Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of Crohn’s disease

Table 1 PCR primers used in this study

Primers	Sequence (5’ to 3’)	Amplified base pairs (bp)1	Amplicon length (bp)
IS900
P90	GTTCGGGGCCGTCGCTTAGG	22-421	400
P91	GAGGTCGATCGCCCACGTGA
AV1	ATGTGGTTGCTGTGTTGGATGG	77-384	308
AV2	CCGCCGCAATCAACTCCAG
rpoB
Efox1	TTGCCGGCCGAACCGACACA	1-721	721
Fox1r	TGTCGACGTCGAACTCCAGC
UCF1	TCGATGTCGCTGTCTTTCTC	373-820	448
UCF2	GCTCGGTGATCTGCTCGTTG
Fox1f	CGGTGTTCATGGGTGACTTC	521-1509	989
DBR	GTAGTGGGACGGGTGCACGTC
Knight1	ACCACTTCGGCAACCGCCGG	1191-1777	587
Knight2	ACTCGACCTCGCCCGCCTTG
Ex1a	AAGGTGGTCGACGGCGTGGT	1621-2340	720
Ex2a	GATCTCGTGCTCCTCGATGT
Ex3	ACGAGGACGCGATCATCCT	2261-2980	720
Ex4	TCGACACGATCTGGTTCGGC
Ex5	GAACATCGACGGCAATCCCG	2901-3597	697
Hrox1	TCCGTCGAGGACCTGGCTTAA

¹Numbers represent nucleotide positions within each gene of MAP.

Table 2 rpoB and susceptibility data for all micro-organization

Microorganism	Strain	RIF MIC (mg/L)	RFB MIC (mg/L)1	Inhib. at 1 mg/L of RIF (%)2	rpoB BLAST result3	Amino acid change4
MAP	ATCC43544	≤ 1.0	≤ 1.0	91 ± 0.71	WT	NC
MAP	UCF3	≤ 1.0		94 ± 2.83	WT	NC
MAP	UCF4	≤ 2.5		79 ± 7.07	A2284C	N762H
MAP	UCF5	≤ 1.0		92.5 ± 0.71	WT	NC
MAP	UCF7	≤ 1.0		88 ± 5.66	WT	NC
MAP	UCF8	≤ 1.0		90 ± 1.41	WT	NC
MAP	18	≥ 30	≤ 5.0	42 ± 1.00	C1367T	T456I
MAP	185	≤ 4.0	≤ 1.0	61 ± 7.07	Silent	NC
MAP	Cow2013	≤ 1.0		95 ± 1.41	WT	NC
MAP	Cow5	≤ 1.0		96.5 ± 0.71	WT	NC
MAP	61a	≤ 1.0		90 ± 3.54	WT	NC
M avium subspecies. avium5	ATCC25291	≤ 1.0		90 ± 1.41
M tuberculosis5	ATCC25177	≤ 1.0		92 ± 1.41
M smegmatis5	ATCC607	≥ 9.0		6.6 ± 1.7
UCF5-RIF16r	Modified UCF5	≥ 30	≥ 10	32 ± 1.41	T1442C	L481P

¹Limited investigation involving MAP18, MAP185, and UCF5-RIF16r;

²Values are expressed as mean ± SD;

³Wild type (WT) indicates identical investigated rpoB sequence compared to MAP K-10 strain, and differentially expressed rpoB mutations are indicated;

⁴Amino acid positions correspond to MAP strain K-10 numbering system from Li et al 2005, and the alteration is indicated with single letter amino acid codes and the corresponding codon number. NC corresponds to no change in beta subunit sequence based on investigated rpoB regions;

⁵rpoB sequence was not investigated.