoms were watery
stools, vomiting and abdominal pain. Sex distribution among the children affected with ETEC
were 12 female (57.14%), and 9 male (42.85%) (Table 2).
Genotypic characterization of ETEC isolates
The ETEC strains analyzed for enterotoxin genes by multiplex PCR lead to detection of LT
(one), STH (four), LT/STH (15) and LT/STP (one) strains with no non specific product. Agarose
gel electrophoresis of PCR products showed the genes for colonization factors with the expected
size. Colonization factor antigen I was the most common CF, followed by CS3, CS2 and CS5.
Some ETEC strains (33.3%) did not express any detectable colonization factors (CFA/I,CS1CS6) (Table 2). Further analysis of colonization factors with RT-PCR showed mRNA production
from genes encoding colonization factor.
6
Phenotypic detection of colonization factors
Dot blot analysis based on Abs for colonization factors was used. Comparisons of strains for
colonization factors by dot blot analysis and PCR showed a high level of agreement. Only one
PCR positive strain for CFA/I was not detected by dot blot assay.
Examination of Caco-2 cell monolayers revealed that ETEC H10407 cells and CFA/I, CS2, CS3,
CS5 positive clinical strains were distributed on the Caco-2cells. Almost all the Caco-2 cells
were observed to bind one or more bacteria with an adhesion index of 14.5 bacteria per cell for
H10407 and 13.5± 0.8, 10.6±0.9, 8.7±0.8 and 6±0.6 bacteria per cell for CFA/I, CS2, CS3, CS5
positive clinical strains respectively.
Phenotypic detection of enterotoxins
ST and LT expression by ST detection kit and GM1-ELISA are given in Table 2. LT/ST
producing ETECs were the most frequently detected strains. One ETEC strain was found to be
eltB positive by multiplex PCR but LT negative by GM1-ELISA.Stimulation of the cAMP levels
in cell culture after addition of bacterial culture supernatant indicated LT release and its toxic
activity (Fig. 1). Fluid accumulation in ligated-gut-loop further confirmed the in vivo entrotoxic
activity of isolated strains. The loops inoculated with clinical strains had a fluid accumulation
ratio of 1.31 - 1.89.
Serotyping
Serotyping of ETEC isolates for detecting somatic antigens are presented in Table 2. A wide
array of O-type were observed among the 16 (76.19 %) of the total ETEC isolates. Serogroups
O20, O128, O6 and O8 typically associated with ETEC, were also detected. The majority of
ETEC isolates were serotyped as O127 and O86.
7
Antimicrobial drug susceptibility
Antimicrobial drug susceptibility test results showed that of ETEC isolates 38% were resistant to
tetracycline, 71.4% to ampicillin, 19% to chloramphenicol, 57.1% to sulfamethoxazoletrimethoprim, 19% to cephalothin, 47.6% amoxicillin–clavulanate and 9.5% to ciprofloxacin.
Most strains were not resistant to Ciprofloxacin and all the tested strains were susceptible to
nalidixic acid and gentamicin. In our study, 57% of the isolates were resistant to more than one
antimicrobial agent and 14.28% of strains were resistant to more than four antibiotics.
Discussion
ETEC is one of the most common bacteria responsible for diarrhea in different parts of the world
.Prevalence of ETEC from Iranian patients with diarrhea referred to hospitals and medical
centers in the central, south and north of IRAN was studied in the present work. Multiplex PCR
findings were supported by toxin ELISA results for all ETEC strains. However, one strain was
found to be eltB positive by multiplex PCR but LT negative by ELISA. It is likely that this strain
contains silent genes or enterotoxin level inadequate for the detection by ELISA. The results
showed that LT/ST-ETEC was the most commonly detected ETEC enterotoxin subgroup. A total
of 50 studies assessing the relative frequency of the three ETEC enterotoxin subgroups indicated
that ST-ETEC was the most common enterotoxin subgroup.7 In Iran, Katouli et al. showed that
ST was the most frequent toxin type (60%) followed by LT/ST and LT.13 In fact, these studies on
ETEC strains isolated shows that there has been a change in the prevalence of enterotoxin toxin
types from 1992 until now.
PCR method for detection of colonization factors is high sensitive and affordable, however the
occurrence of mutations in the primer binding site can cause errors in PCR amplification.9 In
phenotypic detection of colonization factors, phenotypic silencing could be due to storage and
subculture of ETEC isolates. This feature may have a genetic explanation such as loss of
8
regulatory genes.
9
In our ETEC strains, CFA/I and CS3 were the most common CF
combinations detected with the multiplex PCR. Such an observation was reported in a review by
Isidean and co workers that CS3 alone was the predominant phenotype
expressing ETEC.
3
among CFA/II-
CFA/I-expressing strains were common in different parts of the world.3
Shahrokhi et al. showed that CFA/I and CFA/IV were the most common CF types within ETEC
isolates.
14
Although CS6-producing ETEC is the most prevalent strain of ETEC in various
developing countries7,15, none of the ETEC isolates in our study had CS6 antigen. The CFs were
also detected by dot blot indicating that the genes were translated and displayed on bacterial
surface. The local and regional enterotoxin type distributions are related to the estimation of
vaccine coverage against ETEC strains. Using a multivalent vaccine containing LT toxoid, and
the most common CFs as a model and the data from this study, the estimated vaccine coverage
rate in children in IRAN is expected to be 80.95% against toxin and 66.6% against CFs. High
resistance to antibiotics in E.coli has been previously reported in Iranian isolates. 14,16 The pattern
of multidrug resistance in our isolates is similar to those reported by Aslani et.al.16 The results
indicate wide spread antimicrobial property of ETEC strains suggesting limited antimicrobial
treatment to be considered against ETEC diarrhea unless for more severe or persistent cases.
This attempt would hamper further development of antimicrobial resistance. There was a wide
diversity of O antigens represented among the ETEC isolated from children in this study. Of a
great variety of O serogroups identified in ETEC, serotypes O6, O8, O78, O128 and O153 are
the most common antigens.17 This result further confirms that ETEC from different geographical
areas belonged to a relatively large number of serotypes and ETEC serotypes may change over
time.17,18
Ecology and pathogenicity of ETEC help reduce burden of the illness. Effective prophylactic
measures could develop by phenotypic and genotypic characterization of regional ETEC.
9
Acknowledgment
The authors wish to thank Shahed University for the sanction of grants to conduct the present
study. We also declare no conflict of interests.
References
1. Vilchez S, Reyes D, Paniagua M, Bucardo F, Mollby R, Weintraub A. Prevalence of
diarrhoeagenic Escherichia coli in children from Leon, Nicaragua. J Med Microbiol 2009;
58:630-7.
2. Franca EL, Bitencourt RV, Fujimori M, Cristina de Morais T, de Mattos Paranhos
Calderon I, Honorio-Franca AC. Human colostral phagocytes eliminate enterotoxigenic
Escherichia coli opsonized by colostrum supernatant. J Microbiol Immunol Infect 2011;
44:1-7.
3. Isidean SD, Riddle MS, Savarino SJ, Porter CK. A systematic review of ETEC
epidemiology focusing on colonization factor and toxin expression. Vaccine 2011;
29:6167-78.
4. Rodas C, Mamani R, Blanco J, Blanco JE, Wiklund G, Svennerholm AM et al.
Enterotoxins,
colonization
factors,
serotypes
and
antimicrobial
resistance
of
enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with
diarrhea in Bolivia. Braz J Infect Dis 2011; 15:132-7.
5. Svennerholm AM, Lundgren A. Recent progress toward an enterotoxigenic Escherichia
coli vaccine. Expert Rev Vaccines 2012; 11:495-507.
10
6. Garcia PG, Silva VL, Diniz CG. Occurrence and antimicrobial drug susceptibility patterns
of commensal and diarrheagenic Escherichia coli in fecal microbiota from children with
and without acute diarrhea. J Microbiol 2011; 49:46-52.
7. Gupta SK, Keck J, Ram PK, Crump JA, Miller MA, Mintz ED. Part III. Analysis of data
gaps pertaining to enterotoxigenic Escherichia coli infections in low and medium human
development index countries, 1984-2005. Epidemiol Infect 2008; 136:721-38.
8. Clinical Laboratory Standards Institute (CLSI).Performance standards for antimicrobial
susceptibility testing; Eighteenth informational supplement. CLSI document M100-S18 .
Wayne, PA, USA: CLSI; 2008.
9. Sjoling A, Wiklund G, Savarino SJ, Cohen DI, Svennerholm AM. Comparative analyses of
phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins
and colonization factors. J Clin Microbiol 2007; 45:3295-301.
10. De SN, Chatterje DN. An experimental study of the mechanism of action of Vibrio
cholerae on the intestinal mucous membrane. J Pathol Bacteriol 1953; 66:559-62.
11. Tokuhara D, Yuki Y, Nochi T, Kodama T, Mejima M, Kurokawa S et al. Secretory IgAmediated
protection
against
V.
cholerae
and
heat-labile
enterotoxin-producing
enterotoxigenic Escherichia coli by rice-based vaccine. Proc Natl Acad Sci 2010;
107:8794-9.
12. Taniguchi T, Akeda Y, Haba A, et al. Gene cluster for assembly of pilus colonization factor
antigen III of enterotoxigenic Escherichia coli. Infect Immun. 2001; 69:5864-73.
11
13. Katouli M, Jaafari A, Ketabi GR. The role of diarrhoeagenic Escherichia coli in acute
diarrhoea1 diseases in Bandar-Abbas, Iran. J Med Microbiol 1988; 27:71-4.
14. Shahrokhi N, Bouzari S, Jafari A. Comparison of virulence markers and antibiotic
resistance in enterotoxigenic Escherichia coli isolated ten years apart in Tehran. J Infect
Dev Ctries 2011; 5:248-54.
15. Shaheen HI, Abdel Messih IA, Klena JD, Mansour A, El-Wakkeel Z, Wierzba TF et al.
Phenotypic and genotypic analysis of enterotoxigenic Escherichia coli in samples obtained
from Egyptian children presenting to referral hospitals. J Clin Microbiol 2009; 47:189-97.
16. Aslani MM, Ahrabi SS, Alikhani YM, Jafari F, Zali RM, Mani M. Molecular detection and
antimicrobial resistance of diarrheagenic Escherichia coli strains isolated from diarrheal
cases. Saudi Med J 2008; 29:388-92.
17. Wolf MK. Occurrence, distribution, and associations of O and H serogroups, colonization
factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin Microbiol Rev 1997;
10:569-84.
18. Shaheen HI, Khalil SB, Rao MR, Abu ER, Wierzba TF, Peruski LF, Jr. et al. . Phenotypic
profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in
rural Egypt. J Clin Microbiol 2004; 42:5588-95.
12
Figure legends
Figure 1. Toxicity activity detected in cAMP ELISA. Results carried out with H10407 and E.coli
25922 strains as positive and negative controls. WT1-17: ETEC strain containing LT toxin gene.
13
Table 1 Primers sequences used for amplification of toxin and colonization factor of ETEC
virulence
Target
factor
gene
primer sequence
Forward
Product
GenBank
(bp)
accession no
Reverse
LT
eltBI
TCTTTATGATCACGCGAGAGGA
TCTTCTCTCCATAGCTTGGTGATC
412
S60731.1
STH
estA2-4
TGCTAAACCAGTAGAGTCTTCAAAAG
TAATAGCACCCGGTACAAGCAG
157
M29255.1
STP
estA1
AGGTAACATGAAAAAGCTAATGTTG
AGGCAGGATTACAACAAAGTTCA
209
M58746.1
CFA/1
cfaB
ACAATGTTTGTAGCAGTGAGTGC
AGATACTACTCCTGAATAGTTTCCTG
450
M55661.1
CS1
csoA
ACTGTTGACCTTCTGCAATCTGATG
ACGTTGACTGAGTCAGGATAATTG
403
AY513487.1
CS2
cotA
ACTATCGATCTGATGCAATCTGATG
TACAATATTAGTTTGCTGGGTGC
355
Z47800.1
CS3
cstA
TCCTCAGGATAATTTAACATTATCC
ACCTTCAGTGGTAATAAACTTAACTG
304
X16944.1
CS4
csaB
TAGTAGTTTACCTACTGCTGTAGAATTAA
GACGTTCCAAATTTAATTCTGC
252
AF296132.1
CS5
csfA
TCCTTCCGCTCCCGTTA
TAGCTGACGTGTCACGCGT
206
AJ224079.2
CS6
cssA
TGATGATTCCCAATCAATAATCTAC
AGGTATTTCTTTATCTCCGCATG
142
U04844.1
CS21
lngA
TGCTGGAAGTTATCATTGTTCTTG
ACTTAGAATTTTGTCTGCAGTCAAG
569
AF004308.1
14
Table 2 Characterized ETEC strains collected from Iranian children with diarrhea
Patient ID
Age
Children
Sex
19
29m
F
25
28m
F
51
36m
F
56
28m
M
71
22m
F
85
30m
M
89
24m
M
105
20m
F
130
28m
M
141
34m
F
152
20m
M
198
34m
F
210
28m
M
212
24m
F
320
18m
F
354
20m
M
362
26m
M
385
30m
F
401
28m
F
403
24m
F
420
20m
M
Diarrhea
Vomiting
Watery
stool
Watery
stool
Watery
stool
Watery
stool
Watery
stool
Watery
stool
Watery
stool
Mucosal
stool
Watery
stool
Mucosal
stool
Watery
stool
Watery
stool
Watery
stool
Blood
stool
Watery
stool
Watery
stool
Mucosal
stool
Watery
stool
Mucosal
stool
Watery
stool
Watery
stool
+
Characteristics of strains
Toxins
CFs
O
serotypes
LT/STH
CFA/I
O127
+
LT
CFA/I
NT
+
LT/STH
CS2
O6
+
LT/STH
CS3
O86
+
LT/STH
+
LT/STH
CS3
O86
-
LT/STH
CFA/I
NT
-
LT/STH
-
O55
+
LT/STH
+
STH
+
-
CS3
O26
O86
-
O127
LT/STH
CFA/I
O127
-
LT/STH
-
O111
+
STH
CFA/I
NT
+
LT/STH
CFA/I
O78
+
LT/STP
-
O114
-
STH
CFA/I
NT
-
STH
CFA/I
O127
-
LT/STH
CFA/I
O127
-
LT/STH
CS5
O128
+
LT/STH
-
NT
+
LT/STH
-
O20
15