aDepartment of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon
bBacteriology Division, Indian Council of Medical Research-National Institute of Cholera and Enteric Diseases, Kolkata, India
Copyright ©2020, Korea Centers for Disease Control and Prevention
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Target gene | Primers | Primer sequences | Amplicon sizes | References |
---|---|---|---|---|
|
||||
Primers for virulent genes (5′—3′) | ||||
elt/ETEC | LT-F | CACACGGAGCTCCTCAGTC | 508 bp | [19] |
LT-R | CCCCCAGCCTAGCTTAGTTT | |||
|
||||
est/ETEC | ST-F | GCTAAACCAGTAGAGGTCTTCAAAA | 147 bp | [19] |
ST-R | CCCGGTACAGAGCAGGATTACAACA | |||
|
||||
bfpA/EPEC | BFPA-F | GGAAGTCAAATTCATGGGGG | 367 bp | [19] |
BFPA-R | GGAATCAGACGCAGACTGGT | |||
|
||||
eae/EPEC | EAE-F | CCCGAATTCGGCACAAGCATAAGC | 881 bp | [19] |
EAE-R | CCCGGATCCGTCTCGCCAGTATTCG | |||
|
||||
aatA/EAEC | CVD432-F | CTGGCGAAAGACTGTATCAT | 630 bp | [19] |
CVD432-R | CAATGTATAGAAATCCGCTGTT | |||
|
||||
aaic/EAEC | AAIC-F | ATTGTCCTCAGGCATTTCAC | 215 bp | [19] |
AAIC-R | ACGACACCCCTGATAAACAA | |||
|
||||
stx1/STEC | EVT1 | CAACACTGGATGATCTCAG | 349 bp | [20] |
EVT2 | CCCCCTCAACTGCTAATA | |||
|
||||
stx2/STEC | EVS1 | TACAGTCGTCACTCACTGGT | 110 bp | [20] |
EVC2 | CTGCTGTCACAGTGACAAA | |||
|
||||
ial/EIEC | ial-F | CTGGATGGTATGGTGAGG | 700 bp | [21] |
ial-R | GGAGGCCAATTATTTCC |
Antibiotics | Target gene | Primers | Primer sequences | Amplicon sizes | Annealing temperature (times) | References |
---|---|---|---|---|---|---|
|
||||||
Primers for resistance genes (5′—3′) | ||||||
AMP | blaTEM | OT-1 | TTGGGTGCACGAGTGGGT | 503 bp | 58°C (1 min) | [26] |
OT-2 | TAATTGTTGCCGGGAAGC | |||||
blaOxa | OO-1 | ACCAGATTCAACTTTCAA | 598 bp | 57°C (1 min) | [26] | |
OO-2 | TCTTGGCTTTTATGCTTG | |||||
BlaPSE-1 | PSE-1-F | TTT GGT TCC GCG CTA TCT G | 150 | 51°C (1 min) | [27] | |
PSE-1-R | TAC TCC GAG CAC CAA ATC CG | |||||
|
||||||
CHL | cat1 | CAT1-F | TCCCAATGGCATCGTAAAGAAC | 293 bp | 53°C (30 s) | [28] |
CAT1-R | TCGTGGTATTCACTCCAGAGCG | |||||
cat2 | CAT2-F | AACGGCATGATGAACCTGAA | 547 bp | 55°C (30 s) | [29] | |
CAT2-R | ATCCCAATGGCATCGTAAAG | |||||
floR | floR-F | ACTCGGCATGGACATGTACT | 1213 bp | 55°C (40 s) | [29] | |
floR-R | ACGGACTGCGGAATCCATAG | |||||
cmIA | cmIA-F | TGTCATTTACGGCATACTCG | 55°C (30 s) | [26] | ||
cmIA-R | ATCAGGCATCCCATTCCCAT | |||||
|
||||||
TET | tetA | tetA-F | GTAATTCTGAGCACTGTCGC | 956 bp | 58.5°C (1 min) | [30] |
tetA-R | CTGCCTGGACAACATTGCTT | |||||
tetB | tetB-F | CTCAGTATTCCAAGCCTTTG | 415 bp | 55°C (45 s) | [30] | |
tetB-R | ACTCCCCTGAGCTTGAGGGG | |||||
tetG | tetG-F | CAG CTT TCG GAT TCT TAC GG | 884 bp | 60°C (45 s) | [31] | |
tetG-R | GAT TGG TGA GGC TCG TTA GC | |||||
|
||||||
SXT | dfrXII (dfrA12, dfrA13) | dfrXII-F | GGT GSG CAG AAG ATT TTT CGC | 319 bp | 60°C (45 s) | [32] |
dfrXII-R | TGG GAA GAA GGC GTC ACC CTC | |||||
dfrA7, dfrA17 | dfrVII-F | TTG AAA ATT TCA TTG ATT G | 474 bp | 55°C (1 min) | [32] | |
dfrVII-R | TTA GCC TTT TTT CCA AAT CT | |||||
dfrIa (dfrA1, dfrA5, dfrA15) | dfrIa-F | GTG AAA CTA TCA CTA ATG G | 474 bp | 55°C (1 min) | [32] | |
dfrIa-R | TTA ACC CTT TTG CCA GAT TT | |||||
sul1 | sul1-F | CGGCGTGGGCTACCTGAACG | 432 bp | 60°C (30 s) | [33] | |
sul1-R | GCCGATCGCGTGAAGTTCCG | |||||
sul2 | sul2-F | TCA ACA TAA CCT CGG ACA GT | 707 bp | 55°C (45 s) | [34] | |
sul2-R | GAT GAA GTC AGC TCC ACC T | |||||
sul3 | sul3-F | GGAAGAAATCAAAAGACTCAA | 363 bp | 53°C (1 min) | [33] | |
sul3-R | CCTAAAAAGAAGCCCATACC | |||||
|
||||||
DNA gyrase | gyrA | gyrA-F | TGTCCGAGATGGCCTGAAGC | 347 bp | 62°C (45 s) | [35] |
gyrA-R | TACCGTCATAAGTTATCCACG | |||||
gyrB | gyrB-F | CAAACTGGCGGACTGTCAGG | 345 bp | 62°C (45 s) | [35] | |
gyrB-R | TTCCGGCATCTGACGATAGA | |||||
DNA topoisomerase | parC | parC-F | TGTATGCGATGTCTGAACTG | 264 bp | 55°C (30 s) | [36] |
parC-R | CTCAATAGCAGCTCGGAATA |
AMP = ampicillin; CAZ = ceftazidime; CHL = chloramphenicol; CIP = ciprofloxacin; CRO = ceftriaxone; CTX = cefotaxime; EAEC = enteroaggregative E. coli; EIEC = enteroinvasive E. coli; EPEC = enteropathogenic E. coli; ETEC = enterotoxigenic E. coli; GEN = gentamicin; I = intermediate; MEM = meropenem; NA = nalidixic acid; NOR = norfloxacin; R = resistant; S = sensitive; SXT = trimethoprim-sulphamethoxazole; TET = tetracycline.
Virulent genes (%) | Antibiotics (% resistance, % sensitive) | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
||||||||||||||
AMP (78.99, 21.01) | AZM (2.52, 36.13) | CAZ (0.84, 99.16) | CHL (10.08, 87.39) | CIP (0.84, 92.44) | CRO (0.00, 96.64) | CTX (0.00, 92.44) | GEN (1.69, 89.83) | MEM (0.00, 100.00 | NA (24.17, 79.83) | NOR (0.84, 99.16) | OFX (0.84, 99.16 | SXT (89.92, 10.08) | TET (84.87, 10.92) | |
|
||||||||||||||
% of virulent gene among resistance strains - % of virulent gene among sensitive strains p | ||||||||||||||
eae (47.07) | 56.38-12.00 | 0.00-55.81 | 0.00-47.46 | 0.00-52.88 | 0.00-46.36 | 00.00-47.83 | 00-47.27 | 0.00-52.83 | 0.00-47.06 | 29.17-51.58 | 0.00-47.46 | 0.00-47.46 | 52.34-0.00 | 51.49-30.77 |
< 0.001* | 0.002** | 0.002** | < 0.001* | 0.036* | ||||||||||
|
||||||||||||||
aaic (13.45) | 12.77-16.00 | 00.00-6.98 | 00.00-13.56 | 83.33-4.81 | 0.00-14.55 | 0.00-13.04 | 0.00-12.73 | 0.00-6.60 | 0.00-13.45 | 45.83–5.26 | 0.00–13.56 | 0.00–13.56 | 14.02–8.33 | 10.89–23.03 |
< 0.001* | < 0.001** | < 0.001* | ||||||||||||
|
||||||||||||||
CVD432 (33.61) | 27.66-56.66 | 66.67-34.88 | 0.00-33.90 | 83.33-27.88 | 0.00-34.55 | 0.00-33.04 | 0.00-32.73 | 100.00-26.42 | 0.00-33.61 | 58.33-27.37 | 0.00-33.90 | 0.00-33.90 | 30.84-58.33 | 32.67-23.08 |
0.007** | < 0.001* | < 0.001* | 0.004* | |||||||||||
|
||||||||||||||
ial (5.88) | 4.26-12.00 | 33.33-2.33 | 0.00-5.93 | 16.67-4.81 | 100.00-5.45 | 0.00-6.09 | 0.00-6.36 | 0.00-6.60 | 0.00-5.88 | 8.33-5.26 | 100.00-5.08 | 100.00-5.08 | 4.67-16.67 | 5.94-7.69 |
< 0.001* | < 0.001* | < 0.001* | ||||||||||||
|
||||||||||||||
stx1 (0.84) | 1.06-0.00 | 0.00-0.00 | 0.00-0.85 | 0.00-0.96 | 0.00-0.91 | 0.00-0.87 | 0.00-0.91 | 0.00-0.94 | 0.00-0.84 | 0.00-1.05 | 0.00-0.85 | 0.00-0.85 | 0.93-0.00 | 0.99-0.00 |
|
||||||||||||||
Stx2 (10.08) | 11.70-4.00 | 0.00-4.65 | 100.00-9.32 | 0.00-11.54 | 0.00-10.00 | 00.00-9.57 | 0.00-10.00 | 0.00-10.38 | 0.00-10.08 | 4.17-11.58 | 0.00-10.17 | 0.00-10.17 | 9.35-16.67 | 9.90-15.38 |
0.002* |
AMP = ampicillin; CAZ = ceftazidime; CHL = chloramphenicol; CIP = ciprofloxacin; CRO = ceftriaxone; CTX = cefotaxime; GEN = gentamicin; MEM = meropenem; NA = nalidixic acid; NOR = norfloxacin; SXT = trimethoprim-sulphamethoxazole; TET = tetracycline.
(% resistance, % sensitive); % of virulent gene among resistant strains - % of virulent gene among sensitive strains.
* Positive association.
** Negative association.
Virulence gene | BlaOxa | BlaTEM | BlaPSE-1 | cat1 | cat2 | cmIA | floR | tetA | tetB | tetG | dfr12 | dfr7 | dfr1a | Sul1 | Sul2 | Sul3 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
||||||||||||||||
% of virulent gene among strains carry the resistant gene - % of virulent gene among strains not carry the resistant gene p | ||||||||||||||||
eae | 0.00-51.85 | 59.69-33.33 | 0.00-47.46 | 0.00-52.34 | 0.00-52.34 | 0.00-47.06 | 0.00-47.06 | 52.38-41.07 | 45.45-50.00 | 23.81-52.04 | 0.00-47.46 | 0.00-47.06 | 62.50-45.95 | 5.00-55.56 | 54.12-29.41 | 42.86-47.32 |
0.001** | 0.004* | < 0.001** | < 0.001** | 0.018** | < 0.001** | 0.014* | ||||||||||
|
||||||||||||||||
aaic | 72.73-7.41 | 4.84-22.81 | 100.00-12.71 | 75.00-6.54 | 83.33-5.61 | 0.00-13.45 | 0.00-13.45 | 4.76-23.21 | 14.29-11.90 | 19.05-12.24 | 0.00-13.56 | 0.00-13.45 | 0.00-14.41 | 55.00-5.05 | 4.71-35.29 | 0.00-14.29 |
< 0.001** | < 0.001** | 0.010* | < 0.001* | < 0.001* | 0.003** | < 0.001* | < 0.001** | |||||||||
|
||||||||||||||||
CVD432 | 81.82-28.70 | 22.58-45.61 | 0.00-33.90 | 75.00-28.97 | 83.33-28.04 | 0.00-33.61 | 0.00-33.61 | 33.33-33.93 | 37.66-26.19 | 28.57-34.69 | 0.00-33.90 | 0.00-33.61 | 37.50-33.33 | 85.00-23.23 | 24.71-55.88 | 28.57-33.903 |
< 0.001* | 0.007** | 0.001* | < 0.001* | < 0.001* | 0.001** | |||||||||||
|
||||||||||||||||
ial | 18.18-4.63 | 3.23-8.77 | 0.00-5.93 | 16.67-4.67 | 16.67-4.67 | 0.00-5.88 | 0.00-5.88 | 1.59-10.71 | 0.00-16.67 | 28.57-1.02 | 0.00-5.93 | 0.00-5.88 | 0.00-6.31 | 5.00-6.06 | 5.88-5.88 | 28.57-4.46 |
0.034** | < 0.001** | < 0.001* | 0.008* | |||||||||||||
|
||||||||||||||||
stx1 | 0.00-0.93 | 0.00-1.75 | 0.00-0.85 | 0.00-0.93 | 0.00-0.93 | 0.00-0.84 | 0.00-0.84 | 1.59-0.00 | 1.30-0.00 | 0.00-1.02 | 0.00-0.85 | 0.00-0.84 | 0.00-0.90 | 0.00-1.01 | 1.18-0.00 | 0.00-0.89 |
|
||||||||||||||||
Stx2 | 0.00-11.11 | 14.52-5.26 | 0.00-10.17 | 0.00-11.21 | 0.00-11.21 | 0.00-10.08 | 0.00-10.08 | 12.70-7.14 | 14.29-2.38 | 14.29-9.18 | 100.00-9.32 | 0.00-10.08 | 0.00-10.81 | 0.00-12.12 | 12.94-2.94 | 0.00-10.71 |
0.039* | 0.002* |