Antimicrobial Resistance Profiles of Diarrheagenic E. coli (DEC) and Salmonella Species Recovered from Diarrheal Patients in Selected Rural Communities of the Amathole District Municipality, Eastern Cape Province, South Africa

Figures & data

Table 1 Primer Sequences for the Confirmation of Diarrheagenic E. coli and Salmonella Isolates

Bacteria	Primer(s)	Primer Sequence (5ʹ-3ʹ)	Amplicon Size (bp)	PCR Cycling Conditions	Cycles	Reference(s)
Diarrheagenic E. coli	uidA	F: AAA ACG GCA AGA AAAAGC AG R:ACGCGTGGTTAACAGTCTTGCG	147	94°C(5 mins), 95°C(30 secs), 58°C(60 secs), 72°C(60 secs), 72°C(8 mins)	35	[^Citation12]
Salmonella spp.	^ompC invA	F: ATCGCTGAC TTATGCAAT R: CGG GTTGCGTTATAGGTC F: TATCGCCACGTTCGGGCA R:TCGCACCGTCAAAGGAAC	204 275	95°C(1 min), 95°C(20 secs), 57°C(15 secs), 72°C(2 mins), 72°C(7 mins)	35	[^Citation13] [^Citation14]

Abbreviations: Mins, minutes; Secs, seconds.

Table 2 A Panel of Commercial Antimicrobial Disc Used for Susceptibility Testing of Diarrheagenic E. coli Isolates

Antibiotic Class	Antimicrobial Agent (Potency)
Aminoglycosides	Gentamicin (10 µg)
	Amikacin (30 µg)
Fluoroquinolones	Norfloxacin (10 µg)
Carbapenems	Imipenem (10 µg)
	Meropenem (10 µg)
Polymyxins	Polymyxin B (300 units)
Penicillins	Penicillin G (10 µg)
	Ampicillin (10 µg)
	Amoxicillin (10 µg)
Macrolides	Erythromycin (15 µg)
Cephems	Ceftazidime (30 µg)
	Cefotaxime (30 µg)
	Cephalothin (30 µg)
Phenicols	Chloramphenicol (30 µg)
Nitrofurantoin	Nitrofurantoin (200 µg)
Foliate Pathways	Trimethoprim-sulfamethoxazole (25 µg)
Quinolones	Nalidixic Acid (30 µg)
Tetracyclines	Tetracycline (30 µg)
	Doxycycline (30 µg)

Table 3 A Panel of Commercial Antimicrobial Disc Used for Susceptibility Testing of Salmonella Isolates

Antibiotic Class	Antimicrobial Agent (Potency)
Penicillins	Ampicillin (10 µg)
	Amoxicillin (10 µg)
Macrolides	Azithromycin (15 µg)
Cephems	Ceftriaxone (30 µg)
	Cephalothin (30 µg)
	Cefotaxime (30 µg)
Carbapenems	Imipenem (10 µg)
Monobactams	Aztreonam (30 µg)
Folate pathway antagonist	Trimethoprims (25 µg)
Aminoglycoside	Streptomycin (10 µg)
Fluoroquinolones	Ciprofloxacin (5 µg)
Tetracyclines	Tetracycline (30 µg)

Table 4 Oligonucleotides Sequences for the Detection of Resistance Gene Determinants

Antimicrobial Family	Target Gene	Primer Sequence (5ʹ→3ʹ)	Amplicon Size (bp)	PCR Cycling Condition	Cycles	Reference (s)
Beta-lactams	ampC	F:TTCTATCAAMACTGGCAR R:CCYTTTTATGTACCCAYG	550	94°C(4 mins), 94°C(45 secs), 60°C(45 secs), 72°C(45 secs), 72°C(7 mins)	30	[^Citation18]
	BlaTEM	F:TTTCGTGTCGCCCTTATTC R:CCGGCTCCAGATTTATCA	690	95°C(5 Mins), 94°C(30 secs), 60°C(30 secs), 72°C(90 secs), 72°C(5 mins)	30	[^Citation19]
Sulfonamides	sulI	F: TTCGGCATTCTGAATCTC R: ATGATCTAACCCTCGGT	822	94°C(5 mins), 94°C(60 secs), 55°C(60 secs), 72°C(5 mins), 72°C(5 mins)	35	[^Citation20]
	sulII	F: CGGCATCGTCAACATAA R: GTGTGCGGATGAAGTCA	625	94°C(5 mins), 94°C(30 secs), 50°C(30 secs), 72°C(90 secs), 72°C(5 mins)	30	[^Citation21]
Tetracyclines	Tet A	F: GCTACATCCTGCTTGCCT R: CATAGATCGCCGTGAAG	201	94°C(5 mins), 94°C(60 secs), 55°C(60 secs), 72°C(90 secs), 72°C(5 mins)	35	[^Citation22]
Aminoglycosides	aadA	F: GTGGATGGCGGCCTGAA R: AATGCCCAGTCGGCAGC	525	94°C(4 mins), 94°C(45 secs), 50°C(45 secs), 72°C(45 secs), 72°C(7 mins)	30	[^Citation18]

Abbreviations: Mins, minutes; Secs, seconds.

Figure 1 Representative Gel electrophoresis image of the confirmed DEC isolates recovered from diarrheagenic stool specimens. Lane (M) Molecular weight marker (100 bp DNA ladder, Thermo Scientific), lane 1: negative control, lane 2 to 12: Some of the positive diarrheagenic E. coli isolates.

Figure 2 Representative Gel electrophoresis image of the confirmed Salmonella isolates recovered from diarrheagenic stool specimens. Lane (M) Molecular weight marker (100 bp DNA ladder, Thermo Scientific), lane 1: positive control S. enterica subsp. Typhimurium (DSMZ 14028), lane 2: negative control, lane 3 to 13: Some of the positive Salmonella isolates.

Table 5 Patterns of Multiple Antibiotic-Resistance Phenotypes (MARPs) of Diarrheagenic E. coli

Diarrheagenic E. coli (n=324)
No. of Antimicrobials	Resistance Pattern	No. Observed
3	AP-A-PG	4
	AP-PG-TS	2
	AP-PG-E	2
5	KF-PB-E-PG-DXT	3
	AP-PG-E-AM-T	5
	AP-PG-E-T-DXT	1
	AP-PG-A-TS-T	1
6	AP-PG-CIP-E–A-TS	2
	AP-PG-CXM-E-A-T	8
	AP-PG-CIP-CXM-PB-DXT	4
	AP-PG-E-A-TS-T	7
	PG-CXM-PB-TS-T-DXT	1
	AP-NOR-PB-E-DXT	4
	AP-PG-AK-KF-E-CAZ-CTX	11
	AP-PG-KF-CAZ-TS-T	4
	AP-PGAK-E-A-CTX	6
8	AP-PG-E-A-CTX-TS-T-DXT	12
	PG-KF-PB-E-A-TS-T-DXT	1
9	AP-PG-NOR-A-CTX-TS-NA-T-DXT	6
	AP-PG-E-A-PB-C-TS-T-DXT	1
	AP-PG-A-GM-NOR-NI-TS-T-DXT	1
10	AP-PG-KF-NOR-E-A-CTX-TS-T-DXT	1
	AP-PG-KF-E-A-CTX-C-TS-T-DXT	4
	AP-PG-KF-PB-E-CAZ-C-A-TS-NA	1
	AP-PG-KF-NOR-PB-E-CAZ-A-CTX-T	1
	AP-PG-NOR-CXM-E-CAZ-A-CTX-TS-DXT	1
	AP-PG-AK-PG-PB-E-CAZ-A-NA-CTX	3
	AP-PG-AK-PB-E-NI-A-CTX-TS-T-DXT	5
11	PG-KF-PG-A-E-TS-T-DXT-NA-CAZ-CTX	2
13	PG-KF-PG-A-E-T-DXT-NA-C-NI-CTX-PB-TS	1

Abbreviations: AP, ampicillin; GM, gentamycin; AK, amikacin; KF, cephalothin; NOR, norfloxacin; IMI, imipenem; PB, polymyxin B; PG, penicillin G; E, erythromycin; CAZ, ceftazidime; MEM-S, meropenem; CTX, cefotaxime; C, chloramphenicol; NI, nitrofurantoin; TS, trimethoprim/sulphamethaxazole; NA, nalidixic Acid; T, tetracycline; DXT, doxycline; A, amoyxcilin.

Table 6 Patterns of Multiple Antibiotic-Resistance Phenotypes (MARPs) of Salmonella Isolates

Salmonella Isolates (n=62)
No. of Antimicrobials	Resistance Pattern	No. Observed
3	AP-KF-A	2
	S-KF-A	4
	ATH-KF-A	15
	AP-S-KF	1
	ATH-TM-A	1
4	ATH-S-KF-A	1
	ATH-TM-KF-A	1
	ATH-CIP-KF-A	1
	AP-ATH-KF-A	4
	AP-S-KF-A	2
	TM-S-KF-A	1
5	AP-ATH-S-KF-A	2
	AP-TM-S-KF-A	2
	AP-ATH-TM-KF-A	2
6	AP-ATH-TM-S-KF-A	6
	ATH-TM-S-KF-A	1

Abbreviations: AP, ampicillin; ATH, azithromycin; CRO, ceftriaxone; IMI, imipenem; ATM, aztreonam; TM, trimethoprim; CTX, cefotaxime; S, streptomycin; CIP, ciprofloxacin; KF, cephalothin; A, amoyxcilin; T, tetracycline.

Figure 3 Gel electrophoresis representation of β-Lactam resistance genes ampC (198 bp) detected in DEC and Salmonella isolates. Lane (M) Molecular weight marker (100 bp DNA ladder, Thermo Scientific), lane 1: negative control, lane 2 to 13: some of the representatives of the genetic expression of ampC (198 bp) from the β- Lactams -resistant DEC and Salmonella isolates.

Figure 4 A representative gel electrophoresis profile of some β- Lactam resistant genes detected in DEC and Salmonella isolates. Lane (M) molecular weight marker (100 bp DNA ladder, Thermo Scientific), lane 1: negative control, lane 2 to 13: some of the representatives of the genetic expression of bla_TEM (690 bp) from the β-Lactams -resistant DEC.

Figure 5 An illustrative gel electrophoresis image with the genetic expression of the aadA (525 bp) resistant gene among the aminoglycosides-resistant DEC and Salmonella isolates. Lane (M) molecular weight marker (100 bp DNA ladder, Thermo scientific), lane 1: negative control, lane 2 to 11: some of the representatives of the genetic expression of aadA (525 bp) from aminoglycoside-resistant DEC and Salmonella isolates.

Figure 6 An illustrative gel electrophoresis image for the detection of sulI (822 bp) resistance gene from the sulfonamide-resistant DEC. Lane (M) molecular weight marker (100 bp DNA ladder, Thermo scientific), lane 1: negative control, lane 2 to 13: some of the representatives of the genetic expression of sulI (822 bp) from the sulfonamide-resistant DEC.

Figure 7 An illustrative gel electrophoresis for the detection of sulII (625 bp) resistance gene. Lane (M) molecular weight marker (100 bp DNA ladder, Thermo scientific), lane 1: negative control, lane 2 to 10: some of the representatives of the genetic expression of sulII (625 bp) from the sulfonamide-resistant DEC and Salmonella isolates.

Figure 8 A representative gel electrophoresis image for the detection tet A (201 bp) resistance gene determinants. Lane (M) molecular weight marker (100 bp DNA ladder, Thermo scientific), Lane 1: negative control, Lane 2 to 8: some of the representatives of the genetic expression of tet A (201 bp) from the Tetracycline-resistant DEC isolates.

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