Introduction
Materials and methods
Bacterial strains and plasmids
Strains or plasmids | Description | Reference |
---|---|---|
Plasmids | ||
pACYC184 | Cloning vector | |
pacyc184-KPC3 | Insertion of 727-2902 region between BamH I and EcoRI restriction site in pACYC184 | FJ609231 |
pMU-acyc184-KPC3 | Deletion of 1188-1706 in pacyc184-KPC3 | FJ609231 |
pHS70 | Plasmid from E. coli J 53 (pHS70) | This study |
pHS510 | Plasmid from E. coli J 53 (PhS510) | This study |
Strains | ||
Citrobacter freundii
| ||
HS70 | Clinical isolate | This study |
E. coli
| ||
HS510 | Clinical isolate | This study |
E. coli J 53 (pHS70) | E. coli J53 transconjugant derived from HS70 | This study |
E. coli J 53 (pHS510) | E. coli J53 transconjugant derived from HS510 | This study |
EKPC3 | E. coli DH5a containing pacyc184-KPC3 | This study |
EMU-KPC3 | E. coli DH5a containing pMU-acyc184-KPC3 | This study |
DH5α | E. coli reference lab strain | |
J53 | E. coli reference lab strain | |
ATCC25922 | E. coli reference lab strain |
Antimicrobial susceptibility testing
Conjugation experiments and plasmid restriction enzyme digestion analysis
Isoelectric focusing of β-lactamases
PCR analysis and nucleotide sequencing
ESBL or plasmid | Primer | Sequence | Position | GenBank accession number or reference |
---|---|---|---|---|
KPC | KPC-F | 5′-ATGTCACTGTATCGCCGTCT-3′ | 131-150 | AF297554 |
KPC-R | 5′-TTTTCAGAGCCTTACTGCCC-3′ | 1023-1042 | ||
TEM | TEM-F | 5′-ATAAAATTCTTGAAGAC-3′ | 1–17 | X54604.1 |
TEM-R | 5′-TTACCAATGCTTAATCA-3′ | 1075–1059 | ||
SHV | SHV-F | 5′-TGGTTATGCGTTATATTCGCC-3 | 69–89 | X98100.1 |
SHV-R | 5′-GCTTAGCGTTGCCAGTGCT-3′ | 936–918 | ||
CTX-M-1 | M1F | 5′- GGTTAAAAAATCACTGCGTC -3 | 65–84 | X92506 |
M1 R | 5′- TTGGTGACGATTTTAGCCGC-3 | 928–909 | ||
CTX-M-9 | M9F | 5′-ATGGTGACAAAGAGAGTGCA-3 | 1–20 | AF252621.2 |
M9R | 5′-CCCTTCGGCGATGATTCTC-3′ | 870–852 | ||
M2F | 5′-ATGATGACTCAGAGCATTCG-3′ | 304–323 | ||
CTX-M-2 | M2R | 5′-TGGGTTACGATTTTCGCCGC-3′ | 1169–1150 | AJ416343.1 |
pacyc184-KPC3 | KPC-3 F | 5′-GCCTGGTCCGAATTCCCTCGTCATCCGCAGACCAAC-3′ | 727-747 | FJ609231 |
KPC-3R | 5′-GCCTGGTCCGGATCCCGCGCAGACTCCTAGCCTAAA-3′ | 2882-2902 | ||
pMU-acyc184-KPC3 | MU-KPC-3 F | 5′-CTTAACGTGAGTTTTCGTTCCACTGAGCG-3′ | 1816-1844 | FJ609231 |
MU-KPC-3R | 5′-AAGTCATTTTTCAATATTATTGAAGCATTT ATC-3′ | 1112-1144 |
Analysis of the genetic environment of the blaKPC-3 gene and plasmid construction
Results
Antimicrobial resistance
Antimicrobial agent(s) | MIC (μg/mL) | ||||||
---|---|---|---|---|---|---|---|
HS70 b
| E. coli J 53 (pHS70) | HS510 | E. coli J 53 (pHS510) | EKPC3 | EMU-KPC3 | 25922 c
| |
Imipenem | ≥128 | 2 | 16 | 2 | 8 | 32 | ≤0.0625 |
Meropenem | ≥128 | 2 | 16 | 2 | 8 | 32 | ≤0.0625 |
Cefepime | ≥128 | 8 | 64 | 8 | 16 | 64 | ≤0.0625 |
Cefotaxime | ≥128 | 16 | ≥128 | 16 | 16 | 64 | ≤0.0625 |
Ampicillin | ≥128 | ≥128 | ≥128 | ≥128 | ≥128 | ≥128 | 2 |
Ciprofloxacin | ≥128 | ≤0.0625 | ≥128 | ≤0.0625 | ≤0.0625 | ≤0.0625 | ≤0.0625 |
Gentamicin | ≥128 | ≤0.0625 | ≥128 | ≤0.0625 | ≤0.0625 | ≤0.0625 | ≤0.0625 |