TABLE 2.

Correlation between amidase activity and amide drug susceptibility of strains of M. smegmatis, M. tuberculosis, and E. coli

Strain	Amidase activity			MIC (μg/ml)a
	PZA (U/mg)b	Nicotinamide (U/mg)b	Bentamide (+/−)c	PZA	Nicotinamide	Benzamide
M. smegmatis
mc2155	0.210 ± 0.016	0.530 ± 0.040	+	≥2,200	≥2,200	≥500
mc2155(pOam)	6.47 ± 0.30	15.65 ± 0.51	+++	150	150	150
mc2155(pOppncA)				≥2,200	≥2,200	≥500
pzaA::aph	0.0201 ± 0.0006	0.0220 ± 0.009	−	≥2,200	≥2,200	≥500
pzaA::aph(pOncA)	0.0690 ± 0.008	0.0961 ± 0.0154		≥2,200	≥2,200	≥500
pzaA::aph(pOppncA)	0.551 ± 0.025	0.702 ± 0.033		≥2,200	≥2,200	≥500
M. tuberculosis
H37Rv	0.0021 ± 0.0004	0.0042 ± 0.0005	−	20	20	≥500
pncA::hyg	0	0	−	≥2,000	≥2,000	≥500
[pncA::hyg]::pAIncA	0.0022 ± 0.0001	0.0056 ± 0.0002	−	20	20	≥500
[pncA::hyg]::pAIam	0.112 ± 0.0060	0.289 ± 0.010	+	≤10	≥20	20
H37Rv(pOam)	1.84 ± 0.16	7.1 ± 0.2		NDe
E. colid
BL21(DE3)/pLysS	+	+		≥2,000	≥2,000	≥500
BL21(DE3)/pLysS(pTncA)	+++	+++	−	≤200	≤200	≥500
BL21(DE3)/pLysS(pTam)	+++	+++	+++	ND

^aFor M. smegmatis strains, the MIC is defined as the concentration of drug that causes ≥90% growth inhibition (measured by colony counts), since a defined cutoff value was obtained at this point. A 100% growth inhibition of M. smegmatis strains was not obtained, since at higher amide concentrations, the decrease in colony counts was dependent on the initial number of cells plated. For M. tuberculosis strains, the MIC is defined as the concentration of drug at which no visible growth occurs in tests performed at pH 5.8 and/or the concentration that causes >90% reduction in growth index relative to the drug-free control in the BACTEC test. For E. coli strains, the MIC is defined as the drug concentration causing a >99% growth inhibition (measured by colony counts). 

^bSpecific activity calculated from activity data obtained using the HPLC amidase assay and using the previously defined unit (2). The results represent the mean and standard deviation from at least three independent experiments. 

^c+++, high activity; +, detectable activity; −, undetectable activity (as determined by the TLC benzamidase assay and the previously described PZase and nicotinamidase assays) (2). 

^dSince the specific amidase activity in E. coli recombinants carrying pTncA and pTam is dependent on the duration of IPTG induction of gene expression, only a semiquantitative assessment of activity is shown in these cases (see footnote c for a key). 

^eThe pH sensitivity for growth of this strain precluded determination of the MIC of PZA.