TABLE 5.

Amplification of drug resistance of AG112

Continuous growtha	MIC (μg/ml)b					dupIS3 detectedc
	Tet	Chl	Nal	Nor	Amp
None	8	24	12	0.38	6	1
250 generations in LB + tetracycline	128	256	192	2	32	1,590
125 additional generations in LB	24	24	24	0.75	12	1.5

^aAG112 (marR) was sequentially grown in LB broth with tetracycline (5 μg/ml) for about 250 generations and then sequentially grown in the absence of tetracycline for 125 generations (see Materials and Methods).

^bThe susceptibilities to the antibiotics (MICs) were determined by Etests. Tet, tetracycline; Chl, chloramphenicol; Nal, nalidixic acid; Nor, norfloxacin; Amp, ampicillin.

^cThe amplification of acrAB in the whole population was assessed by comparative PCR with primers IS3-1 and IS3-2, which detect the dupIS3 events. The amplification obtained with the template DNA from AG112 before sequential growth in the presence of drug was arbitrarily given the value “1”, and the results after 250 and 125 generations are the number of dupIS3 amplifications detected in similar amounts of template DNA compared to the amounts found in AG112 before the sequential growths.