Levels of antibiotic resistance
of BL21(DE3) cells containing TEM
expression plasmids were determined by measuring their minimum inhibitory
concentrations (MIC90s) using the broth microdilution method according
to the Clinical and Laboratory Standards Institute (CLSI, formerly
the NCCLS) guidelines.38 Strains were grown
to saturation overnight in Luria Miller broth with kanamycin and 1
mM IPTG. Each well of a 96-well microtiter plate was filled with 50
μL of sterile Mueller Hinton II (MHII) medium broth (Sigma).
Antibiotic was dissolved in water making a 20 mM solution and then
diluted with sterile MHII medium broth to 288 μM cefotaxime
(CFX). Exactly 50 μL of the compound solution was added to the
first well of the microtiter plate, and 2-fold serial dilutions were
made down each row of the plate. Exactly 50 μL of bacterial
inoculum (diluted to 5 × 105 CFU mL–1 from the overnight cultures) was then added to each well, giving
a total volume of 100 μL well–1 and compound
concentration gradients of 72–0.04 μM CFX. The plate
was incubated at 37 °C for 17 h, and then each well was examined
for bacterial growth. The MIC90 was recorded as the lowest compound
concentration required to inhibit 90% of bacterial growth as judged
by turbidity of the culture medium relative to a row of wells filled
with a water standard. Gentamicin was included in a control row at
a concentration gradient of 174–0.09 μM.
of BL21(DE3) cells containing TEM
expression plasmids were determined by measuring their minimum inhibitory
concentrations (MIC90s) using the broth microdilution method according
to the Clinical and Laboratory Standards Institute (CLSI, formerly
the NCCLS) guidelines.38 Strains were grown
to saturation overnight in Luria Miller broth with kanamycin and 1
mM IPTG. Each well of a 96-well microtiter plate was filled with 50
μL of sterile Mueller Hinton II (MHII) medium broth (Sigma).
Antibiotic was dissolved in water making a 20 mM solution and then
diluted with sterile MHII medium broth to 288 μM cefotaxime
(CFX). Exactly 50 μL of the compound solution was added to the
first well of the microtiter plate, and 2-fold serial dilutions were
made down each row of the plate. Exactly 50 μL of bacterial
inoculum (diluted to 5 × 105 CFU mL–1 from the overnight cultures) was then added to each well, giving
a total volume of 100 μL well–1 and compound
concentration gradients of 72–0.04 μM CFX. The plate
was incubated at 37 °C for 17 h, and then each well was examined
for bacterial growth. The MIC90 was recorded as the lowest compound
concentration required to inhibit 90% of bacterial growth as judged
by turbidity of the culture medium relative to a row of wells filled
with a water standard. Gentamicin was included in a control row at
a concentration gradient of 174–0.09 μM.