Early genetic diagnosis of clarithromycin resistance in Helicobacter pylori

Table 1 Drug resistance characteristics of three drug-resistant strains (minimal inhibitory concentration: μg/mL)

Strain	Metronidazole	Clarithromycin	Levofloxacin	Amoxicillin
Hpbs1	32	8	8	0.125
Hpbs2	16	8	0.125	0.125
Hpbs3	0.125	8	8	0.125

Table 2 Sequence information of three drug-resistant strains

Sample name	ID name	Sequence type	Sequence topology	Sequence number	Total length (bp)	GC content
Hpbs1	Chromosome1	Chromosome	Circular	1	1563701	38.90
	All	All	-	1	1563701	38.90
Hpbs2	Chromosome1	Chromosome	Circular	1	1534481	38.87
	All	All	-	1	1534481	38.87
Hpbs3	Chromosome1	Chromosome	Circular	1	1534930	38.90
	All	All	-	1	1534930	38.90

Sequence type: Chromosome or plasmid; Sequence topology: Circular or linear.

Table 3 Gene information of three drug-resistant strains

Sample name (#)	Genome size (#)	Total number (#)	Total length (bp)	Average length (#)	Length/genome length (%)	GC content (%)
Hpbs1	1563701	1571	1434202	912.92	91.72	39.49
Hpbs2	1534481	1792	1395399	778.68	90.94	39.44
Hpbs3	1534930	1732	1407495	812.64	91.70	39.49

Total number denotes the count of genes. Total length represents the total length of all genes. Average length refers to the average length of all genes. GC content is the content of G and C in a gene. Length/genome length is the proportion of gene length in the genome.

Table 4 Gene annotation statistics A

Sample name (#)	Total	P450 (#) (%)	VFDB (#) (%)	ARDB (#) (%)	CAZY (#)	SWISSPROT (#) (%)	NOG (#) (%)	COG (#) (%)	CARD (#) (%)	NR (#)
Hpbs1	1571	22 (1.4)	196 (12.47)	0 (0)	14 (0.89)	742 (47.23)	67 (4.26)	1084 (69)	14 (0.89)	1599 (99.23)
Hpbs2	1792	21 (1.17)	177 (9.87)	0 (0)	14 (0.78)	751 (41.9)	125 (6.97)	1111 (61.99)	13 (0.72)	1723 (96.14)
Hpbs3	1732	22 (1.27)	174 (10.04)	0 (0)	14 (0.75)	750 (43.3)	97 (5.6)	1113 (64.26)	15 (0.86)	1698 (98.03)

Table 5 Gene annotation statistics B

Sample name (#)	DBCAN (#) (%)	T3SS (#) (%)	TREMBL (#) (%)	IPR (#)	PHI (#) (%)	KEGG (#) (%)	GO (#) (%)	KOG (#) (%)	Over all (#) (%)
Hpbs1	30 (1.9)	175 (11.13)	1557 (99.1)	1234 (78.54)	54 (3.43)	1026 (65.3)	957 (60.91)	142 (9.03)	1563 (99.49)
Hpbs2	29 (1.61)	197 (10.99)	1706 (95.2)	1372 (76.56)	52 (2.9)	1078 (60.15)	1056 (58.92)	144 (8.03)	1750 (97.65)
Hpbs3	30 (1.73)	209 (12.06)	1688 (97.45)	1340 (77.36)	51 (2.94)	1067 (61.6)	1030 (59.4)	139 (8.02)	1711 (98.78)

Table 6 Analysis of gene resistance in CARD

Gene ID	Subject ID	Align length	Mismatch	Gap	Gene start	Gene end	Subject start	Subject end	E value
GL000175	YP_208874.1	97	39	0	2	98	4	100	6.00E-40
GL000286	YP_006374661.1	398	88	2	1	397	29	421	0
GL000295	NP_312937.1	1389	658	21	8	1371	8	1339	0
GL000296	AAK44936.1	124	35	0	1	124	1	124	4.00E-63
GL000306	NP_207975.1	459	16	0	1	459	1	459	0
GL000309	NP_207972.1	443	10	0	1	443	1	443	0
GL000772	AIL15701	421	220	3	1	420	1	417	4.00E-126
GL000822	YP_002344422.1	853	293	6	3	818	2	851	0
GL000911	NP_415611.1	247	130	2	1	247	1	243	2.00E-66
GL000972	WP_005768149.1	810	390	18	3	773	12	809	0
GL001063	AJF83452.1	287	164	2	1	283	2	288	1.00E-71
GL001265	NP_415804.1	262	141	1	1	261	1	262	2.00E-80
GL001295	YP_001332362.1	222	123	4	1	221	1	216	7.00E-51
GL001455	AJF82049.1	254	141	2	4	255	7	260	2.00E-62

Table 7 Characteristics of drug-resistant genes in CARD

Subject ID	ARO number	Definition of term
YP_208874.1	Neisseria gonorrhoeae FA 1090	rpsJ is a tetracycline resistance protein identified in Neisseria gonorrhoeae. Tetracycline resistance is conferred by binding to the ribosome as a 30S ribosomal protection protein[27]
YP_006374661.1	Enterococcus faecium DO	Sequence variants of Enterococcus faecium elongation factor Tu that can confer resistance to GE2270A[28]
NP_312937.1	Escherichia coli O157•H7 str. Sakai	Point mutation that occurs in Escherichia coli rpoB resulting in resistance to rifampicin[29]
AAK44936.1	Mycobacterium tuberculosis CDC1551	Ribosomal protein S12 stabilizes the highly conserved pseudoknot structure formed by 16S rRNA. Amino acid substitutions in RpsL affect the higher-order structure of 16S rRNA and confer streptomycin resistance by disrupting interactions between 16S rRNA and streptomycin[30-35]
NP_207975.1	Helicobacter pylori 26695	hp1184 is a translocase that belongs to the MATE efflux pump family. It is found in H. pylori and is involved in the active efflux of antibiotics[25,26]
NP_207972.1	Helicobacter pylori 26695	hp1181 is a translocase that is part of the MFS efflux pump family. It is found in H. pylori and plays a role in the active efflux of antibiotics[25]
AIL15701	Escherichia coli ATCC25922	murA or UDP-N-acetylglucosamine enolpyruvy1 transferase catalyzes the initial step in peptidoglycan biosynthesis and is inhibited by Fosfomycin. Over-expression of murA through mutations such as Asp369Asn and Leu370I1e confers fosfomycin resistance. Extensive evidence has shown the significance of C115 mutations in conferring fosfomycin resistance since this residue represents a primary binding site for the antibiotic across many species[36-39]
YP_002344422.1	Campylobacter jejuni subsp. jejuni NCTC 11168	Campylobacter jejuni is a major bacterial infectious agent associated with gastroenteritis. Quinolone resistance is reportedly conferred by a single C-257-T nucleotide substitution in the gyrA gene[40]
NP_415611.1	Escherichia coli str. K-12 substr. MG1655	Fab G is a 3-oxoacyl-acyl carrier protein reductase involved in lipid metabolism and fatty acid biosynthesis. The bacterial biocide Triclosan blocks the final reduction step in fatty acid elongation, inhibiting biosynthesis. Point mutations in fabG can confer resistance to Triclosan[41]
WP_005768149.1	Bartonella bacilliformis KC583	Point mutation in Bartonella bacilliformis results in amino coumarin resistance[42]
AJF83452.1	Acinetobacter baumannii	The LpxC gene is widely known to be involved in the biosynthesis of lipid A in Gram-negative bacteria and mutations to this gene may cause resistance to antimicrobial peptides that target the outer membrane[43,44]
NP_415804.1	Escherichia coli str. K-12 substr. MG1655	fabI is an enoyl-acy1 carrier reductase used in lipid metabolism and fatty acid biosynthesis. The bacterial biocide Triclosan blocks the final reduction step in fatty acid elongation, inhibiting biosynthesis. Point mutations in fabI can confer resistance to Triclosan and Isoniazid[41]
YP_001332362.1	Staphylococcus aureus subsp. aureus str. Newman	Ar1R is a response regulator that binds to the norA promoter to activate expression. Ar1R must first be phosphorylated by Ar1S[45]
AJF82049.1	Acinetobacter baumannii	The LpxA gene is widely known to be involved in the biosynthesis of lipid A in Gram-negative bacteria and mutations to this gene may cause resistance to antimicrobial peptides that target the outer membrane[43,44]

H. pylori: Helicobacter pylori.

Table 8 Mutations in the 23S rRNA genes of Helicobacter pylori strains

Nucleotide position	Ref	Mutation	Hpbs1	Hpbs2	Hpbs3
2143	A	G	+	+	+
2142	A	G	+	+	+
2144	G	T	+	+	+
2302	A	G	-	-	+
2182	T	C	-	+	-
2173	C	T	+	+	+
1513	G	A	-	+	+
2196	C	T	+	-	-
1280	A	G	+	-	-
1023	G	A	-	-	+

Table 9 23S rRNA mutations of Helicobacter pylori strains

Nucleotide position	Ref	Mutation	26695(S)	26695(R)
2142	A	G	-	+
2143	A	G	-	+