Multi-Drug-Resistant Diarrheagenic Escherichia coli Pathotypes in Pediatric Patients with Gastroenteritis from Central Iran

Figures & data

Table 1 The Primers Used in This Study

References	Annealing Temperature	Amplicon Size (bp)	Sequence 5ʹ→3ʹ	Primer	Target Gene Description
^Citation10	67	510	5-GCGTCTGTTGACTGGCAGGTGGTGG-3 5-GTTGCCCGCTTCGAAACCAATGCCT-3	uidA-F uidA-R	E. coli
^Citation11	57.5	630	5-CTGGCGAAAGACTGTATCAT-3 5-CAATGTATAGAAATCCGCTGTT-3	pCVD432-F pCVD432-R	EAEC
^Citation12	48	881	5-CCCGAATTCGGCACAAGCATAAGC-3 5-CCCGGATCCGTCTCGCCAGTATTCG-3	eae-F eae-R	EPEC
^Citation13	55	274	5-ACGGCGTTACTATCCTCTC-3 5-TGGTCTCGGTCAGATATGTG-3	elt-F elt-R	ETEC
^Citation13	55	170	5-TCTTTCCCCTCTTTTAGTCAGTC-3 5-CAGCACAGGCAGGATTAC-3	st-F st-R	ETEC
^Citation4	50	650	5-GGTATGATGATGATGAGTCCA-3 5- GGAGGCCAACAATTATTTCC-3	ial-F ial-R	EIEC
^Citation12	48	518	5-GAGCGAAATAATTTATATGTG-3 5-TGATGATGGCAATTCAGTAT-3	stx-F stx-R	EHEC
^Citation8	55	344	5-AAATCTGCCCGTGTCGTTGGT-3 5-GCCATACCTACGGCGATACC −3	gyrA-F gyrA-R	Fluoroquinolone
^Citation8	55	168	5-CTGAATGCCAGCGCCAAATT-3 5-GCGAACGATTTCGGATCGTC-3	parC-F parC-R
^Citation8	60	656	5-TGGAAACCTACAATCATACATATCG-3 5-TTAGTCAGGATAAACAACAATACCC-3	qnrS-F qnrS-R
^Citation8	60	593	5-GATAAAGTTTTTCAGCAAGAGG-3 5-ATCCAGATCGGCAAAGGTTA-3	qnrA-F qnrA-R
^Citation8	53	264	5-GTTGGCGAAAAAATTGACAGAA-3 5-ACTCCGAATTGGTCAGATCG-3	qnrB-F qnrB-R
^Citation8	55	160	5-CAGTGGACATAAGCCTGTTC-3 5-CCCGAGGCATAGACTGTA-3	Int1-F Int1-R	Integrase1
^Citation8	55	288	5-TTGCGAGTATCCATAACCTG-3 5-TTACCTGCACTGGATTAAGC-3	Int2-F Int2-R	Integrase2
^Citation8	59	979	5-GCCTCCGGCAGCGACTTTCAG-3 5-ACGGATCTGCCAAACCTGACT-3	Int3-F Int3-R	Integrase3
^Citation8	65	331	5-TCACCGAGGACTCCTTCTTC-3 5-CAGTCCGCCTCAGCAATATC-3	Sul1-F Sul1-R	Sulfonamide resistance
^Citation8	58	435	5-CCTGTTTCGTCCGACACAGA-3 5-GAAGCGCAGCCGCAATTCAT-3	Sul2-F Sul2-R
^Citation26	55	370	5-GCCCTACACAAATTGGGAGA-3 5-CTGCGGTACCACTGCCACAA-3	qac-F qac-R	Quaternary ammonium compounds
^Citation8	44	425	5-AAAGATGCTGAAGATCA-3 5-TTTGGTATGGCTTCATTC-3	TEM-F TEM-R	β-Lactamase ESBL+
^Citation8	62	304	5-GCGAAAGCCAGCTGTCGGGC-3 5-GATTGGCGGCGCTGTTATCGC-3	SHV-F SHV-R
^Citation8	52	670	5-AAGACTGGGTGTGGCATTGA-3 5-AGGCTGGGTGAAGTAAGTGA-3	CTX-M1-F CTX-M1-R
^Citation8	60	552	5-CGACGCTACCCCTGCTATT-3 5-CCAGCGTCAGATTTTTCAGG-3	CTX-M2-F CTX-M2-R
^Citation8	55	307	5-CGCTTTGCCATGTGCAGCACC-3 5-GCTCAGTACGATCGAGCC-3	CTX-M8-F CTX-M8-R
^Citation8	50	355	5-TACCGCAGATAATACGCAGGTG-3 5- CAGCGTAGGTTCAGTGCGATCC-3	CTX-M14-F CTX-M14-R
^Citation8	55	955	5-CACACGTGGAATTTAGGGACT-3 5-GCCGTCTAAGGCGATAAACA-3	CTX-M15-F CTX-M15-R
^Citation8	58	758	5′-GCACTTAGCCACCTATACGGCAG-3′ 5′-GCTTTTCAAGAATGCGCCAGG-3′	CMY-2-F CMY-2-R
^Citation8	64	190	5-AACATGGGGTATCAGGGAGATG-3 5-CAAAGCGCGTAACCGGATTGG-3	Fox-F Fox-R	β-Lactamase AmpC+
^Citation8	64	520	5-GCTGCTCAAGGAGCACAGGAT-3 5-CACATTGACATAGGTGTGGTGC-3	Mox-F Mox-R
^Citation8	64	405	5-AACTTTCACAGGTGTGCTGGGT-3 5-CCGTACGCATACTGGCTTTGC-3	DHA-F DHA-R
^Citation8	64	346	5-AACAGCCTCAGCAGCCGGTTA-3 5-TTCGCCGCAATCATCCCTAGC-3	ACC-F ACC-R
^Citation8	64	462	5-TGGCCAGAACTGACAGGCAAA-3 5-TTTCTCCTGAACGTGGCTGGC-3	CIT-F CIT-R

Abbreviations: EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; EIEC, entero-invasive E. coli; EHEC, enterohemorrhagic E. coli.

Table 2 Total Number, Sex, and Age Characteristics of the Patients with E. coli Pathotypes

E. coli Pathotypes	DEC	EACE	EPEC	ETEC		EIEC	Stx Negative EHEC O157:H7
				LT	ST
Total number (%)	32/91 (35.1%)	11/32 (34.3%)	9/32 (28.1%)	6/32 (18.7%)		3/32 (9.3%)	3/32 (9.3%)
				4/6 (66.6%)	2/6 (33.3%)
Male	15/32 (46.8%)	4/11 (36.3%)	5/9 (55.5%)	4/6 (66.6%)		1/3 (33.3%)	1/3 (33.3%)
				3/4 (75%)	1/2 (50%)
Female	17/32 (53.1%)	7/11 (63.6%)	4/9 (44.4%)	2/6(33.3%)		2/3 (66.6%)	2/3 (66.6%)
				1/4 (25%)	1/2 (50%)
Average age	4 years and 6 months	3 years and 1 month	2 years and 9 months	4 years and 6 months		5 years and 8 months	4 years and 1 month
				3 years and 8 months	6 years
Youngest patient’s age	5 months	5 months	8 months	9 months		3 years and 5 months	1 year
				9 months	3 years
Oldest patient’s age	10 years	10 years	10 years	9 years		9 years	6 years
				9 years	9 years

Abbreviation: DEC, diarrheagenic Escherichia coli.

Table 3 Comparison of the Frequency, Clinical Symptoms, and Antibiotic Resistance of DEC and E. coli Pathotypes in This Study and in Other Studies

E. coli Pathotypes		Shiraz (Iran)	India	China (Throughout the Country)	Tehran (Iran)	Sudan	Iran’s North and Northwest Provinces	China (Southeastern)
DEC		43.6%	30.7%	5%	58.9%	48%	27%	14.1%
EAEC		23.6%	6.9%	1.6%	16.6%	43%	0%	62.5%
EPEC		10.9%	21.5%	1.6%	12.6%	29%	10.5%	15%
ETEC		3.6%	10.7%	1%	10.8%	18%	5.8%	17.9%
EIEC		5.5%	–	0.5%	–	9%	0%	0.3%
EHEC		0%	4.6%	0.3%	18.9%	–	3.5%	4%
The most abundant E. coli pathotype		EAEC 84.6%	EPEC 21.5%	EAEC and EPEC 1.6%	EHEC 18.9%	EAEC 43%	EPEC 10.5%	EAEC 62.5%
Clinical symptoms in patients with DEC	Mucus in the stool	–	–	65%	51.5%	–	–	–
	Abdominal pain	–	–	3.4%	–	–	–	–
	Vomiting	–	–	5.1%	52.6%	–	–	–
Antibiotic resistance in patients with DEC	Nalidixic acid	–	–	–	–	34%	–	–
	Ampicillin	93.8%	–	–	–	47%	–	91.8%
	Tetracycline	–	–	–	–	24%	–	57%
	Cotrimoxazole	77.1%	–	–	–	–	–	52.3%
	Cefotaxime	66.7%	–	–	–	–	–	35.7%
	Ceftriaxone	66.7%	–	–	–	–	–	–
	ESBL in DEC isolates	66.7%	–	–	–	–	–	34.5%
The most abundant ESBL genes		–	–	–	–	–	–	blaCTX-M 94.2%
References		^Citation16	^Citation14	^Citation27	^Citation11	^Citation28	^Citation29	^Citation30

Note: Not reviewed.

Table 4 Frequency of Clinical Symptoms in Pediatric Patients with E. coli Pathotypes

E. coli Pathotypes		Mucus in the Stool		Abdominal Pain		Vomiting		Fever		Blood in the Stool
DEC		28/32 (87.5%)		24/32 (75%)		19/32 (59.3%)		17/32 (53.1%)		10/32 (31.2%)
EAEC		10/11 (90.9%)		9/11 (81.8%)		8/11 (72.7%)		7/11 (63.6%)		4/11 (36.3%)
EPEC		9/9 (100%)		4/9 (44.4%)		5/9 (55.5%)		4/9 (44.4%)		1/9 (11.1%)
ETEC	LT	5/6 (83.3%)	3/4 (75%)	5/6 (83.3%)	3/4 (75%)	2/6 (33.3%)	1/4 (25%)	2/6 (33.3%)	1/4 (25%)	0%	0%
	ST		2/2 (100%)		2/2 (100%)		1/2 (50%)		1/2 (50%)		0%
EIEC		3/3 (100%)		3/3 (100%)		3/3 (100%)		3/3 (100%)		2/3 (66.6%)
Stx negative EHEC O157:H7		1/3 (33.3%)		3/3 (100%)		1/3 (33.3%)		1/3 (33.3%)		3/3 (100%)

Table 5 Phenotypic Antibiotic Resistance Rates in E. coli Pathotypes

Antibiotic	DEC n:32	EAEC n:11	EPEC n:9	ETEC n:6	EIEC n:3	Stx Negative EHEC O157:H7 n:3
Nalidixic acid	30 (93.7%)	11 (100%)	8 (88.8%)	5 (83.3%)	3 (100%)	3 (100%)
Ampicillin	29 (90.6%)	11 (100%)	8 (88.8%)	5 (83.3%)	3 (100%)	2 (66.6%)
Tetracycline	25 (78.1%)	10 (90.9%)	5 (55.5%)	5 (83.3%)	3 (100%)	2 (66.6%)
Cotrimoxazole	23 (71.8%)	10 (90.9%)	5 (55.5%)	3 (50%)	2 (66.6%)	3 (100%)
Chloramphenicol	18 (56.2%)	7 (63.6%)	4 (44.4%)	4(66.6%)	2 (66.6%)	1 (33.3%)
Ceftriaxone	18 (56.2%)	9 (81.8%)	5 (55.5%)	2 (33.3%)	1 (33.3%)	1 (33.3%)
Cefixime	18 (56.2%)	9 (81.8%)	5 (55.5%)	2 (33.3%)	1 (33.3%)	1 (33.3%)
Cefotaxime	18(56.2%)	9 (81.8%)	5 (55.5%)	2 (33.3%)	1 (33.3%)	1 (33.3%)
Ceftizoxime	12 (37.5%)	6 (54.5%)	4 (44.4%)	2 (33.3%)	0 (0%)	0 (0%)
Cefoxitin	9 (28.1%)	6 (54.5%)	2 (22.2%)	1 (16.6%)	0 (0%)	0 (0%)
Ceftazidime	9 (28.1%)	5 (45.4%)	4 (44.4%)	0 (0%)	0 (0%)	0 (0%)
Azithromycin	9 (28.1%)	4 (36.3%)	4 (44.4%)	0 (0%)	0 (0%)	1 (33.3%)
Ciprofloxacin	6 (18.7%)	2 (18.1%)	3 (33.3%)	0 (0%)	0 (0%)	1 (33.3%)
Gentamicin	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Imipenem	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
MDR	25 (78.1%)	9 (81.8%)	6 (66.6%)	4 (66.6%)	3 (100%)	3 (100%)
ESBL+	18 (56.2%)	9 (81.8%)	5 (55.5%)	2 (33.3%)	1 (33.3%)	1 (33.3%)
AmpC+	9 (28.1%)	6 (54.5%)	2 (22.2%)	1 (16.6%)	0 (0%)	0 (0%)

Abbreviations: MDR, multi-drug resistance; ESBL, extended spectrum-β-lactamase.

Figure 1 Phenotypic antibiotic resistance rates in E. coli pathotypes.

Abbreviations: MDR, Multi-drug resistance; ESBL, Extended spectrum-β-lactamase.

Table 6 Frequency of Antibiotic Resistance Genes Among E. coli Pathotypes

Resistance	Target Gene	DEC	EAEC	EPEC	ETEC	EIEC	Stx Negative EHEC O157:H7
Sulfonamide	Sul2	18/23 (78.2%)	9/10 (90%)	5/5 (100%)	2/3 (66.6%)	1/2 (50%)	1/3 (33.3%)
	Sul1	14/23(60.8%)	6/10 (60%)	4/5 (80%)	1/3 (33.3%)	1/2 (50%)	2/3 (66.6%)
ESBL+	blaTEM	18/18 (100%)	9/9 (100%)	5/5 (100%)	2/2 (100%)	1/1 (100%)	1/1 (100%)
	blaCTX-M-15	17/18 (94.4%)	8/9 (88.8%)	5/5 (100%)	2/2 (100%)	1/1 (100%)	1/1 (100%)
	blaCTX-M-1	12/18 (66.6%)	6/9 (66.6%)	4/5 (80%)	1/2 (50%)	1/1 (100%)	0%
	blaCTX-M-14	12/18 (66.6%)	7/9 (77.7%)	4/5 (80%)	1/2 (50%)	0%	0%
	blaSHV	6/18 (33.3%)	3/9 (33.3%)	3/5 (60%)	0%	0%	1/1 (100%)
	blaCTX-M-2	6/18 (33.3%)	3/9 (33.3%)	2/5 (40%)	1/2 (50%)	0%	0%
	blaCTX-M-8	0%	0%	0%	0%	0%	0%
	blaCIT	4/9 (44.4%)	2/6 (33.3%)	1/2 (50%)	1/1 (100%)	–	–
	blaDHA	4/9 (44.4%)	2/6 (33.3%)	1/2 (50%)	1/1 (100%)	–	–
	blaCMY-2	3/9 (44.4%)	1/6 (100%)	1/2 (100%)	1/1 (100%)	–	–
	blaACC	1/9 (11.1%)	1/6 (16.6%)	0%	0%	–	–
	blaFOX	0%	0%	0%	0%	–	–
	blaMOX	0%	0%	0%	0%	–	–
Fluoroquinolone	gyrA	20/30 (66.6%)	8/11 (72.7%)	7/8 (87.5%)	2/5 (40%)	1/3 (33.3%)	2/3 (66.6%)
	parC	18/30 (60%)	7/11 (63.6%)	7/8 (87.5%)	2/5 (40%)	1/3 (33.3%)	1/3 (33.3%)
	qnrS	18/30 (60%)	7/11 (63.6%)	6/8 (75%)	2/5 (40%)	1/3 (33.3%)	2/3 (66.6%)
	qnrA	2/30 (6.6%)	1/11 (9%)	1/8 (12.5%)	0%	0%	0%
	qnrB	0%	0%	0%	0%	0%	0%
Integrase	Int1	19/32 (59.3%)	6/11 (54.5%)	6/9 (66.6%)	4/6 (66.6%)	2/3 (66.6%)	1/3 (33.3%)
	Int2	13/32 (40.6%)	4/11 (36.3%)	7/9 (77.7%)	1/6 (16.6%)	0%	1/3 (33.3%)
	Int3	0%	0%	0%	0%	0%	0%
Quaternary Ammonium Compounds	qac	23/32 (71.8%)	8/11 (72.7%)	8/9 (88.8%)	3/6 (50%)	2/3 (66.6%)	2/3 (66.6%)

Figure 2 Frequency of antibiotic resistance genes among E. coli pathotypes.

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