A Newly Discovered Drug Resistance Gene rfaF In Helicobacter pylori

Figures & data

Table 1 PCR Primers Used For Construction In This Study

Primers	Sequence(5ʹ–3ʹ)	Size Of Product (bp)	Positions In 26,695 DNA Sequence(No.NC_000915.1)
F1	CTATAGGGCGAATTGGGTACCCCCATTCAAGCCAATTTTTTCC	718	From 1,261,390 to 1,262,065
R1	TAAAACCGCCCAGTCTCGAG ACGCATGCGTTTGGGTGC
F2	TCTCGAGGCCGCTCACACTCTTTTAGAAGA	777	From 1,263,056 to 1,263,804
R2	AAAGCTGGAGCTCCACCGCGGTTTTAAAGGGCCTAAATGACGC
F3	ATGCGTCTCGAGACTGGGCGGTTTTATGGACA	1214
R3	CTAAAAGAGTGTGAGCGGCCTCGAGAAATTAAAAATGAAGTTTTAGCACG TG
F4	AAGGGCTATTGATGAGCGTA	1066 or 1199	From 1,262,025 to 1,263,090
R4	GGCGTTTAAGGCTCTTCT
F5	TCTCGAGTGCGGGAAAACAAGCATTG	537	From 1,262,771 to 1,263,279
R5	AAAGCTGGAGCTCCACCGCGGACTCAGTGGCCTAATGGCTTTGACT
F6	ATGCGTCTCGAGACTGGGCGGTTTTATGGACA	1214
R6	CAATGCTTGTTTTCCCGCACTCGAGAAATTAAAAATGAAGTTTTAGCACG TG
F7	AAGGGCTATTGATGAGCGTA	1068 or 2257	From 1,262,025 to 1,263,092
R7	AAGGCGTTTAAGGCTCTTC
F8	CTATAGGGCGAATTGGGTACCTTGAGTGTTGAGAATTTAGAAAAATGG	534	From 174,691 to 175,187
R8	GTTTCAGTCGCTCGAGTCACTCTGTGTTTTCTCGCTCCA
F9	CACAGAGTGACTCGAGCGACTGAAACTTTCGCAAGCA	1228	From 1,261,884 to 1,263,085
R9	GTGCCGATCATTAAGGCTCTTCTAAAAGAGTGTGAGC
F10	GACGATATGATCATCTCGAGCCATGAAAACAAAGCCTTAAATTTC	813	From 173,857 to 174,631
R10	AAAGCTGGAGCTCCACCGCGGCGCAAACGGCCAATGATC
F11	AGAGCCTTAATGATCGGCACGTAAGAGGTTC	1050
R11	TCATGGCTCGAGATGATCATATCGTCAATTATTACCTCCA
F12	TGGAATCTTTGGAGCGAG	594 or 2852	From 174,112 to 174,705
R12	AGCGATTGGATGCGAGCTA
F13	CTCAACACTTTAGGGGTAATCAT	1347	From 1,261,953 to 1,263,299
R13	AAACCTAAAAACTCAGTGGTCTAA

Note: Underlined part indicates overlapping DNA sequences.

Table 2 MIC Values Of Various Types Of H. Pylori To Antimicrobial Agents

Antibiotic	11,637			MDR(NO.FZ068)
	WT	ΔrfaF	ΔrfaF/rfaF	WT	ΔrfaF
AMO	0.064	0.128	0.0.096	0.125	0.1875
TET	0.125	0.5	0.25	0.25	0.375
CLA	0.125	0.25	0.1875	128	192
MEM	1	1	1	256	256
LEV	0.5	0.5	0.5	8	8
CHL	1	4	2	2	3

Note: Drug concentration unit is (µg/mL).

Abbreviations: WT, wild strain; ΔrfaF, rfaF knockout strain; ΔrfaF/rfaF, rfaF -complemented Strain; AMO, amoxicillin; TET, tetracycline; CLA, clarithromycin; MEM, metronidazole; LEV, levofloxacin; CHL, chloramphenicol.

Figure 1 Growth curve of various types of H. pylori 11,637 with no drug or with drug.

Notes: (A) Growth curve without drug or with (B) AMO (0.064 µg/mL); (C) TET (0.125 µg/mL); and (D) CLA (0.125 µg/mL).

Figure 2 Comparison of EB accumulation in various types of H. pylori strains.

Notes: The data presented are the means ± standard errors of the means from three separate experiments. *P<0.05; **P<0.01.

Table 3 rfaF Amino Acid Mutation Analysis

Strains	MIC (µg/mL)					Putative Amino Acid Conserved Sequence Variations Of Reducing Sensitivity
	AMO	TET	CLA	MEM	LEV
11,637	0.064	0.125	0.125	0.5	0.25	No found mutation
NO.FZ001	0.125	0.5	128	128	1	K331R
NO.FZ035	0.25	0.5	128	256	4	No found mutation
NO.FZ038	0.25	2	2	32	1	K331R
NO.FZ040	0.125	0.5	128	256	16	I59A, L345F
NO.FZ045	0.25	0.5	16	32	0.125	P192L, K331R
NO.FZ068	0.125	0.25	68	256	8	A341T
NO.FZ076	0.5	0.125	68	16	8	S250N
NO.FZ113	0.5	0.5	256	256	4	A276T
NO.FZ120	1	0.5	0.25	32	4	A342T
N0.FZ202	0.5	0.5	128	128	0.5	E198K, K331R