Gut Commensal Escherichia coli, a High-Risk Reservoir of Transferable Plasmid-Mediated Antimicrobial Resistance Traits

Figures & data

Table 1 Target Genes, Sequences of PCR Oligonucleotide Primers and Expected PCR Product Size

Target Gene	Sequence (5’ – 3’)	Amplicon Size (bp)	Ta/Extension Time	Source
Resistance to beta-lactam antibiotics (β-lactamases encoding genes)
blaTEM	F: ATAAAATTCTTGAAGAC R: TTACCAATGCTTAATCA	1075	42°C/1 min	[66]
blaCTX-M1-like genes	F: TTAATTCGTCTCTTCCAGA R: CAGCGCTTTTGCCGTCTAAG	1042	45°C/1 min
blaCMY-like genes	F: ATGATGAAAAAATCGATATG R: TTATTGCAGTTTTTCAAGAATG	1146	45°C/1 min
Resistance to aminoglycoside antibiotics
aac(3)-II	F: TGAAACGCTGACGGAGCCTC R: GTCGAACAGGTAGCACTGAG	369	55°C/30 s	[66]
aac(6′)-Ib-cr	F: TTGCGATGCTCTATGAGTGGCTA R: CTCGAATGCCTGGCGTGTTT	482	55°C/30 s	[67]
Resistance to quinolones
qnrA	F: ATTTCTCACGCCAGGATTTG R: GATCGGCAAAGGTTAGGTCA	516	52°C/40 s	[67]
qnrB	F: GATCGTGAAAGCCAGAAAGG R: ATGAGCAACGATGCCTGGTA	476
qnrS	F: GCAAGTTCATTGAACAGGGT R: TCTAAACCGTCGAGTTCGGCG	428
Resistance to tetracyclines
tetA	F: GCTACATCCTGCTTGCCT R: CATAGATCGCCGTGAAGA	210	52°C/30 s	[68]
tetB	F: TTGGTTAGGGGCAAGTTTTG R: GTAATGGGCCAATAACACCG	600	52°C/40 s
Resistance to sulfonamides
sulI	F: TGGTGACGGTGTTCGGCATTC R: GCGAAGGTTTCCGAGAAGGTG	790	56°C/50 s	[66]
sulII	F: CGGCATCGTCAACATAACCT R: TGTGCGGATGAAGTCAGCTC	721	56°C/50 s
Type of E. coli pathotype (virulence-associated genes in E. coli)
EPEC: eaeA	F: AAACAGGTGAAACTGTTGCC R: CTCTGCAGATTAACCCTCTGC	454	52°C/30 s	[7]
EAEC: aggR	F: CTAATTGTACAATCGATGTA R: ATGAAGTAATTCTTGAAT	308	42°C/30 s
EAEC: pCVD432	F: CTGGCGAAAGACTGTATCAT R: CAATGTATAGAAATCCGCTGTT	630	55°C/40 s
STEC/EHEC: stx	F: GAGCGAAATAATTTATATGTG R: TGATGATGGCAATTCAGTAT	518	42°C/30 s	[69]
ETEC: est	F: TTAATAGCACCCGGTACAAGCAGG R: CCTGACTCTTCAAAAGAGAAAATTAC	147	52°C/30 s
ETEC: elt	F: TCTCTATGTGCATACGGAGC R: CCATACTGATTGCCGCAAT	322	52°C/30 s
EIEC: ipaH	F: GTTCCTTGACCGCCTTTCCGATACCGTC R: GCCGGTCAGCCACCCTCTGAGAGTAC	619	58°C/40 s

Abbreviations: PCR, polymerase chain reaction; E. coli; Escherichia coli; EPEC, enteropathogenic E. coli; EAEC, enteroaggregative E. coli; STEC, Shiga toxin-producing E. coli; ETEC, enterotoxigenic E. coli; EIEC, enteroinvasive E. coli; EHEC, enterohemorrhagic E. coli; Ta, annealing temperature; bp, base pair.

Table 2 Distribution of Antimicrobial Resistance Among Gut Commensal E. coli Isolates

Antimicrobial Agent	Sensitive No. (%)^a	Resistant No. (%)^a
Ampicillin	14 (25)	42 (75)
Amoxicillin-clavulanate	19 (33.9)	37 (66.1)
Cefoxitin	26 (46.4)	30 (53.6)
Ceftriaxone	15 (26.8)	41 (73.2)
Cefuroxime	16 (28.6)	40 (71.4)
Ceftazidime	16 (28.6)	40 (71.4)
Cefepime	17 (30.4)	39 (69.6)
Aztreonam	16 (28.6)	40 (71.4)
Ciprofloxacin	21 (37.5)	35 (62.5)
Levofloxacin	21 (37.5)	35 (62.5)
Gentamicin	32 (57.1)	24 (42.9)
Amikacin	38 (67.9)	18 (32.1)
Tetracycline	25 (44.6)	31 (55.4)
Trimethoprim/sulfamethoxazole	14 (25)	42 (75)

Note: ^aPercentage correlated to the total number of commensal isolates (n = 56).

Abbreviation: E. coli, Escherichia coli.

Figure 1 Representative agarose gel (0.7%) electrophoresis of the different patterns of extracted plasmids from gut commensal E. coli isolates. The patterns comprised of one band (lanes 2, 3, 6, 8, 10, 12), two bands (lanes 1, 5, 7, 9, 11) and three bands (lane 4) of different sizes. First most left lane, 1 kb DNA molecular weight marker.

Table 3 Distribution of AMR Genes with Corresponding AMR Phenotypes Detected in E. coli Isolates

Target Genes Encoding Diverse Antimicrobial Classes	Prevalence of Target Genes Among E. coli Isolates (n = 56) No. (%)^a	AMR Testing of E. coli Isolates with Corresponding Resistance Genes No. (%)^a
β-Lactams Resistance Genes		Ampicillin	Amoxicillin-Clavulanate	Aztreonam	Cefoxitin	Ceftriaxone	Cefuroxime	Ceftazidime	Cefepime
blaTEM	37 (66.1)	37 (100)	33 (89.1)	35 (94.5)	26 (70.2)	36 (97.3)	35 (94.5)	35 (94.5)	34 (91.9)
blaCTX-M1-like genes	24 (42.8)	24 (100)	19 (79.1)	22 (91.6)	17 (70.8)	24 (100)	24 (100)	24 (100)	24 (100)
blaCMY-like genes	6 (10.7)	6 (100)	6 (100)	6 (100)	4 (66.6)	6 (100)	6 (100)	6 (100)	5 (83.3)
Aminoglycoside Resistance Genes		Gentamicin				Amikacin
aac(3)-II	24 (42.9)	24 (100)				18 (75)
aac(6′)-Ib-cr	14 (25)	14 (100)				12(85.7)
Quinolones Resistance Genes		Ciprofloxacin				Levofloxacin
qnrA	31 (55.4)	31 (100)				31 (100)
qnrB	17 (30.4)	16 (94.1)				16 (94.1)
qnrS	0 (0)	0 (0)				0 (0)
aac(6)-Ib-cr	14 (25)	13 (92.8)				13 (92.8)
Tetracyclines Resistance Genes		Tetracycline
tetA	27 (48.2)	27 (100)
tetB	14 (25)	14 (100)
Sulfonamides Resistance Genes		Trimethoprim/Sulfamethoxazole
sulI	34 (60.7)	34 (100)
sulII	18 (32.1)	18 (100)

Note: ^aPercentage correlated to the total number of commensal isolates (n = 56).

Abbreviations: E. coli, Escherichia coli; AMR, antimicrobial resistance.

Figure 2 Prevalence of AMR-phenotypes and associated genes in commensal E. coli isolates. (A) AMR phenotypes among E. coli isolates; (B) AMR-associated genes harbored by E. coli resistant phenotypes.

Table 4 Genotypic Profiles of AMR Genes Among E. coli Isolates

Genotypic Profile	Frequency^a
blaTEM-blaCTX-M1-like-qnrA-qnrB-tetA-sulI	12/56, 21.4%
blaTEM-aac(3)-II- aac(6′)-Ib-cr-qnrA-tetA-tetB-sulI	8/56, 14.3%
blaCTX-M1-like-aac(3)-II-qnrA-tetB-sulI-sulII	4/56, 7.1%
blaTEM-blaCTX-M1-like-aac(3)-II-aac(6′)-Ib-cr-qnrA-sulI	4/56, 7.1%
blaTEM-qnrA-aac(3)-II-tetA-sulII	3/56, 5.4%
blaTEM-blaCMY-like-qnrB-aac(3)-II-sulI-sulII	3/56, 5.4%
blaTEM-blaCMY-like-sulI-sulII	3/56, 5.4%
blaTEM-blaCTX-M1-like-sulI	2/56, 3.6%
tetA-tetB-sulII	2/56, 3.6%
blaTEM-blaCTX-M1-like-tetA	1/56, 1.8%
blaTEM-aac(3)-II-aac(6′)-Ib-cr-qnrB	1/56, 1.8%
blaCTX-M1-like-aac(3)-II-aac(6′)-Ib-cr-qnrB-tetA-sulII	1/56, 1.8%
No resistance genes	12/56, 21.4%

Note: ^aPercentage correlated to the total number of commensal isolates (n = 56).

Abbreviations: E. coli, Escherichia coli; AMR, antimicrobial resistance.

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