Membrane fecal coliform (mFC) agar with cefotaxime (2 μg/mL) was used to isolate ESBL-producing E. coli. One hundred μL of each sample was inoculated onto mFC agar and incubated at 37 °C for 3 h, then incubated overnight at 44 °C. Up to five suspected E. coli colonies (the ones with black color) on mFC agar were chosen and inoculated on blood agar (5% defibrinated horse blood) and incubated at 37 °C for 24 h. The cefotaxime resistant strains were identified as E. coli by the Phoenix system and subsequently confirmed by polymerase chain reaction (PCR) amplification of the universal stress protein (uspA) gene as described by Chen & Griffiths (1998). Genomic DNA of these isolates was extracted by using a standard boiling method as described by Anastasi et al. (2012). The primer sequences used in the current study and reaction conditions are given in Table 1.
Nucleotide sequence of the primers used in PCR
| Targets . | Sequence (5′–3′) (amplicon sizes) . | PCR conditions . | Reference . |
|---|---|---|---|
| chuA | F: ACGAACCAACGGTCAGGAT R:TGCCGCCAGTACCAAAGACA (Amplicon: 279 bp) | 94 °C 5 s 59 °C 15 s (30 cycles) | Clermont et al. (2013) |
| yjaA | F: TGAAGTGTCAGGAGACGCTG R:TGGAGAATGCGTTCCTCAAC (Amplicon: 211 bp) | ||
| TspE4C | F: GAGTAATGTCGGGGCATTCA R: GCGCCAACAAAGTATTACG (Amplicon: 152 bp) | ||
| arpA | F: GATTCCATCTTGTCAAAATATGCC R: GAAAAGAAAAAGAATTCCCAAGAG (Amplicon: 301 bp) | 94 °C 5 s 57 °C 20 s (30 cycles) | Clermont et al. (2013) |
| trpA | F: AGTTTTATGCCCAGTGCGAG R: TCTGCGCCGGTCACGCCC (Amplicon: 219 bp) | 94 °C 5 s 59 °C 20 s (30 cycles) | Clermont et al. (2013) |
| blaTEM | F: ATAAAATTCTTGAAGACGAAA R: GACAGTTACCAATGCTTAATC (Amplicon: 1080 bp) | 95 °C 30 s 56 °C 30 s (35 cycles) 72 °C 30 s | Ahmed et al. (2007) |
| blaSHV | F: TTATCTCCCTGTTAGCCACC R: GATTTGCTGATTTCGCTCGG (Amplicon: 727 bp) | ||
| Whole blaSHV | F: CGGCCTTCACTCAAGGATGTA R: GTGCTGCGGGCCGGATAAC (Amplicon: 927 bp) | ||
| blaOXA | F: TCAACTTTCAAGATCGCA R: GTGTGTTTAGAATGGTGA (Amplicon: 610 bp) | ||
| Whole blaOXA | F: GGCAATCCAGCCGGGGCCAA R: CGGGCCTGTTCCCGGGTTAA (Amplicon: 891 bp) | ||
| blaCTX-M | F: CGCTTTGCGATGTGCAG R: ACCGCGATATCGTTGGT (Amplicon: 551 bp) | ||
| Whole blaCTX-M | F: CCAGAATAAGGAATCCCATG R: GCCGTCTAAGGCGATAAAC (Amplicon: 948 bp) | ||
| sul1 | F: CGGCGTGGGCTACCTGAACG R: GCCGATCGCGTGAAGTTCCG (Amplicon: 433 bp) | 94 °C 15 s 67 °C 30 s (36 cycles) 72 °C 60 s | Kerrn et al. (2002) |
| sul2 | F: GCGCTCAAGGCAGATGGCATT R: GCGTTTGATACCGGCACCCGT (Amplicon: 293 bp) | ||
| sul3 | F: GAGCAAGATTTTTGGAATCG R: CTAACCTAGGGCTTTGGATAT (Amplicon: 750 bp) | ||
| int1 | F: CAGTGGACATAAGCCTGTTC R: CCCGAGGCATAGACTGTA (Amplicon: 160 bp) | 95 °C 30 s 59 °C 30 s (32 cycles) 72 °C 32 s | Gündoğdu et al. (2011) |
| int2 | F: CACGGATATGCGACAAAAAGGT R: GATGACAACGAGTGACGAAATG (Amplicon: 788 bp) | ||
| int3 | F: GCCTCCGGCAGCGACTTTCAG R: ACGGATCTGCCAAACCTGACT (Amplicon: 979 bp) | ||
| uspA | F: CCGATACGCTGCCAATCAGT R: ACGCAGACCGTAGGCCAGAT (Amplicon: 884 bp) | 94 °C 30 s 56 °C 30 s (30 cycles) 72 °C 30 s | Chen & Griffiths (1998) |
| Targets . | Sequence (5′–3′) (amplicon sizes) . | PCR conditions . | Reference . |
|---|---|---|---|
| chuA | F: ACGAACCAACGGTCAGGAT R:TGCCGCCAGTACCAAAGACA (Amplicon: 279 bp) | 94 °C 5 s 59 °C 15 s (30 cycles) | Clermont et al. (2013) |
| yjaA | F: TGAAGTGTCAGGAGACGCTG R:TGGAGAATGCGTTCCTCAAC (Amplicon: 211 bp) | ||
| TspE4C | F: GAGTAATGTCGGGGCATTCA R: GCGCCAACAAAGTATTACG (Amplicon: 152 bp) | ||
| arpA | F: GATTCCATCTTGTCAAAATATGCC R: GAAAAGAAAAAGAATTCCCAAGAG (Amplicon: 301 bp) | 94 °C 5 s 57 °C 20 s (30 cycles) | Clermont et al. (2013) |
| trpA | F: AGTTTTATGCCCAGTGCGAG R: TCTGCGCCGGTCACGCCC (Amplicon: 219 bp) | 94 °C 5 s 59 °C 20 s (30 cycles) | Clermont et al. (2013) |
| blaTEM | F: ATAAAATTCTTGAAGACGAAA R: GACAGTTACCAATGCTTAATC (Amplicon: 1080 bp) | 95 °C 30 s 56 °C 30 s (35 cycles) 72 °C 30 s | Ahmed et al. (2007) |
| blaSHV | F: TTATCTCCCTGTTAGCCACC R: GATTTGCTGATTTCGCTCGG (Amplicon: 727 bp) | ||
| Whole blaSHV | F: CGGCCTTCACTCAAGGATGTA R: GTGCTGCGGGCCGGATAAC (Amplicon: 927 bp) | ||
| blaOXA | F: TCAACTTTCAAGATCGCA R: GTGTGTTTAGAATGGTGA (Amplicon: 610 bp) | ||
| Whole blaOXA | F: GGCAATCCAGCCGGGGCCAA R: CGGGCCTGTTCCCGGGTTAA (Amplicon: 891 bp) | ||
| blaCTX-M | F: CGCTTTGCGATGTGCAG R: ACCGCGATATCGTTGGT (Amplicon: 551 bp) | ||
| Whole blaCTX-M | F: CCAGAATAAGGAATCCCATG R: GCCGTCTAAGGCGATAAAC (Amplicon: 948 bp) | ||
| sul1 | F: CGGCGTGGGCTACCTGAACG R: GCCGATCGCGTGAAGTTCCG (Amplicon: 433 bp) | 94 °C 15 s 67 °C 30 s (36 cycles) 72 °C 60 s | Kerrn et al. (2002) |
| sul2 | F: GCGCTCAAGGCAGATGGCATT R: GCGTTTGATACCGGCACCCGT (Amplicon: 293 bp) | ||
| sul3 | F: GAGCAAGATTTTTGGAATCG R: CTAACCTAGGGCTTTGGATAT (Amplicon: 750 bp) | ||
| int1 | F: CAGTGGACATAAGCCTGTTC R: CCCGAGGCATAGACTGTA (Amplicon: 160 bp) | 95 °C 30 s 59 °C 30 s (32 cycles) 72 °C 32 s | Gündoğdu et al. (2011) |
| int2 | F: CACGGATATGCGACAAAAAGGT R: GATGACAACGAGTGACGAAATG (Amplicon: 788 bp) | ||
| int3 | F: GCCTCCGGCAGCGACTTTCAG R: ACGGATCTGCCAAACCTGACT (Amplicon: 979 bp) | ||
| uspA | F: CCGATACGCTGCCAATCAGT R: ACGCAGACCGTAGGCCAGAT (Amplicon: 884 bp) | 94 °C 30 s 56 °C 30 s (30 cycles) 72 °C 30 s | Chen & Griffiths (1998) |