Biomarker for Mycobacteriumdownloads.hindawi.com/journals/bmri/2015/271728.pdfof Mycobacterium...
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Research ArticleEvaluation of the Ribosomal Protein S1 Gene (rpsA) as a NovelBiomarker for Mycobacterium Species Identification
Hongfei Duan Guan Liu Xiaobo Wang Yuhong Fu Qian Liang Yuanyuan ShangNaihui Chu and Hairong Huang
National Clinical Laboratory on Tuberculosis Beijing Key Laboratory on Drug-Resistant Tuberculosis Beijing Chest HospitalCapital Medical University Beijing Tuberculosis ampThoracic Tumor Research Institute 97 Beimachang Tongzhou QuBeijing 101149 China
Correspondence should be addressed to Naihui Chu dongchu1994sinacom and Hairong Huang haironghuangcngmailcom
Received 26 July 2014 Accepted 7 November 2014
Academic Editor Om Parkash
Copyright copy 2015 Hongfei Duan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Objectives To evaluate the resolution and reliability of the rpsA gene encoding ribosomal protein S1 as a novel biomarker formycobacteria species identification Methods A segment of the rpsA gene (565 bp) was amplified by PCR from 42 mycobacterialreference strains 172 nontuberculosis mycobacteria clinical isolates and 16 M tuberculosis complex clinical isolates The PCRproducts were sequenced and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and theMEGA program A phylogenetic tree was constructed by the neighbor-joining method Results Comparative sequence analysisof the rpsA gene provided the basis for species differentiation within the genus Mycobacterium Slow- and rapid-growing groupsof mycobacteria were clearly separated and each mycobacterial species was differentiated as a distinct entity in the phylogenetictree The sequences discrepancy was obvious between M kansasii and M gastri M chelonae and M abscessus M avium andM intracellulare and M szulgai and M malmoense which cannot be achieved by 16S ribosomal DNA (rDNA) homologue genescomparison 183 of the 188 (973) clinical isolates consisting of 8 mycobacterial species were identified correctly by rpsA geneblast Conclusions Our study indicates that rpsA sequencing can be used effectively for mycobacteria species identification as asupplement to 16S rDNA sequence analysis
1 Introduction
Members of the genus Mycobacterium are widespread innature and range from harmless saprophytic species tostrict pathogens that cause serious human and animal dis-eases Both slow-growing mycobacteria and rapid-growingmycobacteria can cause human infections Traditionallytaxonomybased onbiochemical characteristics has been usedfor species determination of mycobacteria but this approachis limited due to the overlapping biochemical and phenotypicpatterns among the different mycobacterial species Anotherapproach using analysis of cell-wall fatty acid and mycolicacid composition is also limited by profile similarity amongsome emerging nontuberculosismycobacteria (NTM) [1] 16SrDNA homologue gene sequence comparison has been usedas an important method for the mycobacterial species iden-tification however ambiguous results have been obtainedeither due to the presence of more than one copy of the 16S
rDNA gene within the genome for example in M celatumand M terrae complex [2 3] or due to sequence homol-ogy between species [4] Therefore alternative phylogeneticmarkers which are capable of complementing 16S rRNAgene would be useful for the phylogenetic study and speciesidentification of the genusMycobacterium
Ribosomal protein S1 (rpsA) which is in the 30S ribosomesubunit contains the S1 domain that has been found in a largenumber of RNA-associated proteins RpsA is a vital proteininvolved in protein translation and the ribosome-sparingprocess of translation In addition it has been reported thatRpsA is the target of pyrazinoic acid the active form ofthe antituberculosis drug pyrazinamide [5] Mycobacteriumtuberculosis has a single copy of the rpsA gene in the genome[6] while the sequence homology among different mycobac-teria species in which the gene has already been sequencedis between 867 and 100 This suggests that rpsA may be
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 271728 8 pageshttpdxdoiorg1011552015271728
2 BioMed Research International
suitable for phylogenetic study of the genus MycobacteriumIn this paper we report an evaluation of the rpsA gene as anovel biomarker for mycobacteria species identification
2 Materials and Methods
21 Mycobacterial Reference Strains and Clinical Isolates42 type and reference strains of the genus Mycobacterium(Table 1) 172 clinical NTM isolates and 16 M tuberculosiscomplex (MTC) isolates were investigated All the type andreference strains were purchased from the American TypeCulture Collection (ATCC) and all the clinical isolates usedin this study were obtained from the Clinical Database andSample Bank of tuberculosis of Beijing National ClinicalLab on Tuberculosis Beijing Chest Hospital All the clinicalNTM isolates were identified to the species level by sequencealignment of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoBand hsp65 genes as described before [2 7ndash9] Followingsequencing the 172 NTM clinical strains were found toinclude 10 strains ofM avium 75 strains ofM intracellulare23 strains ofM kansasii 39 strains ofM abscessus 17 strainsofM fortuitum complex 7 strains ofM gordonae and 1 strainofM neoaurum
22 rpsA Gene Amplification and Sequencing DNA wasreleased from cultured mycobacteria by boiling the culturedmycobacterial suspension in TE buffer for 10min After cen-trifugation the supernatant was used for PCR amplification[10] The primers used were forward primer 51015840-CCCTAC-ATCGGCAAGGAG-31015840 position 487ndash504 in the rpsA geneof Mycobacterium tuberculosis GenBank accession num-ber NC 0009622 and reverse primer 51015840-TGTCGATGA-CCTTGACCATC-31015840 position 1032ndash1051 in the rpsA geneof Mycobacterium tuberculosis GenBank accession numberNC 0009622 The amplified product was 565 bp PCR prod-ucts were purified and sequenced by a commercial company(YINGJUN Biotech Company Beijing China) using ABI3730 DNA Analyzer (Applied Biosystems California USA)
23 Sequence Analysis and the Phylogenetic Tree Construc-tions In addition to the 42 reference strains the rpsAsequences of 5 other mycobacterial species were obtaineddirectly from GenBank including M avium subspeciesparatuberculosis (GenBank accession number NC 0029442)M ulcerans (GenBank accession number NC 0086111) Mvanbaalenii (GenBank accession number NC 0087261) Mabscessus subspeciesmassiliense (GenBank accession numberNC 0181501) and M canettii (GenBank accession numberNC 0158481) All sequences were aligned and homologywas calculated by using the multiple alignment algorithm inthe MegAlign package (Windows version 710 DNASTARMadison Wis) 527 bp of sequence (excluding 38 nucleotidesat each end of the amplicon corresponding to the primerbinding sites) was analyzed for the phylogenetic tree con-struction by the neighbor-joining method using the MEGA505 package (httpwwwmegasoftwarenetmegaphp) Abootstrap analysis (1000 repeats) using Rhodococcus equi
(GenBank accession number NC 0063611) as the outgroupwas performed to evaluate the topology of the phylogenetictree
24 Species Identification of the Clinical Isolates The 188clinical isolates were analyzed blindly Sequences minus theknown PCR primer sequences were assembled and edited byusing SeqMan software (version 710 DNASTAR MadisonWI) Isolates were identified by comparing sequences byusing a FASTA BLASTn search with MegAlign (version710 DNASTAR Madison WI) to an in-house databaseof sequences consisting of type and reference strains fromexternal culture collections
3 Results
31 rpsA Sequence Alignment of the Reference StrainsBetween 854 and 100 sequence homology (interspeciesdivergence 0 to 146) was observed among the 42tested reference strains and the 5 additional mycobacterialspecies whose sequences were obtained from the Gen-Bank database (Table 2) All the rpsA gene sequences ofthe analyzed mycobacteria strains were distinct from theoutgroup strain R equi Among the 19 reference strainsof the slow-growing Mycobacterium genus 11 strains weregreater than 97 homology including 5 M tuberculosiscomplex strains Among the 28 reference strains of the rapid-growing Mycobacterium genus 14 strains were greater than97 homology (Table 1) The pathogenic M kansasii waseasily differentiated from the nonpathogenicM gastri (958homology) while those two species were not distinguish-able by the 16S rDNA sequence alignment The sequencehomologies between various species were 918 between Mchelonae and M abscessus 956 between M avium andM intracellulare and 939 between M szulgai and Mmalmoense However the sequence homology between otherspecies was higher for example it was 996 between Mulcerans and M marinum and 987 between M abscessussubspecies massiliense and M abscessus All members ofthe M tuberculosis complex had identical sequences as didM senegalense and M thermoresistibile M parafortuitumand M trivial M diernhoferi and M duvalii and M aus-troafricanum andM terrae
32 Phylogenetic Tree Construction A phylogenetic treewhich provided the basis for species differentiation in thegenus Mycobacterium was constructed (Figure 1) The abso-lute majority tested species showed good separation Therapid-growing species were well defined from the slow-growing species in the tree M chelonae M abscessus sub-speciesmassiliense andMabscessus which are categorized inthe pathogenic taxonomic group of rapid-growing mycobac-teria formed a distinctive cluster which was much closer tothe slow-growing species compared with the nonpathogenicgroup of rapid-growing mycobacteria The reliability of thephylogenetic tree was verified by the bootstrapmethod usingR equi as the outgroup
BioMed Research International 3
Table 1 Percentage rpsA sequence match among type strain
Species Type strain First match ()a Second match ()a
M thermoresistibile ATCC19527 M senegalense (100) M porcinum (987)M senegalense ATCC35796 M thermoresistibile (100) M porcinum (987)M porcinum ATCC33776 M senegalenseM thermoresistibile (987) M fortuitum (983)
M fortuitum ATCC6481 M porcinum (983) M senegalenseM thermoresistibile(981)
M phlei ATCC11758 M porcinum (965) M fortuitum (961)M gadium ATCC27726 M porcinumM fortuitum (957) M smegmatis (953)M flavescens ATCC14474 M smegmatis (966) M vaccae (960)
M smegmatis ATCC19420 M vaccaeM parafortuitumM triviale(966)
M senegalenseM thermoresistibile(963)
M vaccae ATCC15483 M diernhoferiM duvalii (968) M parafortuitumM triviale (966)M parafortuitum ATCC19686 M triviale (100) M terraeM austroafricanum (966)M triviale ATCC23292 M parafortuitum (100) M terraeM austroafricanum (966)M neoaurum ATCC25795 M diernhoferiM duvalii (964) M porcinum (955)M diernhoferi ATCC19340 M duvalii (100) M vaccae (968)M duvalii ATCC43910 M diernhoferi (100) M vaccae (968)M aurum ATCC23366 M gilvum (939) M chubuenseM vanbaalenii (930)M chubuense ATCC27278 M gilvum (966) M vanbaalenii (960)M gilvum ATCC43909 M vanbaalenii (972) M terraeM austroafricanum (970)M vanbaalenii PYR-1 M terraeM austroafricanum (998) M gilvum (972)M terrae ATCC15755 M austroafricanum (100) M vanbaalenii (998)M austroafricanum ATCC33464 M terrae (100) M vanbaalenii (998)M rhodesiae ATCC27024 M terraeM austroafricanum (939) M obuense (937)
M obuense ATCC27023 M porcinum M fortuitum (961) M senegalenseM thermoresistibile(965)
M aichiense ATCC27280 M sphagni (958) M vaccae (947)M sphagni ATCC33027 M aichiense (958) M porcinum (955)M tokaiense ATCC27282 M porcinum (953) M smegmatis (937)M chelonae ATCC35752 M obuense (939) M phlei (935)M abscessus subspeciesmassiliense str GO 06 M abscessus (987) M sphagni (914)
M abscessus ATCC19977 M abscessus subspeciesmassiliense (987) M chubuense (920)
M tuberculosis H37Rv ATCC27294 M tuberculosisH37RaM africanumMbovis BCGM bovis (100) M canettii (998)
M tuberculosis H37Ra ATCC25177 M tuberculosisH37RvM africanumMbovis BCGM bovis (100) M canettii (998)
M africanum ATCC25420 M tuberculosisH37RaM tuberculosisRvM bovis BCGM bovis (100) M canettii (998)
M bovis BCG ATCC35735 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis (100) M canettii (998)
M bovis ATCC19210 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis BCG (100) M canettii (998)
M canettii CIPT140010059 M tuberculosisM africanumM bovisBCGM bovis (998) M asiaticum (932)
M xenopi ATCC19250 M asiaticum (930) M avium subspecies paratuberculosis(924)
M avium ATCC25291 M avium subspecies paratuberculosis(996) M asiaticum (958)
4 BioMed Research International
Table 1 Continued
Species Type strain First match ()a Second match ()a
M avium subspeciesparatuberculosis K-10 M avium (994) M asiaticum (96)
M malmoense ATCC29571 M intracellulare (943) M szulgai (939)
M intracellulare ATCC13950 M avium (956) M avium subspecies paratuberculosis(954)
M scrofulaceum ATCC19981 M asiaticum (954) M szulgai (951)M marinum ATCC927 M ulcerans (996) M szulgai (960)M ulcerans Agy99 M marinum (996) M szulgai (960)M szulgai ATCC25799 M marinumM ulcerans (960) M asiaticum (956)M gordonae ATCC14470 M asiaticum (953) M kansasii (951)M asiaticum ATCC25276 M simiae (977) M kansasii (964)M kansasii ATCC12478 M asiaticum (963) M gastri (958)M gastri ATCC15754 M kansasii (958) M asiaticum (945)Rhodococcus equi 103S M smegmatis (903) M intracellulare (901)aBeside the queried type strain itself the first matched or the second matched type strain
33 Species Identification Outcomes of the Clinical IsolatesrpsA sequence and alignment efficiently identified clinicalisolates representing 8 mycobacterial species We foundthat a criterion of first distinct rpsA sequence match gt97confirmed the identification of 183 of 188 (973) clinicalisolates 20 M abscessus strains 2 M fortuitum strainsand 10 M avium strains were identified to the subspecieslevel Identification discrepancies between rpsA and othersequences were only encountered with 4M gordonae strainsand 1 M kansasii strain (see Table 3) No discrepancies werefound in the species submitted asM intracellulareM aviumM abscessus andM tuberculosis complex
The sequence divergence among MTC members was04 The intraspecies divergence among the 188 clinicalstrains ranged from 06 to 53 (Table 3) The sequencediversity among theM abscessuswas 06 while that amongthe M gordonae clinical isolates was 53 No M gordonaeclinical strain had an identical sequence with that of theM gordonae reference strain (ATCC14470) Interestingly therpsA sequence of one strain among the 23M kansasii isolatesexhibited a relatively low level of sequence similarity (956)to that of the reference strain (ATCC 12478) while those ofall the other M kansasii isolates were identical Since thestrain was confirmed as M kansasii by rpoB and hsp65 genealignment itmight suggest a distant variant or a new subtype
4 Discussion
16S rDNA gene sequence alignment has been used as thereference method for mycobacterial species identificationHowever it has been reported that by using the 16S rDNAgene alone for species identification of clinical NTM 37of such isolates remained unclassified which illustrates theneed for additional molecular tools for proper phylogeneticassignment and accurate NTM identification [11] The com-mon assumption that bacterial isolates belong to the samespecies if they have fewer than 5ndash15 bp differences within
the 16S rDNA gene sequence [12] or if they have more than97 16S rDNA gene sequence identities [13] may not beapplicable to genus of Mycobacterium whose members aremuch more closely related to each other Furthermore somemycobacterial strains shared uniform sequence of 16S rDNAgene such asM intracellulare andM aviumM kansasii andM gastri M abscessus and M chelonae and M szulgai andM malmoense
In this study we found that the rpsA gene is useful as acomplementary method to the 16S rDNA for mycobacterialspecies identification Among the 47 reference strains andtotally 1081 paired comparisons 1081 = [119899119886
1+ 119899(119899 minus 1)1198892]
1198861= 1 119899 = 46 119889 = 1 the rpsA gene alone can
differentiate 972 (1051 out of 1081) of them if using 97sequence homology as cutoff value When referring to thephylogenetic tree the rpsA resolution might increase Forthe most commonly seen pathogenic NTM species suchas M intracellulare M avium M abscessus M chelonaeand M xenopi rpsA gene sequence could differentiate thesespecies easily Furthermore 183 of the 188 (973) clinicalisolates representing 8mycobacterial species were identifiedcorrectly by rpsA gene blast Discrepancies of identificationwere only encountered with 4 M gordonae strains and 1M kansasii strain Both these species have reportedly moreintraspecies sequence divergence [9 14ndash16] Three out of the4 M gordonae strains with discrepant identification still hadthe first distinction asM gordonae by rpsA but the sequenceidentity was lower than 97 which means the identificationmight be correct when a more complete in-house database isbeing developed
rpsA alone cannot differentiate the following betweenmembers of the M tuberculosis complex between M sene-galense and M thermoresistibile between M parafortuitumand M trivial between M diernhoferi and M duvalii andbetween M austroafricanum and M terrae however thesespecies are also difficult to be separated by other markersalone such as 16s rDNA ITS rpoB and hsp65 Several
BioMed Research International 5Ta
ble2Sequ
ence
pairdistanceso
f47type
strainso
fmycob
acteria
and1o
ut-group
strain
determ
ined
byusingthemultip
lealignm
enta
lgorith
min
theMegAlignpackage(versio
n710
DNAST
AR
Madiso
nWI)
Percentidentity
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
1mdash
1000
987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
1Mtherm
oresistibile
200
mdash987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
2Msenegalense
313
13mdash
983
966
958
958
968
968
947
947
956
954
954
920
941
954
949
951
951
930
962
947
956
945
930
894
898
911
911
911
911
911
913
920
945
947
920
934
935
932
932
928
937
937
930
928
894
3Mp
orcin
um
419
1917
mdash962
958
958
962
962
941
941
954
945
945
920
943
945
943
945
945
939
962
949
949
937
937
901
905
909
909
909
909
909
911
920
947
949
913
932
932
930
934
928
935
937
928
930
892
4Mfortuitu
m
541
41
35
39
mdash943
954
964
960
939
939
934
943
943
915
943
943
945
947
947
915
943
941
949
928
911
898
903
901
901
901
901
901
903
911
935
937
913
934
930
935
935
930
937
934
930
920
896
5Mp
hlei
649
49
43
43
59
mdash951
953
941
937
937
947
941
941
913
937
935
934
935
935
932
937
937
937
930
924
886
890
907
907
907
907
907
909
915
943
941
907
932
920
918
922
915
930
932
918
915
880
6Mg
adium
747
47
43
43
47
51
mdash966
960
954
954
947
947
947
930
951
953
956
958
958
932
941
949
947
924
920
901
905
924
924
924
924
924
926
928
949
947
930
941
949
937
937
935
939
943
926
934
903
7Mfl
avescens
837
37
33
39
37
49
35
mdash966
966
966
953
962
962
920
962
962
964
962
962
935
947
945
945
939
915
905
909
911
911
911
911
911
913
922
937
935
920
943
935
932
932
930
934
935
924
922
909
8Msmegmatis
939
39
33
39
41
61
41
35
mdash966
966
951
968
968
924
958
956
962
964
964
930
956
949
947
924
920
915
918
909
909
909
909
909
911
922
943
945
924
939
928
934
934
932
939
935
928
928
892
9Mv
accae
1057
57
55
61
63
65
47
35
35
mdash1000
949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
10Mp
arafortuitu
m
1157
57
55
61
63
65
47
35
35
00
mdash949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
11Mtriv
iale
1251
51
45
47
7055
55
49
51
53
53
mdash964
964
918
945
953
951
953
953
941
951
928
922
926
930
903
907
899
899
899
899
899
901
913
932
928
899
920
920
917
917
911
918
924
909
899
882
12Mn
eoaurum
1357
57
47
57
59
61
55
39
33
39
39
37
mdash1000
926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
13Md
iernh
oferi
1457
57
47
57
59
61
55
39
33
39
39
37
00
mdash926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
14Md
uvalii
1584
84
84
84
9193
7485
80
7474
86
7878
mdash930
939
930
932
932
917
918
911
911
894
911
898
901
892
892
892
892
892
894
888
901
905
915
905
905
896
898
898
892
901
896
890
877
15Ma
urum
1663
63
61
59
59
65
51
39
43
53
53
57
49
49
74mdash
966
960
958
958
939
947
947
935
928
924
918
924
905
905
905
905
905
907
917
932
930
911
926
924
926
930
913
924
935
915
909
884
16Mc
hubu
ense
1761
61
47
57
59
68
49
39
45
43
43
49
43
43
63
35
mdash972
970
970
941
956
945
941
935
934
913
917
903
903
903
903
903
905
913
930
932
920
928
932
926
930
917
928
928
915
911
901
17Mg
ilvum
1859
59
53
59
57
7045
37
39
37
37
51
45
45
7441
29
mdash998
998
941
949
928
934
926
930
907
911
901
901
901
901
901
903
913
928
926
913
924
928
920
920
913
920
924
913
920
892
18Mv
anbaalenii
1957
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
mdash1000
939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
19Mterrae
2057
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
00
mdash939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
20Ma
ustro
afric
anum
2176
7674
63
9172
7267
7474
7461
61
61
89
63
61
61
63
63
mdash937
918
917
911
926
888
892
894
894
894
894
894
896
903
915
917
901
909
909
899
901
896
907
913
909
899
896
21Mrhodesia
e
2245
45
39
39
59
66
61
55
45
59
59
51
53
53
87
55
45
53
51
51
65
mdash937
945
934
941
903
907
903
903
903
903
903
905
905
937
939
903
926
928
922
922
918
926
930
917
909
892
22Mo
buense
2365
65
55
53
61
65
53
57
53
7676
7661
61
9555
57
7674
7486
65
mdash958
939
930
917
920
909
909
909
909
909
911
922
937
939
917
932
926
934
937
922
937
939
924
917
890
23Ma
ichien
se
2455
55
45
53
53
65
55
57
55
7474
82
65
65
9567
61
7067
67
89
57
43
mdash937
934
913
918
915
915
915
915
915
917
915
937
941
922
930
935
928
930
930
934
941
932
924
892
24Msphagni
2572
7257
65
7674
80
63
80
80
80
7876
76115
7667
7876
7695
7063
65
mdash920
888
894
896
896
896
896
896
898
905
945
945
907
930
928
918
922
920
924
934
922
915
882
25Mtokaiense
2680
80
7465
9580
84
9184
89
89
7482
82
9580
7074
7272
7861
7470
84
mdash915
918
896
896
896
896
896
898
898
922
926
894
905
911
907
911
898
920
917
907
903
871
26Mc
helona
e
27121
121
115
106
110
124
106
101
91
110
110
104
9393
110
86
9399
9797
121
104
89
93121
91mdash
987
867
867
867
867
867
869
884
884
888
884
886
894
896
896
884
901
898
882
871
871
27Ma
bscessu
s
28117
117
110
101
104
119
101
9786
106
106
9989
89
106
80
89
9593
93117
9984
86
115
87
13mdash
875
875
875
875
875
877
888
888
892
888
890
898
899
899
890
905
901
886
875
877
28Ma
bscessu
s
2995
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
mdash1000
1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
29Mtub
erculosis
3095
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
mdash1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
30Mtub
erculosis
3195
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
mdash1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
31Mb
ovis
3295
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
mdash1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
32Ma
fricanu
m
3395
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
00
mdash998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
33Mb
ovis
3493
9393
95104
9778
9395
106
106
106
112
112
115
99102
104
102
102
112
102
9589
111
110
144
135
02
02
02
02
02
mdash911
918
922
907
918
924
918
917
918
926
932
920
917
880
34Mc
anettii
3591
9184
84
9591
7682
82
9191
9399
99122
89
9393
9191
104
102
82
91102
110
126
121
9898
9898
9895
mdash922
924
907
917
920
911
911
905
918
930
915
909
877
35Mx
enopi
3663
63
57
55
67
59
53
65
59
7878
7284
84
106
7274
7674
7491
65
65
65
57
82
126
121
89
89
89
89
89
87
82
mdash994
930
956
947
939
943
941
951
958
947
937
886
36Ma
vium
3761
61
55
53
65
61
55
67
57
80
80
7686
86
101
7472
7876
7689
63
63
61
57
78121
117
84
84
84
84
84
82
80
06
mdash935
954
951
945
949
945
954
960
953
939
888
37Ma
vium
3893
9384
9393
9974
84
80
9393
108
9595
9195
84
9391
91106
104
89
82
99115
126
121
9797
9797
97100
9974
67
mdash943
937
935
934
924
939
930
937
930
890
38Mm
almoense
3974
7470
7270
7261
59
63
7272
84
7474
102
7876
80
7878
9778
7274
74102
123
119
89
89
89
89
89
87
89
45
47
59
mdash945
941
941
934
951
947
943
943
907
39Mintracellu
lare
4076
7667
7274
84
53
67
7684
84
84
86
86
101
80
7276
7474
9776
7867
7695
115
110
82
82
82
82
82
80
84
55
51
65
57
mdash943
943
949
951
954
939
934
896
40Mscrofulaceum
4178
7872
7468
86
65
7270
9595
89
9191
112
7878
84
82
82
108
82
7076
87
99112
108
84
84
84
84
84
87
9563
57
68
61
59
mdash996
945
960
956
956
937
896
41Mm
arinum
4278
7872
7068
82
65
7270
9595
89
9191
110
7474
84
82
82
106
82
65
7482
95112
108
87
87
87
87
87
89
9559
53
7061
59
04
mdash943
960
956
954
939
894
42Mu
lcerans
4380
80
7676
7491
67
7472
9393
9593
93110
9389
9391
91112
86
82
7484
110
126
119
84
84
84
84
84
87
101
61
57
80
7053
57
59
mdash943
953
951
934
890
43Mg
ordona
e
4470
7065
67
65
7463
7063
86
86
86
89
89
117
80
7684
82
82
9978
65
7080
84
106
101
7676
7676
7678
86
51
47
63
51
51
41
41
59
mdash956
954
945
901
44Mszulga
i
4570
7065
65
7072
59
67
67
9191
80
86
86
106
67
7680
7878
9374
63
61
7089
110
106
7070
7070
7072
7443
41
7455
47
45
45
49
45
mdash964
947
888
45Ma
siaticum
4680
80
7476
7486
7880
76104
104
9793
93112
9191
9391
9197
89
80
7282
99128
123
82
82
82
82
82
85
9155
49
65
59
63
45
47
51
47
37
mdash958
888
46Mk
ansasii
4776
7676
7484
9170
82
7689
89
108
101
101
119
9795
84
82
82
108
9789
80
91104
142
137
87
87
87
87
87
89
9765
63
7459
7066
63
7057
55
43
mdash879
47Mg
astri
48117
117
115
117
112
130
104
97117
119
119
128
110
110
135
126
106
117
119
119
112
117
119
117
128
142
142
135
130
130
130
130
130
130
135
124
121
119
99112
112
115
119
106
121
121
132
mdash48
Rhodococcusequi
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
6 BioMed Research International
001
99
78
93
37
11
2
10
0
1
1
1
19
99
9 99
99
99
99
99
76
52
5
17
35
67
23
23
60
57
10
32
38
32
23
99
9
66
13
16
13
24
55
M thermoresistibileM senegalense
M porcinumM fortuitum
M phleiM gadium
M flavescensM smegmatisM vaccae
M parafortuitumM triviale
M neoaurumM diernhoferiM duvalii
M aurumM chubuense
M gilvumM vanbaalenii
M terraeM austroafricanum
M rhodesiaeM obuenseM aichienseM sphagni
M tokaienseM chelonae
M abscessusM abscessus subspecies massiliense
M tuberculosis H37RvM tuberculosis H37Ra
M bovisM bovis BCG
M canettiiM xenopi
M aviumM avium subspecies paratuberculosis
M malmoenseM intracellulareM scrofulaceum
M marinumM ulcerans
M szulgaiM gordonae
M asiaticumM kansassii
M gastri
M africanum
Rhodococcus equi
Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them
BioMed Research International 7
Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa
Primary identificationb (119899)Primary identificationb (119899)
First choice Second choiceIdentification (119899) match Identification (119899) match
M intracellulare (75)
M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961
M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991
M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960
M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970
M fortuitum complex (17)
M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981
M gordonae (7)
M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947
M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994
M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes
reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]
In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for
Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65
Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has
8 BioMed Research International
the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]
Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
HongfeiDuan andGuanLiu contributed equally to this study
Acknowledgments
The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital
References
[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006
[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996
[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998
[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007
[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011
[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998
[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998
[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999
[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999
[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002
[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003
[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992
[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004
[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992
[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003
[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009
[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013
[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Microbiology
2 BioMed Research International
suitable for phylogenetic study of the genus MycobacteriumIn this paper we report an evaluation of the rpsA gene as anovel biomarker for mycobacteria species identification
2 Materials and Methods
21 Mycobacterial Reference Strains and Clinical Isolates42 type and reference strains of the genus Mycobacterium(Table 1) 172 clinical NTM isolates and 16 M tuberculosiscomplex (MTC) isolates were investigated All the type andreference strains were purchased from the American TypeCulture Collection (ATCC) and all the clinical isolates usedin this study were obtained from the Clinical Database andSample Bank of tuberculosis of Beijing National ClinicalLab on Tuberculosis Beijing Chest Hospital All the clinicalNTM isolates were identified to the species level by sequencealignment of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoBand hsp65 genes as described before [2 7ndash9] Followingsequencing the 172 NTM clinical strains were found toinclude 10 strains ofM avium 75 strains ofM intracellulare23 strains ofM kansasii 39 strains ofM abscessus 17 strainsofM fortuitum complex 7 strains ofM gordonae and 1 strainofM neoaurum
22 rpsA Gene Amplification and Sequencing DNA wasreleased from cultured mycobacteria by boiling the culturedmycobacterial suspension in TE buffer for 10min After cen-trifugation the supernatant was used for PCR amplification[10] The primers used were forward primer 51015840-CCCTAC-ATCGGCAAGGAG-31015840 position 487ndash504 in the rpsA geneof Mycobacterium tuberculosis GenBank accession num-ber NC 0009622 and reverse primer 51015840-TGTCGATGA-CCTTGACCATC-31015840 position 1032ndash1051 in the rpsA geneof Mycobacterium tuberculosis GenBank accession numberNC 0009622 The amplified product was 565 bp PCR prod-ucts were purified and sequenced by a commercial company(YINGJUN Biotech Company Beijing China) using ABI3730 DNA Analyzer (Applied Biosystems California USA)
23 Sequence Analysis and the Phylogenetic Tree Construc-tions In addition to the 42 reference strains the rpsAsequences of 5 other mycobacterial species were obtaineddirectly from GenBank including M avium subspeciesparatuberculosis (GenBank accession number NC 0029442)M ulcerans (GenBank accession number NC 0086111) Mvanbaalenii (GenBank accession number NC 0087261) Mabscessus subspeciesmassiliense (GenBank accession numberNC 0181501) and M canettii (GenBank accession numberNC 0158481) All sequences were aligned and homologywas calculated by using the multiple alignment algorithm inthe MegAlign package (Windows version 710 DNASTARMadison Wis) 527 bp of sequence (excluding 38 nucleotidesat each end of the amplicon corresponding to the primerbinding sites) was analyzed for the phylogenetic tree con-struction by the neighbor-joining method using the MEGA505 package (httpwwwmegasoftwarenetmegaphp) Abootstrap analysis (1000 repeats) using Rhodococcus equi
(GenBank accession number NC 0063611) as the outgroupwas performed to evaluate the topology of the phylogenetictree
24 Species Identification of the Clinical Isolates The 188clinical isolates were analyzed blindly Sequences minus theknown PCR primer sequences were assembled and edited byusing SeqMan software (version 710 DNASTAR MadisonWI) Isolates were identified by comparing sequences byusing a FASTA BLASTn search with MegAlign (version710 DNASTAR Madison WI) to an in-house databaseof sequences consisting of type and reference strains fromexternal culture collections
3 Results
31 rpsA Sequence Alignment of the Reference StrainsBetween 854 and 100 sequence homology (interspeciesdivergence 0 to 146) was observed among the 42tested reference strains and the 5 additional mycobacterialspecies whose sequences were obtained from the Gen-Bank database (Table 2) All the rpsA gene sequences ofthe analyzed mycobacteria strains were distinct from theoutgroup strain R equi Among the 19 reference strainsof the slow-growing Mycobacterium genus 11 strains weregreater than 97 homology including 5 M tuberculosiscomplex strains Among the 28 reference strains of the rapid-growing Mycobacterium genus 14 strains were greater than97 homology (Table 1) The pathogenic M kansasii waseasily differentiated from the nonpathogenicM gastri (958homology) while those two species were not distinguish-able by the 16S rDNA sequence alignment The sequencehomologies between various species were 918 between Mchelonae and M abscessus 956 between M avium andM intracellulare and 939 between M szulgai and Mmalmoense However the sequence homology between otherspecies was higher for example it was 996 between Mulcerans and M marinum and 987 between M abscessussubspecies massiliense and M abscessus All members ofthe M tuberculosis complex had identical sequences as didM senegalense and M thermoresistibile M parafortuitumand M trivial M diernhoferi and M duvalii and M aus-troafricanum andM terrae
32 Phylogenetic Tree Construction A phylogenetic treewhich provided the basis for species differentiation in thegenus Mycobacterium was constructed (Figure 1) The abso-lute majority tested species showed good separation Therapid-growing species were well defined from the slow-growing species in the tree M chelonae M abscessus sub-speciesmassiliense andMabscessus which are categorized inthe pathogenic taxonomic group of rapid-growing mycobac-teria formed a distinctive cluster which was much closer tothe slow-growing species compared with the nonpathogenicgroup of rapid-growing mycobacteria The reliability of thephylogenetic tree was verified by the bootstrapmethod usingR equi as the outgroup
BioMed Research International 3
Table 1 Percentage rpsA sequence match among type strain
Species Type strain First match ()a Second match ()a
M thermoresistibile ATCC19527 M senegalense (100) M porcinum (987)M senegalense ATCC35796 M thermoresistibile (100) M porcinum (987)M porcinum ATCC33776 M senegalenseM thermoresistibile (987) M fortuitum (983)
M fortuitum ATCC6481 M porcinum (983) M senegalenseM thermoresistibile(981)
M phlei ATCC11758 M porcinum (965) M fortuitum (961)M gadium ATCC27726 M porcinumM fortuitum (957) M smegmatis (953)M flavescens ATCC14474 M smegmatis (966) M vaccae (960)
M smegmatis ATCC19420 M vaccaeM parafortuitumM triviale(966)
M senegalenseM thermoresistibile(963)
M vaccae ATCC15483 M diernhoferiM duvalii (968) M parafortuitumM triviale (966)M parafortuitum ATCC19686 M triviale (100) M terraeM austroafricanum (966)M triviale ATCC23292 M parafortuitum (100) M terraeM austroafricanum (966)M neoaurum ATCC25795 M diernhoferiM duvalii (964) M porcinum (955)M diernhoferi ATCC19340 M duvalii (100) M vaccae (968)M duvalii ATCC43910 M diernhoferi (100) M vaccae (968)M aurum ATCC23366 M gilvum (939) M chubuenseM vanbaalenii (930)M chubuense ATCC27278 M gilvum (966) M vanbaalenii (960)M gilvum ATCC43909 M vanbaalenii (972) M terraeM austroafricanum (970)M vanbaalenii PYR-1 M terraeM austroafricanum (998) M gilvum (972)M terrae ATCC15755 M austroafricanum (100) M vanbaalenii (998)M austroafricanum ATCC33464 M terrae (100) M vanbaalenii (998)M rhodesiae ATCC27024 M terraeM austroafricanum (939) M obuense (937)
M obuense ATCC27023 M porcinum M fortuitum (961) M senegalenseM thermoresistibile(965)
M aichiense ATCC27280 M sphagni (958) M vaccae (947)M sphagni ATCC33027 M aichiense (958) M porcinum (955)M tokaiense ATCC27282 M porcinum (953) M smegmatis (937)M chelonae ATCC35752 M obuense (939) M phlei (935)M abscessus subspeciesmassiliense str GO 06 M abscessus (987) M sphagni (914)
M abscessus ATCC19977 M abscessus subspeciesmassiliense (987) M chubuense (920)
M tuberculosis H37Rv ATCC27294 M tuberculosisH37RaM africanumMbovis BCGM bovis (100) M canettii (998)
M tuberculosis H37Ra ATCC25177 M tuberculosisH37RvM africanumMbovis BCGM bovis (100) M canettii (998)
M africanum ATCC25420 M tuberculosisH37RaM tuberculosisRvM bovis BCGM bovis (100) M canettii (998)
M bovis BCG ATCC35735 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis (100) M canettii (998)
M bovis ATCC19210 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis BCG (100) M canettii (998)
M canettii CIPT140010059 M tuberculosisM africanumM bovisBCGM bovis (998) M asiaticum (932)
M xenopi ATCC19250 M asiaticum (930) M avium subspecies paratuberculosis(924)
M avium ATCC25291 M avium subspecies paratuberculosis(996) M asiaticum (958)
4 BioMed Research International
Table 1 Continued
Species Type strain First match ()a Second match ()a
M avium subspeciesparatuberculosis K-10 M avium (994) M asiaticum (96)
M malmoense ATCC29571 M intracellulare (943) M szulgai (939)
M intracellulare ATCC13950 M avium (956) M avium subspecies paratuberculosis(954)
M scrofulaceum ATCC19981 M asiaticum (954) M szulgai (951)M marinum ATCC927 M ulcerans (996) M szulgai (960)M ulcerans Agy99 M marinum (996) M szulgai (960)M szulgai ATCC25799 M marinumM ulcerans (960) M asiaticum (956)M gordonae ATCC14470 M asiaticum (953) M kansasii (951)M asiaticum ATCC25276 M simiae (977) M kansasii (964)M kansasii ATCC12478 M asiaticum (963) M gastri (958)M gastri ATCC15754 M kansasii (958) M asiaticum (945)Rhodococcus equi 103S M smegmatis (903) M intracellulare (901)aBeside the queried type strain itself the first matched or the second matched type strain
33 Species Identification Outcomes of the Clinical IsolatesrpsA sequence and alignment efficiently identified clinicalisolates representing 8 mycobacterial species We foundthat a criterion of first distinct rpsA sequence match gt97confirmed the identification of 183 of 188 (973) clinicalisolates 20 M abscessus strains 2 M fortuitum strainsand 10 M avium strains were identified to the subspecieslevel Identification discrepancies between rpsA and othersequences were only encountered with 4M gordonae strainsand 1 M kansasii strain (see Table 3) No discrepancies werefound in the species submitted asM intracellulareM aviumM abscessus andM tuberculosis complex
The sequence divergence among MTC members was04 The intraspecies divergence among the 188 clinicalstrains ranged from 06 to 53 (Table 3) The sequencediversity among theM abscessuswas 06 while that amongthe M gordonae clinical isolates was 53 No M gordonaeclinical strain had an identical sequence with that of theM gordonae reference strain (ATCC14470) Interestingly therpsA sequence of one strain among the 23M kansasii isolatesexhibited a relatively low level of sequence similarity (956)to that of the reference strain (ATCC 12478) while those ofall the other M kansasii isolates were identical Since thestrain was confirmed as M kansasii by rpoB and hsp65 genealignment itmight suggest a distant variant or a new subtype
4 Discussion
16S rDNA gene sequence alignment has been used as thereference method for mycobacterial species identificationHowever it has been reported that by using the 16S rDNAgene alone for species identification of clinical NTM 37of such isolates remained unclassified which illustrates theneed for additional molecular tools for proper phylogeneticassignment and accurate NTM identification [11] The com-mon assumption that bacterial isolates belong to the samespecies if they have fewer than 5ndash15 bp differences within
the 16S rDNA gene sequence [12] or if they have more than97 16S rDNA gene sequence identities [13] may not beapplicable to genus of Mycobacterium whose members aremuch more closely related to each other Furthermore somemycobacterial strains shared uniform sequence of 16S rDNAgene such asM intracellulare andM aviumM kansasii andM gastri M abscessus and M chelonae and M szulgai andM malmoense
In this study we found that the rpsA gene is useful as acomplementary method to the 16S rDNA for mycobacterialspecies identification Among the 47 reference strains andtotally 1081 paired comparisons 1081 = [119899119886
1+ 119899(119899 minus 1)1198892]
1198861= 1 119899 = 46 119889 = 1 the rpsA gene alone can
differentiate 972 (1051 out of 1081) of them if using 97sequence homology as cutoff value When referring to thephylogenetic tree the rpsA resolution might increase Forthe most commonly seen pathogenic NTM species suchas M intracellulare M avium M abscessus M chelonaeand M xenopi rpsA gene sequence could differentiate thesespecies easily Furthermore 183 of the 188 (973) clinicalisolates representing 8mycobacterial species were identifiedcorrectly by rpsA gene blast Discrepancies of identificationwere only encountered with 4 M gordonae strains and 1M kansasii strain Both these species have reportedly moreintraspecies sequence divergence [9 14ndash16] Three out of the4 M gordonae strains with discrepant identification still hadthe first distinction asM gordonae by rpsA but the sequenceidentity was lower than 97 which means the identificationmight be correct when a more complete in-house database isbeing developed
rpsA alone cannot differentiate the following betweenmembers of the M tuberculosis complex between M sene-galense and M thermoresistibile between M parafortuitumand M trivial between M diernhoferi and M duvalii andbetween M austroafricanum and M terrae however thesespecies are also difficult to be separated by other markersalone such as 16s rDNA ITS rpoB and hsp65 Several
BioMed Research International 5Ta
ble2Sequ
ence
pairdistanceso
f47type
strainso
fmycob
acteria
and1o
ut-group
strain
determ
ined
byusingthemultip
lealignm
enta
lgorith
min
theMegAlignpackage(versio
n710
DNAST
AR
Madiso
nWI)
Percentidentity
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
1mdash
1000
987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
1Mtherm
oresistibile
200
mdash987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
2Msenegalense
313
13mdash
983
966
958
958
968
968
947
947
956
954
954
920
941
954
949
951
951
930
962
947
956
945
930
894
898
911
911
911
911
911
913
920
945
947
920
934
935
932
932
928
937
937
930
928
894
3Mp
orcin
um
419
1917
mdash962
958
958
962
962
941
941
954
945
945
920
943
945
943
945
945
939
962
949
949
937
937
901
905
909
909
909
909
909
911
920
947
949
913
932
932
930
934
928
935
937
928
930
892
4Mfortuitu
m
541
41
35
39
mdash943
954
964
960
939
939
934
943
943
915
943
943
945
947
947
915
943
941
949
928
911
898
903
901
901
901
901
901
903
911
935
937
913
934
930
935
935
930
937
934
930
920
896
5Mp
hlei
649
49
43
43
59
mdash951
953
941
937
937
947
941
941
913
937
935
934
935
935
932
937
937
937
930
924
886
890
907
907
907
907
907
909
915
943
941
907
932
920
918
922
915
930
932
918
915
880
6Mg
adium
747
47
43
43
47
51
mdash966
960
954
954
947
947
947
930
951
953
956
958
958
932
941
949
947
924
920
901
905
924
924
924
924
924
926
928
949
947
930
941
949
937
937
935
939
943
926
934
903
7Mfl
avescens
837
37
33
39
37
49
35
mdash966
966
966
953
962
962
920
962
962
964
962
962
935
947
945
945
939
915
905
909
911
911
911
911
911
913
922
937
935
920
943
935
932
932
930
934
935
924
922
909
8Msmegmatis
939
39
33
39
41
61
41
35
mdash966
966
951
968
968
924
958
956
962
964
964
930
956
949
947
924
920
915
918
909
909
909
909
909
911
922
943
945
924
939
928
934
934
932
939
935
928
928
892
9Mv
accae
1057
57
55
61
63
65
47
35
35
mdash1000
949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
10Mp
arafortuitu
m
1157
57
55
61
63
65
47
35
35
00
mdash949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
11Mtriv
iale
1251
51
45
47
7055
55
49
51
53
53
mdash964
964
918
945
953
951
953
953
941
951
928
922
926
930
903
907
899
899
899
899
899
901
913
932
928
899
920
920
917
917
911
918
924
909
899
882
12Mn
eoaurum
1357
57
47
57
59
61
55
39
33
39
39
37
mdash1000
926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
13Md
iernh
oferi
1457
57
47
57
59
61
55
39
33
39
39
37
00
mdash926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
14Md
uvalii
1584
84
84
84
9193
7485
80
7474
86
7878
mdash930
939
930
932
932
917
918
911
911
894
911
898
901
892
892
892
892
892
894
888
901
905
915
905
905
896
898
898
892
901
896
890
877
15Ma
urum
1663
63
61
59
59
65
51
39
43
53
53
57
49
49
74mdash
966
960
958
958
939
947
947
935
928
924
918
924
905
905
905
905
905
907
917
932
930
911
926
924
926
930
913
924
935
915
909
884
16Mc
hubu
ense
1761
61
47
57
59
68
49
39
45
43
43
49
43
43
63
35
mdash972
970
970
941
956
945
941
935
934
913
917
903
903
903
903
903
905
913
930
932
920
928
932
926
930
917
928
928
915
911
901
17Mg
ilvum
1859
59
53
59
57
7045
37
39
37
37
51
45
45
7441
29
mdash998
998
941
949
928
934
926
930
907
911
901
901
901
901
901
903
913
928
926
913
924
928
920
920
913
920
924
913
920
892
18Mv
anbaalenii
1957
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
mdash1000
939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
19Mterrae
2057
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
00
mdash939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
20Ma
ustro
afric
anum
2176
7674
63
9172
7267
7474
7461
61
61
89
63
61
61
63
63
mdash937
918
917
911
926
888
892
894
894
894
894
894
896
903
915
917
901
909
909
899
901
896
907
913
909
899
896
21Mrhodesia
e
2245
45
39
39
59
66
61
55
45
59
59
51
53
53
87
55
45
53
51
51
65
mdash937
945
934
941
903
907
903
903
903
903
903
905
905
937
939
903
926
928
922
922
918
926
930
917
909
892
22Mo
buense
2365
65
55
53
61
65
53
57
53
7676
7661
61
9555
57
7674
7486
65
mdash958
939
930
917
920
909
909
909
909
909
911
922
937
939
917
932
926
934
937
922
937
939
924
917
890
23Ma
ichien
se
2455
55
45
53
53
65
55
57
55
7474
82
65
65
9567
61
7067
67
89
57
43
mdash937
934
913
918
915
915
915
915
915
917
915
937
941
922
930
935
928
930
930
934
941
932
924
892
24Msphagni
2572
7257
65
7674
80
63
80
80
80
7876
76115
7667
7876
7695
7063
65
mdash920
888
894
896
896
896
896
896
898
905
945
945
907
930
928
918
922
920
924
934
922
915
882
25Mtokaiense
2680
80
7465
9580
84
9184
89
89
7482
82
9580
7074
7272
7861
7470
84
mdash915
918
896
896
896
896
896
898
898
922
926
894
905
911
907
911
898
920
917
907
903
871
26Mc
helona
e
27121
121
115
106
110
124
106
101
91
110
110
104
9393
110
86
9399
9797
121
104
89
93121
91mdash
987
867
867
867
867
867
869
884
884
888
884
886
894
896
896
884
901
898
882
871
871
27Ma
bscessu
s
28117
117
110
101
104
119
101
9786
106
106
9989
89
106
80
89
9593
93117
9984
86
115
87
13mdash
875
875
875
875
875
877
888
888
892
888
890
898
899
899
890
905
901
886
875
877
28Ma
bscessu
s
2995
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
mdash1000
1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
29Mtub
erculosis
3095
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
mdash1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
30Mtub
erculosis
3195
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
mdash1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
31Mb
ovis
3295
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
mdash1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
32Ma
fricanu
m
3395
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
00
mdash998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
33Mb
ovis
3493
9393
95104
9778
9395
106
106
106
112
112
115
99102
104
102
102
112
102
9589
111
110
144
135
02
02
02
02
02
mdash911
918
922
907
918
924
918
917
918
926
932
920
917
880
34Mc
anettii
3591
9184
84
9591
7682
82
9191
9399
99122
89
9393
9191
104
102
82
91102
110
126
121
9898
9898
9895
mdash922
924
907
917
920
911
911
905
918
930
915
909
877
35Mx
enopi
3663
63
57
55
67
59
53
65
59
7878
7284
84
106
7274
7674
7491
65
65
65
57
82
126
121
89
89
89
89
89
87
82
mdash994
930
956
947
939
943
941
951
958
947
937
886
36Ma
vium
3761
61
55
53
65
61
55
67
57
80
80
7686
86
101
7472
7876
7689
63
63
61
57
78121
117
84
84
84
84
84
82
80
06
mdash935
954
951
945
949
945
954
960
953
939
888
37Ma
vium
3893
9384
9393
9974
84
80
9393
108
9595
9195
84
9391
91106
104
89
82
99115
126
121
9797
9797
97100
9974
67
mdash943
937
935
934
924
939
930
937
930
890
38Mm
almoense
3974
7470
7270
7261
59
63
7272
84
7474
102
7876
80
7878
9778
7274
74102
123
119
89
89
89
89
89
87
89
45
47
59
mdash945
941
941
934
951
947
943
943
907
39Mintracellu
lare
4076
7667
7274
84
53
67
7684
84
84
86
86
101
80
7276
7474
9776
7867
7695
115
110
82
82
82
82
82
80
84
55
51
65
57
mdash943
943
949
951
954
939
934
896
40Mscrofulaceum
4178
7872
7468
86
65
7270
9595
89
9191
112
7878
84
82
82
108
82
7076
87
99112
108
84
84
84
84
84
87
9563
57
68
61
59
mdash996
945
960
956
956
937
896
41Mm
arinum
4278
7872
7068
82
65
7270
9595
89
9191
110
7474
84
82
82
106
82
65
7482
95112
108
87
87
87
87
87
89
9559
53
7061
59
04
mdash943
960
956
954
939
894
42Mu
lcerans
4380
80
7676
7491
67
7472
9393
9593
93110
9389
9391
91112
86
82
7484
110
126
119
84
84
84
84
84
87
101
61
57
80
7053
57
59
mdash943
953
951
934
890
43Mg
ordona
e
4470
7065
67
65
7463
7063
86
86
86
89
89
117
80
7684
82
82
9978
65
7080
84
106
101
7676
7676
7678
86
51
47
63
51
51
41
41
59
mdash956
954
945
901
44Mszulga
i
4570
7065
65
7072
59
67
67
9191
80
86
86
106
67
7680
7878
9374
63
61
7089
110
106
7070
7070
7072
7443
41
7455
47
45
45
49
45
mdash964
947
888
45Ma
siaticum
4680
80
7476
7486
7880
76104
104
9793
93112
9191
9391
9197
89
80
7282
99128
123
82
82
82
82
82
85
9155
49
65
59
63
45
47
51
47
37
mdash958
888
46Mk
ansasii
4776
7676
7484
9170
82
7689
89
108
101
101
119
9795
84
82
82
108
9789
80
91104
142
137
87
87
87
87
87
89
9765
63
7459
7066
63
7057
55
43
mdash879
47Mg
astri
48117
117
115
117
112
130
104
97117
119
119
128
110
110
135
126
106
117
119
119
112
117
119
117
128
142
142
135
130
130
130
130
130
130
135
124
121
119
99112
112
115
119
106
121
121
132
mdash48
Rhodococcusequi
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
6 BioMed Research International
001
99
78
93
37
11
2
10
0
1
1
1
19
99
9 99
99
99
99
99
76
52
5
17
35
67
23
23
60
57
10
32
38
32
23
99
9
66
13
16
13
24
55
M thermoresistibileM senegalense
M porcinumM fortuitum
M phleiM gadium
M flavescensM smegmatisM vaccae
M parafortuitumM triviale
M neoaurumM diernhoferiM duvalii
M aurumM chubuense
M gilvumM vanbaalenii
M terraeM austroafricanum
M rhodesiaeM obuenseM aichienseM sphagni
M tokaienseM chelonae
M abscessusM abscessus subspecies massiliense
M tuberculosis H37RvM tuberculosis H37Ra
M bovisM bovis BCG
M canettiiM xenopi
M aviumM avium subspecies paratuberculosis
M malmoenseM intracellulareM scrofulaceum
M marinumM ulcerans
M szulgaiM gordonae
M asiaticumM kansassii
M gastri
M africanum
Rhodococcus equi
Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them
BioMed Research International 7
Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa
Primary identificationb (119899)Primary identificationb (119899)
First choice Second choiceIdentification (119899) match Identification (119899) match
M intracellulare (75)
M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961
M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991
M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960
M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970
M fortuitum complex (17)
M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981
M gordonae (7)
M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947
M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994
M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes
reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]
In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for
Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65
Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has
8 BioMed Research International
the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]
Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
HongfeiDuan andGuanLiu contributed equally to this study
Acknowledgments
The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital
References
[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006
[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996
[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998
[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007
[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011
[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998
[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998
[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999
[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999
[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002
[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003
[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992
[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004
[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992
[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003
[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009
[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013
[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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BioMed Research International 3
Table 1 Percentage rpsA sequence match among type strain
Species Type strain First match ()a Second match ()a
M thermoresistibile ATCC19527 M senegalense (100) M porcinum (987)M senegalense ATCC35796 M thermoresistibile (100) M porcinum (987)M porcinum ATCC33776 M senegalenseM thermoresistibile (987) M fortuitum (983)
M fortuitum ATCC6481 M porcinum (983) M senegalenseM thermoresistibile(981)
M phlei ATCC11758 M porcinum (965) M fortuitum (961)M gadium ATCC27726 M porcinumM fortuitum (957) M smegmatis (953)M flavescens ATCC14474 M smegmatis (966) M vaccae (960)
M smegmatis ATCC19420 M vaccaeM parafortuitumM triviale(966)
M senegalenseM thermoresistibile(963)
M vaccae ATCC15483 M diernhoferiM duvalii (968) M parafortuitumM triviale (966)M parafortuitum ATCC19686 M triviale (100) M terraeM austroafricanum (966)M triviale ATCC23292 M parafortuitum (100) M terraeM austroafricanum (966)M neoaurum ATCC25795 M diernhoferiM duvalii (964) M porcinum (955)M diernhoferi ATCC19340 M duvalii (100) M vaccae (968)M duvalii ATCC43910 M diernhoferi (100) M vaccae (968)M aurum ATCC23366 M gilvum (939) M chubuenseM vanbaalenii (930)M chubuense ATCC27278 M gilvum (966) M vanbaalenii (960)M gilvum ATCC43909 M vanbaalenii (972) M terraeM austroafricanum (970)M vanbaalenii PYR-1 M terraeM austroafricanum (998) M gilvum (972)M terrae ATCC15755 M austroafricanum (100) M vanbaalenii (998)M austroafricanum ATCC33464 M terrae (100) M vanbaalenii (998)M rhodesiae ATCC27024 M terraeM austroafricanum (939) M obuense (937)
M obuense ATCC27023 M porcinum M fortuitum (961) M senegalenseM thermoresistibile(965)
M aichiense ATCC27280 M sphagni (958) M vaccae (947)M sphagni ATCC33027 M aichiense (958) M porcinum (955)M tokaiense ATCC27282 M porcinum (953) M smegmatis (937)M chelonae ATCC35752 M obuense (939) M phlei (935)M abscessus subspeciesmassiliense str GO 06 M abscessus (987) M sphagni (914)
M abscessus ATCC19977 M abscessus subspeciesmassiliense (987) M chubuense (920)
M tuberculosis H37Rv ATCC27294 M tuberculosisH37RaM africanumMbovis BCGM bovis (100) M canettii (998)
M tuberculosis H37Ra ATCC25177 M tuberculosisH37RvM africanumMbovis BCGM bovis (100) M canettii (998)
M africanum ATCC25420 M tuberculosisH37RaM tuberculosisRvM bovis BCGM bovis (100) M canettii (998)
M bovis BCG ATCC35735 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis (100) M canettii (998)
M bovis ATCC19210 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis BCG (100) M canettii (998)
M canettii CIPT140010059 M tuberculosisM africanumM bovisBCGM bovis (998) M asiaticum (932)
M xenopi ATCC19250 M asiaticum (930) M avium subspecies paratuberculosis(924)
M avium ATCC25291 M avium subspecies paratuberculosis(996) M asiaticum (958)
4 BioMed Research International
Table 1 Continued
Species Type strain First match ()a Second match ()a
M avium subspeciesparatuberculosis K-10 M avium (994) M asiaticum (96)
M malmoense ATCC29571 M intracellulare (943) M szulgai (939)
M intracellulare ATCC13950 M avium (956) M avium subspecies paratuberculosis(954)
M scrofulaceum ATCC19981 M asiaticum (954) M szulgai (951)M marinum ATCC927 M ulcerans (996) M szulgai (960)M ulcerans Agy99 M marinum (996) M szulgai (960)M szulgai ATCC25799 M marinumM ulcerans (960) M asiaticum (956)M gordonae ATCC14470 M asiaticum (953) M kansasii (951)M asiaticum ATCC25276 M simiae (977) M kansasii (964)M kansasii ATCC12478 M asiaticum (963) M gastri (958)M gastri ATCC15754 M kansasii (958) M asiaticum (945)Rhodococcus equi 103S M smegmatis (903) M intracellulare (901)aBeside the queried type strain itself the first matched or the second matched type strain
33 Species Identification Outcomes of the Clinical IsolatesrpsA sequence and alignment efficiently identified clinicalisolates representing 8 mycobacterial species We foundthat a criterion of first distinct rpsA sequence match gt97confirmed the identification of 183 of 188 (973) clinicalisolates 20 M abscessus strains 2 M fortuitum strainsand 10 M avium strains were identified to the subspecieslevel Identification discrepancies between rpsA and othersequences were only encountered with 4M gordonae strainsand 1 M kansasii strain (see Table 3) No discrepancies werefound in the species submitted asM intracellulareM aviumM abscessus andM tuberculosis complex
The sequence divergence among MTC members was04 The intraspecies divergence among the 188 clinicalstrains ranged from 06 to 53 (Table 3) The sequencediversity among theM abscessuswas 06 while that amongthe M gordonae clinical isolates was 53 No M gordonaeclinical strain had an identical sequence with that of theM gordonae reference strain (ATCC14470) Interestingly therpsA sequence of one strain among the 23M kansasii isolatesexhibited a relatively low level of sequence similarity (956)to that of the reference strain (ATCC 12478) while those ofall the other M kansasii isolates were identical Since thestrain was confirmed as M kansasii by rpoB and hsp65 genealignment itmight suggest a distant variant or a new subtype
4 Discussion
16S rDNA gene sequence alignment has been used as thereference method for mycobacterial species identificationHowever it has been reported that by using the 16S rDNAgene alone for species identification of clinical NTM 37of such isolates remained unclassified which illustrates theneed for additional molecular tools for proper phylogeneticassignment and accurate NTM identification [11] The com-mon assumption that bacterial isolates belong to the samespecies if they have fewer than 5ndash15 bp differences within
the 16S rDNA gene sequence [12] or if they have more than97 16S rDNA gene sequence identities [13] may not beapplicable to genus of Mycobacterium whose members aremuch more closely related to each other Furthermore somemycobacterial strains shared uniform sequence of 16S rDNAgene such asM intracellulare andM aviumM kansasii andM gastri M abscessus and M chelonae and M szulgai andM malmoense
In this study we found that the rpsA gene is useful as acomplementary method to the 16S rDNA for mycobacterialspecies identification Among the 47 reference strains andtotally 1081 paired comparisons 1081 = [119899119886
1+ 119899(119899 minus 1)1198892]
1198861= 1 119899 = 46 119889 = 1 the rpsA gene alone can
differentiate 972 (1051 out of 1081) of them if using 97sequence homology as cutoff value When referring to thephylogenetic tree the rpsA resolution might increase Forthe most commonly seen pathogenic NTM species suchas M intracellulare M avium M abscessus M chelonaeand M xenopi rpsA gene sequence could differentiate thesespecies easily Furthermore 183 of the 188 (973) clinicalisolates representing 8mycobacterial species were identifiedcorrectly by rpsA gene blast Discrepancies of identificationwere only encountered with 4 M gordonae strains and 1M kansasii strain Both these species have reportedly moreintraspecies sequence divergence [9 14ndash16] Three out of the4 M gordonae strains with discrepant identification still hadthe first distinction asM gordonae by rpsA but the sequenceidentity was lower than 97 which means the identificationmight be correct when a more complete in-house database isbeing developed
rpsA alone cannot differentiate the following betweenmembers of the M tuberculosis complex between M sene-galense and M thermoresistibile between M parafortuitumand M trivial between M diernhoferi and M duvalii andbetween M austroafricanum and M terrae however thesespecies are also difficult to be separated by other markersalone such as 16s rDNA ITS rpoB and hsp65 Several
BioMed Research International 5Ta
ble2Sequ
ence
pairdistanceso
f47type
strainso
fmycob
acteria
and1o
ut-group
strain
determ
ined
byusingthemultip
lealignm
enta
lgorith
min
theMegAlignpackage(versio
n710
DNAST
AR
Madiso
nWI)
Percentidentity
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
1mdash
1000
987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
1Mtherm
oresistibile
200
mdash987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
2Msenegalense
313
13mdash
983
966
958
958
968
968
947
947
956
954
954
920
941
954
949
951
951
930
962
947
956
945
930
894
898
911
911
911
911
911
913
920
945
947
920
934
935
932
932
928
937
937
930
928
894
3Mp
orcin
um
419
1917
mdash962
958
958
962
962
941
941
954
945
945
920
943
945
943
945
945
939
962
949
949
937
937
901
905
909
909
909
909
909
911
920
947
949
913
932
932
930
934
928
935
937
928
930
892
4Mfortuitu
m
541
41
35
39
mdash943
954
964
960
939
939
934
943
943
915
943
943
945
947
947
915
943
941
949
928
911
898
903
901
901
901
901
901
903
911
935
937
913
934
930
935
935
930
937
934
930
920
896
5Mp
hlei
649
49
43
43
59
mdash951
953
941
937
937
947
941
941
913
937
935
934
935
935
932
937
937
937
930
924
886
890
907
907
907
907
907
909
915
943
941
907
932
920
918
922
915
930
932
918
915
880
6Mg
adium
747
47
43
43
47
51
mdash966
960
954
954
947
947
947
930
951
953
956
958
958
932
941
949
947
924
920
901
905
924
924
924
924
924
926
928
949
947
930
941
949
937
937
935
939
943
926
934
903
7Mfl
avescens
837
37
33
39
37
49
35
mdash966
966
966
953
962
962
920
962
962
964
962
962
935
947
945
945
939
915
905
909
911
911
911
911
911
913
922
937
935
920
943
935
932
932
930
934
935
924
922
909
8Msmegmatis
939
39
33
39
41
61
41
35
mdash966
966
951
968
968
924
958
956
962
964
964
930
956
949
947
924
920
915
918
909
909
909
909
909
911
922
943
945
924
939
928
934
934
932
939
935
928
928
892
9Mv
accae
1057
57
55
61
63
65
47
35
35
mdash1000
949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
10Mp
arafortuitu
m
1157
57
55
61
63
65
47
35
35
00
mdash949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
11Mtriv
iale
1251
51
45
47
7055
55
49
51
53
53
mdash964
964
918
945
953
951
953
953
941
951
928
922
926
930
903
907
899
899
899
899
899
901
913
932
928
899
920
920
917
917
911
918
924
909
899
882
12Mn
eoaurum
1357
57
47
57
59
61
55
39
33
39
39
37
mdash1000
926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
13Md
iernh
oferi
1457
57
47
57
59
61
55
39
33
39
39
37
00
mdash926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
14Md
uvalii
1584
84
84
84
9193
7485
80
7474
86
7878
mdash930
939
930
932
932
917
918
911
911
894
911
898
901
892
892
892
892
892
894
888
901
905
915
905
905
896
898
898
892
901
896
890
877
15Ma
urum
1663
63
61
59
59
65
51
39
43
53
53
57
49
49
74mdash
966
960
958
958
939
947
947
935
928
924
918
924
905
905
905
905
905
907
917
932
930
911
926
924
926
930
913
924
935
915
909
884
16Mc
hubu
ense
1761
61
47
57
59
68
49
39
45
43
43
49
43
43
63
35
mdash972
970
970
941
956
945
941
935
934
913
917
903
903
903
903
903
905
913
930
932
920
928
932
926
930
917
928
928
915
911
901
17Mg
ilvum
1859
59
53
59
57
7045
37
39
37
37
51
45
45
7441
29
mdash998
998
941
949
928
934
926
930
907
911
901
901
901
901
901
903
913
928
926
913
924
928
920
920
913
920
924
913
920
892
18Mv
anbaalenii
1957
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
mdash1000
939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
19Mterrae
2057
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
00
mdash939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
20Ma
ustro
afric
anum
2176
7674
63
9172
7267
7474
7461
61
61
89
63
61
61
63
63
mdash937
918
917
911
926
888
892
894
894
894
894
894
896
903
915
917
901
909
909
899
901
896
907
913
909
899
896
21Mrhodesia
e
2245
45
39
39
59
66
61
55
45
59
59
51
53
53
87
55
45
53
51
51
65
mdash937
945
934
941
903
907
903
903
903
903
903
905
905
937
939
903
926
928
922
922
918
926
930
917
909
892
22Mo
buense
2365
65
55
53
61
65
53
57
53
7676
7661
61
9555
57
7674
7486
65
mdash958
939
930
917
920
909
909
909
909
909
911
922
937
939
917
932
926
934
937
922
937
939
924
917
890
23Ma
ichien
se
2455
55
45
53
53
65
55
57
55
7474
82
65
65
9567
61
7067
67
89
57
43
mdash937
934
913
918
915
915
915
915
915
917
915
937
941
922
930
935
928
930
930
934
941
932
924
892
24Msphagni
2572
7257
65
7674
80
63
80
80
80
7876
76115
7667
7876
7695
7063
65
mdash920
888
894
896
896
896
896
896
898
905
945
945
907
930
928
918
922
920
924
934
922
915
882
25Mtokaiense
2680
80
7465
9580
84
9184
89
89
7482
82
9580
7074
7272
7861
7470
84
mdash915
918
896
896
896
896
896
898
898
922
926
894
905
911
907
911
898
920
917
907
903
871
26Mc
helona
e
27121
121
115
106
110
124
106
101
91
110
110
104
9393
110
86
9399
9797
121
104
89
93121
91mdash
987
867
867
867
867
867
869
884
884
888
884
886
894
896
896
884
901
898
882
871
871
27Ma
bscessu
s
28117
117
110
101
104
119
101
9786
106
106
9989
89
106
80
89
9593
93117
9984
86
115
87
13mdash
875
875
875
875
875
877
888
888
892
888
890
898
899
899
890
905
901
886
875
877
28Ma
bscessu
s
2995
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
mdash1000
1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
29Mtub
erculosis
3095
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
mdash1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
30Mtub
erculosis
3195
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
mdash1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
31Mb
ovis
3295
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
mdash1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
32Ma
fricanu
m
3395
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
00
mdash998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
33Mb
ovis
3493
9393
95104
9778
9395
106
106
106
112
112
115
99102
104
102
102
112
102
9589
111
110
144
135
02
02
02
02
02
mdash911
918
922
907
918
924
918
917
918
926
932
920
917
880
34Mc
anettii
3591
9184
84
9591
7682
82
9191
9399
99122
89
9393
9191
104
102
82
91102
110
126
121
9898
9898
9895
mdash922
924
907
917
920
911
911
905
918
930
915
909
877
35Mx
enopi
3663
63
57
55
67
59
53
65
59
7878
7284
84
106
7274
7674
7491
65
65
65
57
82
126
121
89
89
89
89
89
87
82
mdash994
930
956
947
939
943
941
951
958
947
937
886
36Ma
vium
3761
61
55
53
65
61
55
67
57
80
80
7686
86
101
7472
7876
7689
63
63
61
57
78121
117
84
84
84
84
84
82
80
06
mdash935
954
951
945
949
945
954
960
953
939
888
37Ma
vium
3893
9384
9393
9974
84
80
9393
108
9595
9195
84
9391
91106
104
89
82
99115
126
121
9797
9797
97100
9974
67
mdash943
937
935
934
924
939
930
937
930
890
38Mm
almoense
3974
7470
7270
7261
59
63
7272
84
7474
102
7876
80
7878
9778
7274
74102
123
119
89
89
89
89
89
87
89
45
47
59
mdash945
941
941
934
951
947
943
943
907
39Mintracellu
lare
4076
7667
7274
84
53
67
7684
84
84
86
86
101
80
7276
7474
9776
7867
7695
115
110
82
82
82
82
82
80
84
55
51
65
57
mdash943
943
949
951
954
939
934
896
40Mscrofulaceum
4178
7872
7468
86
65
7270
9595
89
9191
112
7878
84
82
82
108
82
7076
87
99112
108
84
84
84
84
84
87
9563
57
68
61
59
mdash996
945
960
956
956
937
896
41Mm
arinum
4278
7872
7068
82
65
7270
9595
89
9191
110
7474
84
82
82
106
82
65
7482
95112
108
87
87
87
87
87
89
9559
53
7061
59
04
mdash943
960
956
954
939
894
42Mu
lcerans
4380
80
7676
7491
67
7472
9393
9593
93110
9389
9391
91112
86
82
7484
110
126
119
84
84
84
84
84
87
101
61
57
80
7053
57
59
mdash943
953
951
934
890
43Mg
ordona
e
4470
7065
67
65
7463
7063
86
86
86
89
89
117
80
7684
82
82
9978
65
7080
84
106
101
7676
7676
7678
86
51
47
63
51
51
41
41
59
mdash956
954
945
901
44Mszulga
i
4570
7065
65
7072
59
67
67
9191
80
86
86
106
67
7680
7878
9374
63
61
7089
110
106
7070
7070
7072
7443
41
7455
47
45
45
49
45
mdash964
947
888
45Ma
siaticum
4680
80
7476
7486
7880
76104
104
9793
93112
9191
9391
9197
89
80
7282
99128
123
82
82
82
82
82
85
9155
49
65
59
63
45
47
51
47
37
mdash958
888
46Mk
ansasii
4776
7676
7484
9170
82
7689
89
108
101
101
119
9795
84
82
82
108
9789
80
91104
142
137
87
87
87
87
87
89
9765
63
7459
7066
63
7057
55
43
mdash879
47Mg
astri
48117
117
115
117
112
130
104
97117
119
119
128
110
110
135
126
106
117
119
119
112
117
119
117
128
142
142
135
130
130
130
130
130
130
135
124
121
119
99112
112
115
119
106
121
121
132
mdash48
Rhodococcusequi
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
6 BioMed Research International
001
99
78
93
37
11
2
10
0
1
1
1
19
99
9 99
99
99
99
99
76
52
5
17
35
67
23
23
60
57
10
32
38
32
23
99
9
66
13
16
13
24
55
M thermoresistibileM senegalense
M porcinumM fortuitum
M phleiM gadium
M flavescensM smegmatisM vaccae
M parafortuitumM triviale
M neoaurumM diernhoferiM duvalii
M aurumM chubuense
M gilvumM vanbaalenii
M terraeM austroafricanum
M rhodesiaeM obuenseM aichienseM sphagni
M tokaienseM chelonae
M abscessusM abscessus subspecies massiliense
M tuberculosis H37RvM tuberculosis H37Ra
M bovisM bovis BCG
M canettiiM xenopi
M aviumM avium subspecies paratuberculosis
M malmoenseM intracellulareM scrofulaceum
M marinumM ulcerans
M szulgaiM gordonae
M asiaticumM kansassii
M gastri
M africanum
Rhodococcus equi
Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them
BioMed Research International 7
Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa
Primary identificationb (119899)Primary identificationb (119899)
First choice Second choiceIdentification (119899) match Identification (119899) match
M intracellulare (75)
M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961
M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991
M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960
M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970
M fortuitum complex (17)
M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981
M gordonae (7)
M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947
M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994
M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes
reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]
In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for
Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65
Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has
8 BioMed Research International
the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]
Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
HongfeiDuan andGuanLiu contributed equally to this study
Acknowledgments
The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital
References
[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006
[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996
[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998
[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007
[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011
[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998
[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998
[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999
[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999
[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002
[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003
[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992
[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004
[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992
[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003
[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009
[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013
[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
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BioinformaticsAdvances in
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Signal TransductionJournal of
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Evolutionary BiologyInternational Journal of
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Microbiology
4 BioMed Research International
Table 1 Continued
Species Type strain First match ()a Second match ()a
M avium subspeciesparatuberculosis K-10 M avium (994) M asiaticum (96)
M malmoense ATCC29571 M intracellulare (943) M szulgai (939)
M intracellulare ATCC13950 M avium (956) M avium subspecies paratuberculosis(954)
M scrofulaceum ATCC19981 M asiaticum (954) M szulgai (951)M marinum ATCC927 M ulcerans (996) M szulgai (960)M ulcerans Agy99 M marinum (996) M szulgai (960)M szulgai ATCC25799 M marinumM ulcerans (960) M asiaticum (956)M gordonae ATCC14470 M asiaticum (953) M kansasii (951)M asiaticum ATCC25276 M simiae (977) M kansasii (964)M kansasii ATCC12478 M asiaticum (963) M gastri (958)M gastri ATCC15754 M kansasii (958) M asiaticum (945)Rhodococcus equi 103S M smegmatis (903) M intracellulare (901)aBeside the queried type strain itself the first matched or the second matched type strain
33 Species Identification Outcomes of the Clinical IsolatesrpsA sequence and alignment efficiently identified clinicalisolates representing 8 mycobacterial species We foundthat a criterion of first distinct rpsA sequence match gt97confirmed the identification of 183 of 188 (973) clinicalisolates 20 M abscessus strains 2 M fortuitum strainsand 10 M avium strains were identified to the subspecieslevel Identification discrepancies between rpsA and othersequences were only encountered with 4M gordonae strainsand 1 M kansasii strain (see Table 3) No discrepancies werefound in the species submitted asM intracellulareM aviumM abscessus andM tuberculosis complex
The sequence divergence among MTC members was04 The intraspecies divergence among the 188 clinicalstrains ranged from 06 to 53 (Table 3) The sequencediversity among theM abscessuswas 06 while that amongthe M gordonae clinical isolates was 53 No M gordonaeclinical strain had an identical sequence with that of theM gordonae reference strain (ATCC14470) Interestingly therpsA sequence of one strain among the 23M kansasii isolatesexhibited a relatively low level of sequence similarity (956)to that of the reference strain (ATCC 12478) while those ofall the other M kansasii isolates were identical Since thestrain was confirmed as M kansasii by rpoB and hsp65 genealignment itmight suggest a distant variant or a new subtype
4 Discussion
16S rDNA gene sequence alignment has been used as thereference method for mycobacterial species identificationHowever it has been reported that by using the 16S rDNAgene alone for species identification of clinical NTM 37of such isolates remained unclassified which illustrates theneed for additional molecular tools for proper phylogeneticassignment and accurate NTM identification [11] The com-mon assumption that bacterial isolates belong to the samespecies if they have fewer than 5ndash15 bp differences within
the 16S rDNA gene sequence [12] or if they have more than97 16S rDNA gene sequence identities [13] may not beapplicable to genus of Mycobacterium whose members aremuch more closely related to each other Furthermore somemycobacterial strains shared uniform sequence of 16S rDNAgene such asM intracellulare andM aviumM kansasii andM gastri M abscessus and M chelonae and M szulgai andM malmoense
In this study we found that the rpsA gene is useful as acomplementary method to the 16S rDNA for mycobacterialspecies identification Among the 47 reference strains andtotally 1081 paired comparisons 1081 = [119899119886
1+ 119899(119899 minus 1)1198892]
1198861= 1 119899 = 46 119889 = 1 the rpsA gene alone can
differentiate 972 (1051 out of 1081) of them if using 97sequence homology as cutoff value When referring to thephylogenetic tree the rpsA resolution might increase Forthe most commonly seen pathogenic NTM species suchas M intracellulare M avium M abscessus M chelonaeand M xenopi rpsA gene sequence could differentiate thesespecies easily Furthermore 183 of the 188 (973) clinicalisolates representing 8mycobacterial species were identifiedcorrectly by rpsA gene blast Discrepancies of identificationwere only encountered with 4 M gordonae strains and 1M kansasii strain Both these species have reportedly moreintraspecies sequence divergence [9 14ndash16] Three out of the4 M gordonae strains with discrepant identification still hadthe first distinction asM gordonae by rpsA but the sequenceidentity was lower than 97 which means the identificationmight be correct when a more complete in-house database isbeing developed
rpsA alone cannot differentiate the following betweenmembers of the M tuberculosis complex between M sene-galense and M thermoresistibile between M parafortuitumand M trivial between M diernhoferi and M duvalii andbetween M austroafricanum and M terrae however thesespecies are also difficult to be separated by other markersalone such as 16s rDNA ITS rpoB and hsp65 Several
BioMed Research International 5Ta
ble2Sequ
ence
pairdistanceso
f47type
strainso
fmycob
acteria
and1o
ut-group
strain
determ
ined
byusingthemultip
lealignm
enta
lgorith
min
theMegAlignpackage(versio
n710
DNAST
AR
Madiso
nWI)
Percentidentity
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
1mdash
1000
987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
1Mtherm
oresistibile
200
mdash987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
2Msenegalense
313
13mdash
983
966
958
958
968
968
947
947
956
954
954
920
941
954
949
951
951
930
962
947
956
945
930
894
898
911
911
911
911
911
913
920
945
947
920
934
935
932
932
928
937
937
930
928
894
3Mp
orcin
um
419
1917
mdash962
958
958
962
962
941
941
954
945
945
920
943
945
943
945
945
939
962
949
949
937
937
901
905
909
909
909
909
909
911
920
947
949
913
932
932
930
934
928
935
937
928
930
892
4Mfortuitu
m
541
41
35
39
mdash943
954
964
960
939
939
934
943
943
915
943
943
945
947
947
915
943
941
949
928
911
898
903
901
901
901
901
901
903
911
935
937
913
934
930
935
935
930
937
934
930
920
896
5Mp
hlei
649
49
43
43
59
mdash951
953
941
937
937
947
941
941
913
937
935
934
935
935
932
937
937
937
930
924
886
890
907
907
907
907
907
909
915
943
941
907
932
920
918
922
915
930
932
918
915
880
6Mg
adium
747
47
43
43
47
51
mdash966
960
954
954
947
947
947
930
951
953
956
958
958
932
941
949
947
924
920
901
905
924
924
924
924
924
926
928
949
947
930
941
949
937
937
935
939
943
926
934
903
7Mfl
avescens
837
37
33
39
37
49
35
mdash966
966
966
953
962
962
920
962
962
964
962
962
935
947
945
945
939
915
905
909
911
911
911
911
911
913
922
937
935
920
943
935
932
932
930
934
935
924
922
909
8Msmegmatis
939
39
33
39
41
61
41
35
mdash966
966
951
968
968
924
958
956
962
964
964
930
956
949
947
924
920
915
918
909
909
909
909
909
911
922
943
945
924
939
928
934
934
932
939
935
928
928
892
9Mv
accae
1057
57
55
61
63
65
47
35
35
mdash1000
949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
10Mp
arafortuitu
m
1157
57
55
61
63
65
47
35
35
00
mdash949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
11Mtriv
iale
1251
51
45
47
7055
55
49
51
53
53
mdash964
964
918
945
953
951
953
953
941
951
928
922
926
930
903
907
899
899
899
899
899
901
913
932
928
899
920
920
917
917
911
918
924
909
899
882
12Mn
eoaurum
1357
57
47
57
59
61
55
39
33
39
39
37
mdash1000
926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
13Md
iernh
oferi
1457
57
47
57
59
61
55
39
33
39
39
37
00
mdash926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
14Md
uvalii
1584
84
84
84
9193
7485
80
7474
86
7878
mdash930
939
930
932
932
917
918
911
911
894
911
898
901
892
892
892
892
892
894
888
901
905
915
905
905
896
898
898
892
901
896
890
877
15Ma
urum
1663
63
61
59
59
65
51
39
43
53
53
57
49
49
74mdash
966
960
958
958
939
947
947
935
928
924
918
924
905
905
905
905
905
907
917
932
930
911
926
924
926
930
913
924
935
915
909
884
16Mc
hubu
ense
1761
61
47
57
59
68
49
39
45
43
43
49
43
43
63
35
mdash972
970
970
941
956
945
941
935
934
913
917
903
903
903
903
903
905
913
930
932
920
928
932
926
930
917
928
928
915
911
901
17Mg
ilvum
1859
59
53
59
57
7045
37
39
37
37
51
45
45
7441
29
mdash998
998
941
949
928
934
926
930
907
911
901
901
901
901
901
903
913
928
926
913
924
928
920
920
913
920
924
913
920
892
18Mv
anbaalenii
1957
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
mdash1000
939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
19Mterrae
2057
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
00
mdash939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
20Ma
ustro
afric
anum
2176
7674
63
9172
7267
7474
7461
61
61
89
63
61
61
63
63
mdash937
918
917
911
926
888
892
894
894
894
894
894
896
903
915
917
901
909
909
899
901
896
907
913
909
899
896
21Mrhodesia
e
2245
45
39
39
59
66
61
55
45
59
59
51
53
53
87
55
45
53
51
51
65
mdash937
945
934
941
903
907
903
903
903
903
903
905
905
937
939
903
926
928
922
922
918
926
930
917
909
892
22Mo
buense
2365
65
55
53
61
65
53
57
53
7676
7661
61
9555
57
7674
7486
65
mdash958
939
930
917
920
909
909
909
909
909
911
922
937
939
917
932
926
934
937
922
937
939
924
917
890
23Ma
ichien
se
2455
55
45
53
53
65
55
57
55
7474
82
65
65
9567
61
7067
67
89
57
43
mdash937
934
913
918
915
915
915
915
915
917
915
937
941
922
930
935
928
930
930
934
941
932
924
892
24Msphagni
2572
7257
65
7674
80
63
80
80
80
7876
76115
7667
7876
7695
7063
65
mdash920
888
894
896
896
896
896
896
898
905
945
945
907
930
928
918
922
920
924
934
922
915
882
25Mtokaiense
2680
80
7465
9580
84
9184
89
89
7482
82
9580
7074
7272
7861
7470
84
mdash915
918
896
896
896
896
896
898
898
922
926
894
905
911
907
911
898
920
917
907
903
871
26Mc
helona
e
27121
121
115
106
110
124
106
101
91
110
110
104
9393
110
86
9399
9797
121
104
89
93121
91mdash
987
867
867
867
867
867
869
884
884
888
884
886
894
896
896
884
901
898
882
871
871
27Ma
bscessu
s
28117
117
110
101
104
119
101
9786
106
106
9989
89
106
80
89
9593
93117
9984
86
115
87
13mdash
875
875
875
875
875
877
888
888
892
888
890
898
899
899
890
905
901
886
875
877
28Ma
bscessu
s
2995
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
mdash1000
1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
29Mtub
erculosis
3095
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
mdash1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
30Mtub
erculosis
3195
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
mdash1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
31Mb
ovis
3295
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
mdash1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
32Ma
fricanu
m
3395
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
00
mdash998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
33Mb
ovis
3493
9393
95104
9778
9395
106
106
106
112
112
115
99102
104
102
102
112
102
9589
111
110
144
135
02
02
02
02
02
mdash911
918
922
907
918
924
918
917
918
926
932
920
917
880
34Mc
anettii
3591
9184
84
9591
7682
82
9191
9399
99122
89
9393
9191
104
102
82
91102
110
126
121
9898
9898
9895
mdash922
924
907
917
920
911
911
905
918
930
915
909
877
35Mx
enopi
3663
63
57
55
67
59
53
65
59
7878
7284
84
106
7274
7674
7491
65
65
65
57
82
126
121
89
89
89
89
89
87
82
mdash994
930
956
947
939
943
941
951
958
947
937
886
36Ma
vium
3761
61
55
53
65
61
55
67
57
80
80
7686
86
101
7472
7876
7689
63
63
61
57
78121
117
84
84
84
84
84
82
80
06
mdash935
954
951
945
949
945
954
960
953
939
888
37Ma
vium
3893
9384
9393
9974
84
80
9393
108
9595
9195
84
9391
91106
104
89
82
99115
126
121
9797
9797
97100
9974
67
mdash943
937
935
934
924
939
930
937
930
890
38Mm
almoense
3974
7470
7270
7261
59
63
7272
84
7474
102
7876
80
7878
9778
7274
74102
123
119
89
89
89
89
89
87
89
45
47
59
mdash945
941
941
934
951
947
943
943
907
39Mintracellu
lare
4076
7667
7274
84
53
67
7684
84
84
86
86
101
80
7276
7474
9776
7867
7695
115
110
82
82
82
82
82
80
84
55
51
65
57
mdash943
943
949
951
954
939
934
896
40Mscrofulaceum
4178
7872
7468
86
65
7270
9595
89
9191
112
7878
84
82
82
108
82
7076
87
99112
108
84
84
84
84
84
87
9563
57
68
61
59
mdash996
945
960
956
956
937
896
41Mm
arinum
4278
7872
7068
82
65
7270
9595
89
9191
110
7474
84
82
82
106
82
65
7482
95112
108
87
87
87
87
87
89
9559
53
7061
59
04
mdash943
960
956
954
939
894
42Mu
lcerans
4380
80
7676
7491
67
7472
9393
9593
93110
9389
9391
91112
86
82
7484
110
126
119
84
84
84
84
84
87
101
61
57
80
7053
57
59
mdash943
953
951
934
890
43Mg
ordona
e
4470
7065
67
65
7463
7063
86
86
86
89
89
117
80
7684
82
82
9978
65
7080
84
106
101
7676
7676
7678
86
51
47
63
51
51
41
41
59
mdash956
954
945
901
44Mszulga
i
4570
7065
65
7072
59
67
67
9191
80
86
86
106
67
7680
7878
9374
63
61
7089
110
106
7070
7070
7072
7443
41
7455
47
45
45
49
45
mdash964
947
888
45Ma
siaticum
4680
80
7476
7486
7880
76104
104
9793
93112
9191
9391
9197
89
80
7282
99128
123
82
82
82
82
82
85
9155
49
65
59
63
45
47
51
47
37
mdash958
888
46Mk
ansasii
4776
7676
7484
9170
82
7689
89
108
101
101
119
9795
84
82
82
108
9789
80
91104
142
137
87
87
87
87
87
89
9765
63
7459
7066
63
7057
55
43
mdash879
47Mg
astri
48117
117
115
117
112
130
104
97117
119
119
128
110
110
135
126
106
117
119
119
112
117
119
117
128
142
142
135
130
130
130
130
130
130
135
124
121
119
99112
112
115
119
106
121
121
132
mdash48
Rhodococcusequi
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
6 BioMed Research International
001
99
78
93
37
11
2
10
0
1
1
1
19
99
9 99
99
99
99
99
76
52
5
17
35
67
23
23
60
57
10
32
38
32
23
99
9
66
13
16
13
24
55
M thermoresistibileM senegalense
M porcinumM fortuitum
M phleiM gadium
M flavescensM smegmatisM vaccae
M parafortuitumM triviale
M neoaurumM diernhoferiM duvalii
M aurumM chubuense
M gilvumM vanbaalenii
M terraeM austroafricanum
M rhodesiaeM obuenseM aichienseM sphagni
M tokaienseM chelonae
M abscessusM abscessus subspecies massiliense
M tuberculosis H37RvM tuberculosis H37Ra
M bovisM bovis BCG
M canettiiM xenopi
M aviumM avium subspecies paratuberculosis
M malmoenseM intracellulareM scrofulaceum
M marinumM ulcerans
M szulgaiM gordonae
M asiaticumM kansassii
M gastri
M africanum
Rhodococcus equi
Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them
BioMed Research International 7
Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa
Primary identificationb (119899)Primary identificationb (119899)
First choice Second choiceIdentification (119899) match Identification (119899) match
M intracellulare (75)
M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961
M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991
M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960
M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970
M fortuitum complex (17)
M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981
M gordonae (7)
M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947
M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994
M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes
reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]
In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for
Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65
Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has
8 BioMed Research International
the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]
Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
HongfeiDuan andGuanLiu contributed equally to this study
Acknowledgments
The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital
References
[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006
[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996
[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998
[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007
[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011
[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998
[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998
[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999
[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999
[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002
[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003
[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992
[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004
[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992
[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003
[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009
[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013
[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 5Ta
ble2Sequ
ence
pairdistanceso
f47type
strainso
fmycob
acteria
and1o
ut-group
strain
determ
ined
byusingthemultip
lealignm
enta
lgorith
min
theMegAlignpackage(versio
n710
DNAST
AR
Madiso
nWI)
Percentidentity
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
1mdash
1000
987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
1Mtherm
oresistibile
200
mdash987
981
960
953
954
964
962
945
945
951
945
945
920
939
941
943
945
945
928
956
937
947
932
924
888
892
911
911
911
911
911
913
915
939
941
913
930
928
926
926
924
934
934
924
928
892
2Msenegalense
313
13mdash
983
966
958
958
968
968
947
947
956
954
954
920
941
954
949
951
951
930
962
947
956
945
930
894
898
911
911
911
911
911
913
920
945
947
920
934
935
932
932
928
937
937
930
928
894
3Mp
orcin
um
419
1917
mdash962
958
958
962
962
941
941
954
945
945
920
943
945
943
945
945
939
962
949
949
937
937
901
905
909
909
909
909
909
911
920
947
949
913
932
932
930
934
928
935
937
928
930
892
4Mfortuitu
m
541
41
35
39
mdash943
954
964
960
939
939
934
943
943
915
943
943
945
947
947
915
943
941
949
928
911
898
903
901
901
901
901
901
903
911
935
937
913
934
930
935
935
930
937
934
930
920
896
5Mp
hlei
649
49
43
43
59
mdash951
953
941
937
937
947
941
941
913
937
935
934
935
935
932
937
937
937
930
924
886
890
907
907
907
907
907
909
915
943
941
907
932
920
918
922
915
930
932
918
915
880
6Mg
adium
747
47
43
43
47
51
mdash966
960
954
954
947
947
947
930
951
953
956
958
958
932
941
949
947
924
920
901
905
924
924
924
924
924
926
928
949
947
930
941
949
937
937
935
939
943
926
934
903
7Mfl
avescens
837
37
33
39
37
49
35
mdash966
966
966
953
962
962
920
962
962
964
962
962
935
947
945
945
939
915
905
909
911
911
911
911
911
913
922
937
935
920
943
935
932
932
930
934
935
924
922
909
8Msmegmatis
939
39
33
39
41
61
41
35
mdash966
966
951
968
968
924
958
956
962
964
964
930
956
949
947
924
920
915
918
909
909
909
909
909
911
922
943
945
924
939
928
934
934
932
939
935
928
928
892
9Mv
accae
1057
57
55
61
63
65
47
35
35
mdash1000
949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
10Mp
arafortuitu
m
1157
57
55
61
63
65
47
35
35
00
mdash949
962
962
930
949
958
964
966
966
930
943
928
930
924
917
898
901
899
899
899
899
899
901
915
926
924
913
932
920
911
911
913
918
915
903
917
890
11Mtriv
iale
1251
51
45
47
7055
55
49
51
53
53
mdash964
964
918
945
953
951
953
953
941
951
928
922
926
930
903
907
899
899
899
899
899
901
913
932
928
899
920
920
917
917
911
918
924
909
899
882
12Mn
eoaurum
1357
57
47
57
59
61
55
39
33
39
39
37
mdash1000
926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
13Md
iernh
oferi
1457
57
47
57
59
61
55
39
33
39
39
37
00
mdash926
953
958
956
958
958
941
949
941
937
928
922
913
917
894
894
894
894
894
896
907
920
918
911
930
918
915
915
913
917
918
913
905
898
14Md
uvalii
1584
84
84
84
9193
7485
80
7474
86
7878
mdash930
939
930
932
932
917
918
911
911
894
911
898
901
892
892
892
892
892
894
888
901
905
915
905
905
896
898
898
892
901
896
890
877
15Ma
urum
1663
63
61
59
59
65
51
39
43
53
53
57
49
49
74mdash
966
960
958
958
939
947
947
935
928
924
918
924
905
905
905
905
905
907
917
932
930
911
926
924
926
930
913
924
935
915
909
884
16Mc
hubu
ense
1761
61
47
57
59
68
49
39
45
43
43
49
43
43
63
35
mdash972
970
970
941
956
945
941
935
934
913
917
903
903
903
903
903
905
913
930
932
920
928
932
926
930
917
928
928
915
911
901
17Mg
ilvum
1859
59
53
59
57
7045
37
39
37
37
51
45
45
7441
29
mdash998
998
941
949
928
934
926
930
907
911
901
901
901
901
901
903
913
928
926
913
924
928
920
920
913
920
924
913
920
892
18Mv
anbaalenii
1957
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
mdash1000
939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
19Mterrae
2057
57
51
57
55
68
43
39
37
35
35
49
43
43
7243
31
02
00
mdash939
951
930
935
928
932
909
913
903
903
903
903
903
905
915
930
928
915
926
930
922
922
915
922
926
915
922
890
20Ma
ustro
afric
anum
2176
7674
63
9172
7267
7474
7461
61
61
89
63
61
61
63
63
mdash937
918
917
911
926
888
892
894
894
894
894
894
896
903
915
917
901
909
909
899
901
896
907
913
909
899
896
21Mrhodesia
e
2245
45
39
39
59
66
61
55
45
59
59
51
53
53
87
55
45
53
51
51
65
mdash937
945
934
941
903
907
903
903
903
903
903
905
905
937
939
903
926
928
922
922
918
926
930
917
909
892
22Mo
buense
2365
65
55
53
61
65
53
57
53
7676
7661
61
9555
57
7674
7486
65
mdash958
939
930
917
920
909
909
909
909
909
911
922
937
939
917
932
926
934
937
922
937
939
924
917
890
23Ma
ichien
se
2455
55
45
53
53
65
55
57
55
7474
82
65
65
9567
61
7067
67
89
57
43
mdash937
934
913
918
915
915
915
915
915
917
915
937
941
922
930
935
928
930
930
934
941
932
924
892
24Msphagni
2572
7257
65
7674
80
63
80
80
80
7876
76115
7667
7876
7695
7063
65
mdash920
888
894
896
896
896
896
896
898
905
945
945
907
930
928
918
922
920
924
934
922
915
882
25Mtokaiense
2680
80
7465
9580
84
9184
89
89
7482
82
9580
7074
7272
7861
7470
84
mdash915
918
896
896
896
896
896
898
898
922
926
894
905
911
907
911
898
920
917
907
903
871
26Mc
helona
e
27121
121
115
106
110
124
106
101
91
110
110
104
9393
110
86
9399
9797
121
104
89
93121
91mdash
987
867
867
867
867
867
869
884
884
888
884
886
894
896
896
884
901
898
882
871
871
27Ma
bscessu
s
28117
117
110
101
104
119
101
9786
106
106
9989
89
106
80
89
9593
93117
9984
86
115
87
13mdash
875
875
875
875
875
877
888
888
892
888
890
898
899
899
890
905
901
886
875
877
28Ma
bscessu
s
2995
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
mdash1000
1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
29Mtub
erculosis
3095
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
mdash1000
1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
30Mtub
erculosis
3195
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
mdash1000
1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
31Mb
ovis
3295
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
mdash1000
998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
32Ma
fricanu
m
3395
9595
97106
100
80
9597
108
108
108
115
115
117
101
104
106
104
104
115
104
9791
113
113
146
137
00
00
00
00
mdash998
909
917
920
909
917
922
920
918
920
928
934
922
918
880
33Mb
ovis
3493
9393
95104
9778
9395
106
106
106
112
112
115
99102
104
102
102
112
102
9589
111
110
144
135
02
02
02
02
02
mdash911
918
922
907
918
924
918
917
918
926
932
920
917
880
34Mc
anettii
3591
9184
84
9591
7682
82
9191
9399
99122
89
9393
9191
104
102
82
91102
110
126
121
9898
9898
9895
mdash922
924
907
917
920
911
911
905
918
930
915
909
877
35Mx
enopi
3663
63
57
55
67
59
53
65
59
7878
7284
84
106
7274
7674
7491
65
65
65
57
82
126
121
89
89
89
89
89
87
82
mdash994
930
956
947
939
943
941
951
958
947
937
886
36Ma
vium
3761
61
55
53
65
61
55
67
57
80
80
7686
86
101
7472
7876
7689
63
63
61
57
78121
117
84
84
84
84
84
82
80
06
mdash935
954
951
945
949
945
954
960
953
939
888
37Ma
vium
3893
9384
9393
9974
84
80
9393
108
9595
9195
84
9391
91106
104
89
82
99115
126
121
9797
9797
97100
9974
67
mdash943
937
935
934
924
939
930
937
930
890
38Mm
almoense
3974
7470
7270
7261
59
63
7272
84
7474
102
7876
80
7878
9778
7274
74102
123
119
89
89
89
89
89
87
89
45
47
59
mdash945
941
941
934
951
947
943
943
907
39Mintracellu
lare
4076
7667
7274
84
53
67
7684
84
84
86
86
101
80
7276
7474
9776
7867
7695
115
110
82
82
82
82
82
80
84
55
51
65
57
mdash943
943
949
951
954
939
934
896
40Mscrofulaceum
4178
7872
7468
86
65
7270
9595
89
9191
112
7878
84
82
82
108
82
7076
87
99112
108
84
84
84
84
84
87
9563
57
68
61
59
mdash996
945
960
956
956
937
896
41Mm
arinum
4278
7872
7068
82
65
7270
9595
89
9191
110
7474
84
82
82
106
82
65
7482
95112
108
87
87
87
87
87
89
9559
53
7061
59
04
mdash943
960
956
954
939
894
42Mu
lcerans
4380
80
7676
7491
67
7472
9393
9593
93110
9389
9391
91112
86
82
7484
110
126
119
84
84
84
84
84
87
101
61
57
80
7053
57
59
mdash943
953
951
934
890
43Mg
ordona
e
4470
7065
67
65
7463
7063
86
86
86
89
89
117
80
7684
82
82
9978
65
7080
84
106
101
7676
7676
7678
86
51
47
63
51
51
41
41
59
mdash956
954
945
901
44Mszulga
i
4570
7065
65
7072
59
67
67
9191
80
86
86
106
67
7680
7878
9374
63
61
7089
110
106
7070
7070
7072
7443
41
7455
47
45
45
49
45
mdash964
947
888
45Ma
siaticum
4680
80
7476
7486
7880
76104
104
9793
93112
9191
9391
9197
89
80
7282
99128
123
82
82
82
82
82
85
9155
49
65
59
63
45
47
51
47
37
mdash958
888
46Mk
ansasii
4776
7676
7484
9170
82
7689
89
108
101
101
119
9795
84
82
82
108
9789
80
91104
142
137
87
87
87
87
87
89
9765
63
7459
7066
63
7057
55
43
mdash879
47Mg
astri
48117
117
115
117
112
130
104
97117
119
119
128
110
110
135
126
106
117
119
119
112
117
119
117
128
142
142
135
130
130
130
130
130
130
135
124
121
119
99112
112
115
119
106
121
121
132
mdash48
Rhodococcusequi
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
2930
3132
3334
3536
3738
3940
4142
4344
4546
4748
6 BioMed Research International
001
99
78
93
37
11
2
10
0
1
1
1
19
99
9 99
99
99
99
99
76
52
5
17
35
67
23
23
60
57
10
32
38
32
23
99
9
66
13
16
13
24
55
M thermoresistibileM senegalense
M porcinumM fortuitum
M phleiM gadium
M flavescensM smegmatisM vaccae
M parafortuitumM triviale
M neoaurumM diernhoferiM duvalii
M aurumM chubuense
M gilvumM vanbaalenii
M terraeM austroafricanum
M rhodesiaeM obuenseM aichienseM sphagni
M tokaienseM chelonae
M abscessusM abscessus subspecies massiliense
M tuberculosis H37RvM tuberculosis H37Ra
M bovisM bovis BCG
M canettiiM xenopi
M aviumM avium subspecies paratuberculosis
M malmoenseM intracellulareM scrofulaceum
M marinumM ulcerans
M szulgaiM gordonae
M asiaticumM kansassii
M gastri
M africanum
Rhodococcus equi
Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them
BioMed Research International 7
Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa
Primary identificationb (119899)Primary identificationb (119899)
First choice Second choiceIdentification (119899) match Identification (119899) match
M intracellulare (75)
M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961
M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991
M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960
M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970
M fortuitum complex (17)
M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981
M gordonae (7)
M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947
M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994
M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes
reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]
In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for
Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65
Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has
8 BioMed Research International
the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]
Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
HongfeiDuan andGuanLiu contributed equally to this study
Acknowledgments
The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital
References
[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006
[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996
[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998
[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007
[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011
[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998
[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998
[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999
[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999
[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002
[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003
[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992
[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004
[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992
[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003
[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009
[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013
[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 BioMed Research International
001
99
78
93
37
11
2
10
0
1
1
1
19
99
9 99
99
99
99
99
76
52
5
17
35
67
23
23
60
57
10
32
38
32
23
99
9
66
13
16
13
24
55
M thermoresistibileM senegalense
M porcinumM fortuitum
M phleiM gadium
M flavescensM smegmatisM vaccae
M parafortuitumM triviale
M neoaurumM diernhoferiM duvalii
M aurumM chubuense
M gilvumM vanbaalenii
M terraeM austroafricanum
M rhodesiaeM obuenseM aichienseM sphagni
M tokaienseM chelonae
M abscessusM abscessus subspecies massiliense
M tuberculosis H37RvM tuberculosis H37Ra
M bovisM bovis BCG
M canettiiM xenopi
M aviumM avium subspecies paratuberculosis
M malmoenseM intracellulareM scrofulaceum
M marinumM ulcerans
M szulgaiM gordonae
M asiaticumM kansassii
M gastri
M africanum
Rhodococcus equi
Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them
BioMed Research International 7
Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa
Primary identificationb (119899)Primary identificationb (119899)
First choice Second choiceIdentification (119899) match Identification (119899) match
M intracellulare (75)
M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961
M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991
M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960
M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970
M fortuitum complex (17)
M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981
M gordonae (7)
M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947
M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994
M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes
reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]
In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for
Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65
Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has
8 BioMed Research International
the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]
Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
HongfeiDuan andGuanLiu contributed equally to this study
Acknowledgments
The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital
References
[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006
[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996
[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998
[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007
[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011
[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998
[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998
[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999
[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999
[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002
[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003
[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992
[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004
[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992
[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003
[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009
[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013
[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 7
Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa
Primary identificationb (119899)Primary identificationb (119899)
First choice Second choiceIdentification (119899) match Identification (119899) match
M intracellulare (75)
M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961
M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991
M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960
M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970
M fortuitum complex (17)
M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981
M gordonae (7)
M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947
M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994
M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes
reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]
In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for
Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65
Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has
8 BioMed Research International
the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]
Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
HongfeiDuan andGuanLiu contributed equally to this study
Acknowledgments
The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital
References
[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006
[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996
[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998
[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007
[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011
[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998
[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998
[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999
[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999
[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002
[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003
[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992
[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004
[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992
[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003
[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009
[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013
[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 BioMed Research International
the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]
Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
HongfeiDuan andGuanLiu contributed equally to this study
Acknowledgments
The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital
References
[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006
[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996
[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998
[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007
[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011
[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998
[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998
[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999
[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999
[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002
[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003
[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992
[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004
[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992
[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003
[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009
[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013
[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology