MIC relative

Growth/Fitness

Minimum Inhibitory Concentration (MIC)

unitless

Broth Dilution/Turbidity

Mueller Hinton II broth

37°C

7.0

N/A

N/A

None

MIC assay. E. coli ElectroMAX DH10B cells (Invitrogen, Grand Island, NY) transformed with the pBC SK(+) phagemid harboring the
blaIMP-1-V67A, blaIMP-1-V67I, or blaIMP-10 gene were used for susceptibility
assays. MIC assays with DH10B cells producing the various MBLs in the
presence of 10 antibacterials were performed by a broth microdilution
method following the guidelines of the Clinical and Laboratory Standards
Institute (37), using antibiotic concentrations in the range of 0.03125
microg/ml to 1,024 microg/ml. An inoculum of 10^4 E. coli DH10B cells containing the corresponding blaIMP-carrying phagemid was grown in Mueller-Hinton II broth at 37°C for 20 h in the presence of one of the antibacterials,
supplied in serial dilutions in a 96-well plate assay format. DH10B cells
transformed with the pBC SK(+) phagemid lacking the MBL gene and
cells lacking any phagemid served as negative controls, while DH10B cells
with no antibacterials served as a positive control. Six independent measurements were carried out, and the median was designated the MIC.

MICs are expressed as x-fold relative to the negative control (cells with empty pBCSK(+) plasmid).
The absolute MICs for the negative control were as follows:
Cephalothin (CE): 16 ug/ml
Cefotaxime (CT): 0.125 ug/ml
Ceftazidime (CA): 0.5 ug/ml
Cefoxitin (FO): 8 ug/ml
Benzylpenicillin (PE): 32 ug/ml
Ampicillin (AM): 4 ug/ml
Imipenem (IP): 0.25 ug/ml
Meropenem (ME): 0.03125 ug/ml
Doripenem (DO): 0.0625 ug/ml
Aztreonam (AZ): 0.25 ug/ml