ARG-ANNOT, a New Bioinformatic Tool To Discover Antibiotic Resistance Genes in Bacterial Genomes

Sushim Kumar Gupta; Babu Roshan Padmanabhan; Seydina M. Diene; Rafael Lopez-Rojas; Marie Kempf; Luce Landraud; Jean-Marc Rolain

doi:10.1128/AAC.01310-13

DOI: 10.1128/AAC.01310-13

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FIG 1
ARG-ANNOT database browser on Bio-Edit. (A) Paste the query sequences and select type of database, select the different parameters, and search. (B) BLAST output.
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FIG 2
(A) Topographical representation of BLAST results for rmtF after curing it from the database. (B) BLAST analysis of rmtF gene using ARG-ANNOT.

TABLE 1

Position of the mutation in the reference gene which leads to antibiotic resistance in different chromosomal genes

Gene^{^a}	Mutational point(s)	Reference sequence (5′–3′)^{^b}
rpoB	C1564(64)A:A1565(65)T:A1565(65)G:G1573(73)T:A1574(74)T:C1603(103)G:C1603(103)T:A1604(104)T:C1619(119)A:C1619(119)T:C1696(196)T:A1741(241)T	1501(1):TTGATTAATGCAAAACCAGTTGCTGCTGCAATCAAAGAATTCTTTGGTTCAAGCCAGTTATCTCAGTTTATGGACCAAAACAACCCACTATCTGAGATTACACATAAACGTCGTGTATCTGCGCTTGGTCCTGGTGGTTTAACACGTGAACGTGCGGGCTTCGAAGTACGTGACGTACACCAAACTCACTATGGTCGTGTTTGTCCAATTGAAACTCCTGAAGGTCCAAACATTGGTTTGATCAACTCGCTTTCTGTATACGCAAAAGCGAATGACTTCGGTTTCTTGGAAACTCCATAC:1800(300)
gyrA1	T175(175)A:G224(224)C:C198(198)T:G259(259)T:G259(259)A	1(1):ATGAGCGACCTTGCGAGAGAAATTACACCGGTCAACATTGAGGAAGAGCTGAAGAGCTCCTATCTGGATTATGCGATGTCGGTCATTGTTGGCCGTGCGCTGCCAGATGTCCGAGATGGCCTGAAGCCGGTACACCGTCGCGTACTTTACGCCATGAACGTACTAGGCAATGACTGGAACAAAGCCTATAAAAAATCTGCCCGTGTCGTTGGTGACGTAATCGGTAAATACCATCCCCATGGTGACTCGGCGGTCTATGACACGATCGTCCGCATGGCGCAGCCATTCTCGCTGCGTTAT:300(300)
gyrA2	G2599(299)T	2301(1):GACGAAAGGGGTTATCTCCATCAAGGTTACCGAACGTAACGGTTTAGTTGTTGGCGCGGTACAGGTAGATGACTGCGACCAGATCATGATGATCACCGATGCCGGTACGCTGGTACGTACTCGCGTTTCGGAAATCAGCATCGTGGGCCGTAACACCCAGGGCGTGATCCTCATCCGTACTGCGGAAGATGAAAACGTAGTGGGTCTGCAACGTGTTGCTGAACCGGTTGACGAGGAAGATCTGGATACCATCGACGGCAGTGCCGCGGAAGGGGACGATGAAATCGCTCCGGAAGTGGA:2600(300)
parC	G233(33)A:A238(38)C:G239(39)T:C240(40)A:G250(50)A: A682(482)G:G757(557)A	201(1):CGGTGACGTACTGGGTAAATACCATCCGCACGGCGATAGCGCCTGTTATGAAGCGATGGTCCTGATGGCGCAACCGTTCTCTTACCGTTATCCGCTGGTTGATGGTCAGGGGAACTGGGGCGCGCCGGACGATCCGAAATCGTTCGCGGCAATGCGTTACACCGAATCCCGGTTGTCGAAATATTCCGAGCTGCTATTGAGCGAGCTGGGGCAGGGGACGGCTGACTGGGTGCCAAACTTCGACGGCACTTTGCAGGAGCCGAAAATGCTACCTGCCCGTCTGCCAAACATTTTGCTTAACGGCACCACCGGTATTGCCGTCGGCATGGCGACCGATATTCCACCGCATAACCTGCGTGAAGTGGCTCAGGCGGCAATCGCATTAATCGACCAGCCGAAAACCACGCTCGATCAGCTGCTGGATATCGTGCAGGGGCCGGATTATCCGACTGAAGCGGAAATTATCACTTCGCGCGCCGAGATCCGTAAAATCTACGAGAACGGACGTGGTTCAGTGCGTATGCGCGCGGTGTGGAAGAAAGAAGATGGCGCGGTGGTTATCAGCGCATTGCCGCATCAGGTTTCAGGTGCGCGCGTACT:800(600)
16S rRNA	T1406(206)A:A1408(208)G:C1409(209)T:G1491(291)T	1201(1):ATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAA:1500(300)
23S rRNA	A2058(8)G:A2059(9)G:C2611(561)T	2051(1):AAGACGGAAAGACCCCGTGAACCTTTACTATAGCTTGACACTGAACATTGAGCCTTGATGTGTAGGATAGGTGGGAGGCTTTGAAGTGTGGACGCCAGTCTGCATGGAGCCGACCTTGAAATACCACCCTTTAATGTTTGATGTTCTAACGTTGACCCGTAATCCGGGTTGCGGACAGTGTCTGGTGGGTAGTTTGACTGGGGCGGTCTCCTCCTAAAGAGTAACGGAGGAGCACGAAGGTTGGCTAATCCTGGTCGGACATCAGGAGGTTAGTGCAATGGCATAAGCCAGCTTGACTGCGAGCGTGACGGCGCGAGCAGGTGCGAAAGCAGGTCATAGTGATCCGGTGGTTCTGAATGGAAGGGCCATCGCTCAACGGATAAAAGGTACTCCGGGGATAACAGGCTGATACCGCCCAAGAGTTCATATCGACGGCGGTGTTTGGCACCTCGATGTCGGCTCATCACATCCTGGGGCTGAAGTAGGTCCCAAGGGTATGGCTGTTCGCCATTTAAAGTGGTACGCGAGCTGGGTTTAGAACGTCGTGAGACAGTTCGGTCCCTATCTGCCGTGGGCGCTGGAGAACTGAGGGGGGCTGCT:2650(300)
katG	G871(171)A:G900(200)C:G944(244)C:G985(285)GG	701(1):GGCCGAACGGCAACCCGGACCCCATGGCCGCGGCGGTCGACATTCGCGAGACGTTTCGGCGCATGGCCATGAACGACGTCGAAACAGCGGCGCTGATCGTCGGCGGTCACACTTTCGGTAAGACCCATGGCGCCGGCCCGGCCGATCTGGTCGGCCCCGAACCCGAGGCTGCTCCGCTGGAGCAGATGGGCTTGGGCTGGAAGAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATCACCAGCGGCATCGAGGTCGTATGGACGAACACCCCGACGAAATGGGACAACAGTTTCCTCG:1000(300)
pncA	A139(39)G:T192(92)TA	1(1):ATGCGGGCGTTGATCATCGTCGACGTGCAGAACGACTTCTGCGAGGGTGGCTCGCTGGCGGTAACCGGTGGCGCCGCGCTGGCCCGCGCCATCAGCGACTACCTGGCCGAAGCGGCGGACTACCATCACGTCGTGGCAACCAAGGACTTCCACATCGACCCGGGTGACCACTTCTCCGGCACACCGGACTATTCCTCGTCGTGGCCACCGCATTGCGTCAGCGGTACTCCCGGCGCGGACTTCCATCCCAGTCTGGACACGTCGGCAATCGAGGCGGTGTTCTACAAGGGTGCCTACACC:300(300)
embB	A916(16)G:A916(16)C:G918(18)T:G918(18)A:G918(18)C	901(1):GGCTACATCCTGGGCATGGCCCGAGTCGCCGACCACGCCGGCTACATGTCCAACTATTTCCGCTGGTTCGGCAGCCCGGAGGATCCCTTCGGCTGGTATTACAACCTGCTGGCGCTGATGACCCATGTCAGCGACGCCAGTCTGTGGATGCGCCTGCCAGACCTGGCCGCCGGGCTAGTGTGCTGGCTGCTGCTGTCGCGTGAGGTGCTGCCCCGCCTCGGGCCGGCGGTGGAGGCCAGCAAACCCGCCTACTGGGCGGCGGCCATGGTCTTGCTGACCGCGTGGATGCCGTTCAACAAC:1200(300)
rpsL	A128(128)G:A263(263)G	1(1):ATGGCAACAGTTAACCAGCTGGTACGCAAACCACGTGCTCGCAAAGTTGCGAAAAGCAACGTGCCTGCGCTGGAAGCATGCCCGCAAAAACGTGGCGTATGTACTCGTGTATATACTACCACTCCTAAAAAACCGAACTCCGCGCTGCGTAAAGTATGCCGTGTTCGTCTGACTAACGGTTTCGAAGTGACTTCCTACATCGGTGGTGAAGGTCACAACCTGCAGGAGCACTCCGTGATCCTGATCCGTGGCGGTCGTGTTAAAGACCTCCCGGGTGTTCGTTACCACACCGTACGTGGT:300(300)
pmrA	G305(155)A	151(1):GAGTTGCTTGCCCGTATTCATGCCTTACTACGCCGTAGTGGAGTAGAAGCTCAACTTGCGAGTCAAGATCAACTATTAGAAAGTGGTGATCTGGTTTTAAATGTTGAACAGCATATTGCGACGTTTAAAGGCCAACGCATTGATTTATCAAATCGTGAATGGGCAATCTTTYKAATTCCACTTATGACTCACCCAAATAAAATCTTTTCTAAAGCCAACTTAGAAGATAAGTTATATGATTTTGATAGTGATGTGACCAGTAATACTATTGAAGTATATGTTCACCATTTAAGAGCGAAG:450(300)
pmrB1	C38(38)A:C41(41)T:G261(261)C:G261(261)T	1(1):GTGCATTATTCATTAAAAAAACGACTGATTTGGGGCACCTCAATTTTCAGTGTCATCTTAGGTTGTATTTTAATTTTTAGTGCTTATAAGGTTGCACTTCAAGAAGTCGATGAAATTCTAGATACTCAAATGAAGTATTTAGCGGAAAGAACAGCTGAGCACCCTTTAAAAACTGTAAGCAGTAAGTTTGATTTTCATAAAACTTACCACGAAGAAGATCTGTTTATCGATATCTGGGCTTATAAGGATCAGGCCCATTTGTCTCATCATTTACATTTGCTGGTTCCACCTGTTGAGCAA:300(300)
pmrB2	T434(14)A:C680(260)T:C697(277)T:C697(277)A:G784(364)C	321(1):CTTGCGGGCAGTATGTTTATTCCGTATTTAATTATTTTACCTTTTGCAATATTTGCCTTAGCAGCCATTATTCGTCGTGGTTTAAAACCAATAGATGATTTTAAAAATGAGTTAAAAGAACGCGATTCCGAAGAACTCACACCAATTGAAGTACATGATTATCCTCAAGAGCTTTTACCTACTATTGACGAAATGAACCGTCTTTTTGAGCGCATTTCTAAAGCGCAAAATGAACAGAAGCAATTTATTGCCGATGCTGCTCATGAATTACGAACACCTGTGACTGCATTGAACTTACAAACCAAGATTTTGCTAAGCCAGTTTCCTGAGCATGAATCATTGCAAAACTTAAGCAAGGGTTTGGCGCGTATTCAGCATTT:700(380)
pmrB3	T1057(7)A:T1160(110)A:C1208(158)T	1051(1):GTTATTAATATTTCAGTTTATACCGATCCAGATCACTACGCATGTATTCAAATTGAAGATAGCGGTGCAGGAATAGACCCTGAAAATTACGATAAAGTCCTTAAGCGTTTTTATCGCGTGCATCACCATCTTGAGGTGGGAAGTGGTCTAGGTTTATCTATTGTAGATCGTGCAACTCAAAGGCTTGGTGGGACTTTAACTCTCGATAAGAGCTTAGAGCTTGGCGGTCTTTCTGTATTAGTGAAATTAC:1300(250)

↵a The nucleotide sequences for gyrA, parC, rpsL, 16s rRNA, and 23s rRNA were obtained from Escherichia coli sp. strain K-12 substrain MG1655; for rpoB, pmrA, and pmrB genes from Acinetobacter baumannii 17978; and for katG, pncA, and embB genes from Mycobacterium tuberculosis H37Rv.
↵b The first number and the second number in parentheses represent the nucleotide position of the reference gene in NCBI GenBank and the nucleotide position in the table, respectively.

TABLE 2

BLAST analyses to validate the system under different stringent conditions using gene fragments of different percentages of length^{^a}

% length e-value	Parameter	Value(s) for gene fragment of length:
		100%		70%		50%		25%		10%		5%
		X	Y	X	Y	X	Y	X	Y	X	Y	X	Y
1E-100	Sp (%)	100	0	96 (4)	0	90 (10)	0	74 (26)	No	No	No	No	No
1E-100	Sn (%)	0	100	0	100	0	100	0	No	No	No	No	No
1E-50	Sp (%)	100	0	95 (5)	0	89 (11)	0	86 (14)	0	81 (19)	No	No	No
1E-50	Sn (%)	0	100	0	100	0	100	0	100	0	No	No	No
1E-25	Sp (%)	100	0	94 (6)	0	89 (11)	0	86 (14)	0	77 (23)	0	66 (34)	No
1E-25	Sn (%)	0	100	0	100	0	100	0	100	0	100	0	No
1E-10	Sp (%)	100	0	94 (6)	0	89 (11)	0	86 (14)	0	81 (19)	0	77 (23)	0
1E-10	Sn (%)	0	100	0	100	0	100	0	100	0	100	0	100
1E-5	Sp (%)	100	0	94 (6)	0	89 (11)	0	84 (16)	0	76 (24)	0	74 (26)	0
1E-5	Sn (%)	0	100	0	100	0	100	0	100	0	100	0	100

↵a The table shows the specificity and sensitivity analysis of 100 randomly selected sequences (1.0E-100 to 1.0E-5). The e-values represent the different stringent conditions. X, randomly selected antibiotic resistance gene sequences from the database (see Fig. S8 in the supplemental material); Y, randomly selected gene sequences (no antibiotic resistance gene) from Escherichia coli, Acinetobacter baumannii, and Staphylococcus aureus genomes; Sp, specificity; Sn, sensitivity; No, no output. Values in parentheses show the numbers of specific hits at the group level.