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Guidelines for interpretation of 16S rRNA gene

Guidelines for interpretation of16S rRNA gene sequence-based results for identification of medically important aerobic Gram-positive bacteria Patrick C.Y.Woo,1,2,33Jade L.L.Teng,33Jeff K.L.Wu,3

Fion P.S.Leung,3Herman Tse,1,2,3Ami M.Y.Fung,3

Susanna http://www.wendangku.net/doc/341eb6fb941ea76e58fa04ef.htmlu1,2,3and Kwok-yung Yuen1,2,3

Correspondence Kwok-yung Yuen hkumicro@hkucc.hku.hk 1State Key Laboratory of Emerging Infectious Diseases,University of Hong Kong,Hong Kong SAR 2Research Centre of Infection and Immunology,University of Hong Kong,Hong Kong SAR

3Department of Microbiology,University of Hong Kong,Hong Kong SAR

Received10December2008 Accepted3May2009This study is believed to be the first to provide guidelines for facilitating interpretation of results based on full and527bp16S rRNA gene sequencing and MicroSeq databases used for identifying medically important aerobic Gram-positive bacteria.Overall,full and527bp16S rRNA gene sequencing can identify24and40%of medically important Gram-positive cocci(GPC), and21and34%of medically important Gram-positive rods(GPR)confidently to the species level,whereas the full-MicroSeq and500-MicroSeq databases can identify15and34%of medically important GPC and14and25%of medically important GPR confidently to the species level.Among staphylococci,streptococci,enterococci,mycobacteria,corynebacteria,nocardia and members of Bacillus and related taxa(Paenibacillus,Brevibacillus,Geobacillus and Virgibacillus),the methods and databases are least useful for identification of staphylococci and nocardia.Only0–2and2–13%of staphylococci,and0and0–10%of nocardia,can be confidently and doubtfully identified,respectively.However,these methods and databases are most useful for identification of Bacillus and related taxa,with36–56and11–14%of Bacillus and related taxa confidently and doubtfully identified,respectively.A total of15medically important GPC and18medically important GPR that should be confidently identified by full16S rRNA gene sequencing are not included in the full-MicroSeq database.A total of9medically important GPC and21medically important GPR that should be confidently identified by527bp 16S rRNA gene sequencing are not included in the500-MicroSeq database.16S rRNA gene sequence results of Gram-positive bacteria should be interpreted with basic phenotypic tests results.Additional biochemical tests or sequencing of additional gene loci are often required for definitive identification.To improve the usefulness of the MicroSeq databases,bacterial species that can be confidently identified by16S rRNA gene sequencing but are not found in the MicroSeq databases should be included.

INTRODUCTION

Since its first use three decades ago,16S rRNA gene sequencing has become one of the indispensable pillars in the polyphasic approach to bacterial classification and novel bacteria discovery(Woo et al.,2008a).In the last decade,the technology has further developed beyond the research realm and matured into a clinical application for bacterial identification.Despite its increasing use in reference laboratories,one of the major limitations to its wider use is the difficulty associated with interpretation of 16S rRNA gene sequence results.The use of16S rRNA gene sequencing for bacterial identification depends on a significant inter-species difference and a small intra-species difference in16S rRNA gene sequences.Therefore,one of the major limitations is that when two different bacterial species share almost the same16S rRNA gene sequence; this technique alone would not be useful for distinguishing them confidently.

Aimed at facilitating16S rRNA gene sequence interpreta-tion,the MicroSeq50016S rRNA gene bacterial iden-tification system(MicroSeq;Perkin-Elmer Applied

Abbreviations:GPC,Gram-positive cocci;GPR,Gram-positive rods.

3These authors contributed equally to this work.

Tables of sequence comparison and identification data are available as

supplementary material with the online version of this paper.

Journal of Medical Microbiology(2009),58,1030–1036DOI10.1099/jmm.0.008615-0 1030008615G2009SGM Printed in Great Britain

Biosystems)has been designed,using a527bp fragment in the59end of the16S rRNA gene(Cloud et al.,2004; Fontana et al.,2005;Lau et al.,2006;Patel et al.,2000;Tang et al.,1998,2000;Woo et al.,2003c).Recently,a full16S rRNA gene sequence(full-MicroSeq)database(version 2.0)has also been generated.However,the MicroSeq databases do not take into account bacterial species sharing similar16S rRNA gene sequences that would not be confidently identified by16S rRNA gene sequence analysis. Furthermore,the accuracy of identification is further limited by the bacterial species included in the database, as bacterial species that are not included in the database would never be determined as the identity of an isolate. In view of these limitations,3years ago we started to systematically evaluate the usefulness of full and527bp 16S rRNA gene sequencing and the MicroSeq databases for identification of various groups of medically important bacteria.Recently,we have published the results for medically important anaerobes(Woo et al.,2007).In this article,we describe the results of an evaluation of the usefulness of full and527bp16S rRNA gene sequencing and the MicroSeq databases for identification of409 medically important aerobic Gram-positive bacteria. Practical points when using such technologies for iden-tification of these bacteria are also discussed. METHODS

16S rRNA gene sequences of medically important aerobic Gram-positive bacteria.The species of medically important aerobic Gram-positive bacteria included in this study include all the409 species of the59genera of medically important aerobic Gram-positive cocci(GPC)and Gram-positive rods(GPR)listed in the most recent edition of the Manual of Clinical Microbiology(Murray et al.,2007). For each bacterial species,a list of the16S rRNA gene sequence was retrieved from the GenBank database.In the list,the most representative16S rRNA gene sequence for each species was chosen for analysis according to the following criteria:(a)strains with good phenotypic characterization,such as the type strains,were preferred to those without good phenotypic characterization;(b)strains isolated from humans were preferred to those isolated from animals or the environment;(c)sequences with fewer undetermined bases were preferred to those with more undetermined bases;and(d) longer sequences,especially those with better coverage of the59end, were preferred to shorter sequences and those with poorer coverage of the59end.

Comparison of full16S rRNA gene sequences of medically important aerobic Gram-positive bacteria.The percentage differences of the16S rRNA gene sequences among the different species in the same group/genus of medically important GPC and GPR were determined by pairwise alignment using CLUSTAL W (Thompson et al.,1994).For sequences with undetermined bases, other16S rRNA gene sequences of the same species were retrieved and the undetermined bases were manually amended.If there was no other16S rRNA gene sequence for the same species,the positions of the undetermined bases were deleted in the analysis.

Comparison of527bp16S rRNA gene sequences of medically important aerobic Gram-positive bacteria.The527bp sequences that should be amplified by the primers of MicroSeq were extracted from the full16S rRNA gene sequences.The percentage differences of the resultant partial16S rRNA gene sequences among the different species in the same group/genus of medically important GPC and GPR were determined by pairwise alignment using CLUSTAL W (Thompson et al.,1994).

Definitions.A bacterium species is defined as‘confidently identified by16S rRNA gene sequencing’if there is.3%difference between the 16S rRNA gene sequence of the species and those of other medically important bacteria species.A bacterium species is defined as‘not confidently identified by16S rRNA gene sequencing’if there is,2% difference between the16S rRNA gene sequence of the species and that of one or more medically important aerobic Gram-positive bacterium species.A bacterium species is defined as‘only doubtfully identified by16S rRNA gene sequencing’if there is2–3%difference between the16S rRNA gene sequence of the species and that of one or more medically important aerobic Gram-positive bacterium species. RESULTS

The percentage differences of the full and527bp16S rRNA gene sequences among the different groups of medically important aerobic Gram-positive bacteria are shown in Supplementary Table S1(a–k)(available with the online journal).

Full16S rRNA gene sequencing for identification of medically important aerobic Gram-positive bacteria

None and1(2%)of46staphylococci,8(20%)and9 (22%)of41streptococci,none and5(17%)of30 enterococci,32(65%)and7(14%)of49GPC other than staphylococci,streptococci and enterococci,2(3%)and7 (9%)of74mycobacteria,9(24%)and12(32%)of38 corynebacteria,none and1(3%)of31nocardia,16(44%) and4(11%)of36members of Bacillus and related taxa (Paenibacillus,Brevibacillus,Geobacillus and Virgibacillus), and24(38%)and11(17%)of64GPR other than mycobacteria,corynebacteria,nocardia and members of Bacillus and related taxa can be confidently and doubtfully identified by full16S rRNA gene sequencing,respectively (Table1,Supplementary Table S2a–k,available with the online journal).

Full-MicroSeq database for identification of medically important aerobic Gram-positive bacteria

None and1(2%)of46staphylococci,7(17%)and8 (20%)of41streptococci,none and4(13%)of30 enterococci,18(37%)and4(8%)of49GPC other than staphylococci,streptococci and enterococci,2(3%)and5 (7%)of74mycobacteria,3(8%)and8(21%)of38 corynebacteria,none of31nocardia,13(36%)and4 (11%)of36members of Bacillus and related taxa (Paenibacillus,Brevibacillus,Geobacillus and Virgibacillus), and15(23%)and5(8%)of64GPR other than mycobacteria,corynebacteria,nocardia and members of Bacillus and related taxa can be confidently and doubtfully 16S rRNA gene sequencing for Gram-positive bacteria

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identified by the full-MicroSeq database,respectively (Table1,Supplementary Table S2a–k,available with the online journal).

527bp16S rRNA gene sequencing for identification of medically important aerobic Gram-positive bacteria

One(2%)and6(13%)of46staphylococci,19(46%)and 5(12%)of41streptococci,8(27%)and1(3%)of30 enterococci,38(78%)and4(8%)of49GPC other than staphylococci,streptococci and enterococci,9(12%)and 15(20%)of74mycobacteria,15(39%)and5(13%)of38 corynebacteria,none and3(10%)of31nocardia,20 (56%)and5(14%)of36members of Bacillus and related taxa(Paenibacillus,Brevibacillus,Geobacillus and Virgibacillus),and38(59%)and6(9%)of64GPR other than mycobacteria,corynebacteria,nocardia and members of Bacillus and related taxa can be confidently and doubtfully identified by527bp16S rRNA gene sequencing, respectively(Table1,Supplementary Table S2a–k, available with the online journal).

500-MicroSeq database for identification of medically important aerobic Gram-positive bacteria

One(2%)and5(11%)of46staphylococci,18(44%)and 3(7%)of41streptococci,7(23%)and none of30 enterococci,31(63%)and3(6%)of49GPC other than staphylococci,streptococci and enterococci,8(11%)and 11(15%)of74mycobacteria,11(29%)and3(8%)of38 corynebacteria,none and2(6%)of31nocardia,17(47%) and5(14%)of36members of Bacillus and related taxa (Paenibacillus,Brevibacillus,Geobacillus and Virgibacillus), and25(39%)and4(6%)of64GPR other than mycobacteria,corynebacteria,nocardia and members of Bacillus and related taxa can be confidently and doubtfully identified by the500-MicroSeq database,respectively (Table1,Supplementary Table S2a–k available with the online journal).

DISCUSSION

The major difficulties and controversies in interpreting16S rRNA gene sequence data relate to the assignment of bacterial species based on similarity search results,as no threshold values are available as for DNA–DNA hybrid-ization(Petti,2007).As different bacterial species are likely to evolve at different rates,there has been no agreement on a universal cut-off for bacterial species delineation.In1994, Stackebrandt and Goebel proposed a97%similarity level for bacterial speciation using16S rRNA gene sequence (Stackebrandt&Goebel,1994).Although in the study by Drancourt et al.(2000)a.99and.97%sequence similarity was used as the cut-off for species and genus identification,respectively,the authors indicated that these sharp values were mainly made for practical purposes for interpretation of their large sequence dataset.On the other hand,Janda and Abbott suggested that a minimum of .99%,and ideally.99.5%,sequence similarity be used for species identification in their recommended guidelines (Janda&Abbott,2007).Although different studies have identified groups of bacteria for which16S rRNA gene sequences are not discriminative enough,no study has attempted to solve this problem in a systematic way. Moreover,the use of public databases such as GenBank can be problematic in that errors in sequence data are not uncommon.For example,the strain with GenBank accession number Z26896was claimed to be Staphylococcus haemolyticus.However,the sequence has

Table1.Number and percentage of species of major groups of medically important Gram-positive bacteria confidently identified by full16S rRNA gene sequence,527bp16S rRNA gene sequence and the MicroSeq databases

Bacterial genera Total no.of species No.(%)of species confidently identified

Full16S rRNA gene sequencing Full-MicroSeq

database

527bp16S rRNA

gene sequencing

500-MicroSeq

database

Gram-positive cocci

Staphylococcus460(0)0(0)1(2)1(2) Streptococcus418(20)7(17)19(46)18(44) Enterococcus300(0)0(0)8(27)7(23) Abiotrophia and Granulicatella41(25)1(25)2(50)1(25) Gemella41(25)0(0)2(50)2(50) Gram-positive rods

Mycobacterium742(3)2(3)9(12)8(11) Corynebacterium389(24)3(8)15(39)11(29) Nocardia310(0)0(0)0(0)0(0) Bacillus and related taxa*3616(44)13(36)20(56)17(47)

*Paenibacillus,Brevibacillus,Geobacillus and Virgibacillus.

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highest nucleotide identities to sequences of16S rRNA genes of Staphylococcus aureus,rather than other strains of Staphylococcus haemolyticus,and no results of phenotypic characterization were given for this strain.Therefore,the true identity of this strain is difficult to ascertain.In the present study and a previous one(Woo et al.,2007),we used.3,2–3and,2%differences between the most representative16S rRNA gene sequence of species obtained from GenBank and those of other medically important bacteria to indicate the degree of confidence when using 16S rRNA sequence for identification of medically important bacterial species.When there is2–3%difference between the16S rRNA gene sequence of a bacterial species and a closely related one,the degree of16S rRNA gene sequence homogeneity in different strains of that species becomes very important in determining whether16S rRNA gene sequencing is able to identify the bacterium to the species level.More importantly,medically important bacterial species with similar16S rRNA gene sequences were also identified(Supplementary Table S2a–k available with the online journal),so that the users can easily note the species that may be potentially confused based on16S rRNA gene sequence analysis.Further phenotypic tests and/or sequencing of other gene loci can be performed to differentiate these species.

For species identification,16S rRNA gene sequence results of aerobic Gram-positive bacteria should be interpreted cautiously with basic phenotypic tests results,and addi-tional biochemical tests or sequencing of additional gene loci are often required for definitive identification.Overall, using3%base difference as the cut-off,full and527bp16S rRNA gene sequencing can only identify24and40%of medically important GPC and21and34%of medically important GPR confidently to the species level.This is apparently different from the results reported in some other studies,in which up to83and92%of the isolates can be assigned to species level(Mignard&Flandrois, 2006;Bosshard et al.,2006).In some studies,the difference was due to the use of different cut-offs for confident identification;whereas in others,the difference was a result of a larger proportion of bacterial strains,other than Gram-positive bacteria,that can be confidently identified. The higher percentage of bacteria that can be confidently identified by527bp16S rRNA gene sequencing than full 16S rRNA gene sequencing is a result of the non-random distribution of base difference along the16S rRNA gene. For some genera,such as staphylococci,mycobacteria and nocardia,both full and527bp16S rRNA gene sequencing can identify,13%of bacteria confidently.Therefore,in order to prevent misinterpretation of16S rRNA gene sequence results,they should always be interpreted in the context of basic phenotypic test results.Moreover,key phenotypic tests,that can help distinguish between‘the closest match’by16S rRNA gene sequencing and medically important bacterial species with similar16S rRNA gene sequences as listed in Supplementary Table S2(a–k) (available with the online journal),should be performed for differentiation of these possibilities(Woo et al.,2001, 2003d;Lau et al.,2002;Cloud et al.,2005).For example,in our previous study on Tsukamurella conjunctivitis,16S rRNA gene sequencing was able to identify the various isolates as Tsukamurella to the genus level,but all Tsukamurella species possess.99%nucleotide identities with each other.Phenotypic tests have to be performed to distinguish among the various Tsukamurella species(Woo et al.,2003d).Alternatively,sequencing of other gene loci can be performed for identification of species in these genera.In fact,another gene locus,groEL,has been shown to be better than16S rRNA gene for identification of staphylococci and mycobacteria(Kwok et al.,1999;Goh et al.,1996;Ringuet et al.,1999).Other genes,such as tuf, dnaJ and rpoB,and secA1have also been used for identification of staphylococci,mycobacteria and nocardia, respectively(Heikens et al.,2005;Yamada-Noda et al., 2007;Ade′kambi et al.,2003;Conville et al.,2006).This conclusion of the necessity for support of16S rRNA gene sequence results by other identification methodologies, such as phenotypic tests and/or sequencing other gene targets,is in line with the recommendations given by the Clinical and Laboratory Standards Institute,although different cut-offs were used in their recommendations (CLSI,2008).One has to be particularly cautious if16S rRNA gene sequencing is used for culture-negative infections,such as infective endocarditis,as no isolate is available for further phenotypic testing(Gauduchon et al., 2003;Kotilainen et al.,2006).Furthermore,some medically important Gram-positive bacteria,such as Streptococcus sinensis,have been reported as a cause of infective endocarditis in Asia and Europe with the oral cavity documented to be the reservoir,but are still not yet included in the Manual of Clinical Microbiology(Woo et al., 2002,2004b,2008b;Uc?kay et al.,2007;Faibis et al.,2008). One must bear in mind that these bacteria not yet included in the Manual of Clinical Microbiology may also be the identity of an isolate.Particular attention has to be paid if there is discrepancy between16S rRNA gene sequence and phenotypic test results.

Despite its relatively low confidence for species identifica-tion,16S rRNA gene sequencing is very useful for identification of medically important Gram-positive bac-teria to the genus level,especially for some groups of Gram-positive bacteria notoriously difficult to identify by phenotypic methods.Although some genera of Gram-positive bacteria,such as Mycobacterium and Nocardia, possess unique phenotypic characteristics,such as acid-fastness,for their easy identification to the genus level, most streptococcus-like Gram-positive cocci,such as Granulicatella,Abiotrophia,Gemella and Helcococcus,are particularly difficult for genus identification using pheno-typic tests.In our experience,most commercial systems failed to identify most of our clinical isolates of Granulicatella,Abiotrophia,Gemella and Helcococcus to the genus level(Woo et al.,2003a,b,2005).Gram-positive bacterial isolates that possess unusual phenotypic profiles 16S rRNA gene sequencing for Gram-positive bacteria

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also led to the same problem(Woo et al.,2003e,2004a).In these cases,16S rRNA gene sequencing would be extremely useful for genus identification and narrowing down the identification to a few possible species that possess similar 16S rRNA gene sequences.When using the MicroSeq databases for bacterial iden-tification,additional attention has to be paid to possible medically important bacterial species that are not included in the database,and the accuracies reported for MicroSeq for various groups of Gram-positive bacteria might have

Table2.Medically important Gram-positive bacteria that can be confidently identified by full or 527bp16S rRNA gene sequencing but are not included in the MicroSeq databases

Bacterial genera Bacterial species

Not included in full-MicroSeq database

Not included in 500-MicroSeq database

Gram-positive cocci

Aerococcus A.christensenii A.sanguinicola

A.sanguinicola A.urinaehominis

A.urinaehominis

Dolosicoccus D.paucivorans

Dolosigranulum D.pigrum D.pigrum

Enterococcus E.italicus

Facklamia F.hominis

F.ignava

http://www.wendangku.net/doc/341eb6fb941ea76e58fa04ef.htmlnguida

F.sourekii

Gemella G.bergeri

Granulicatella G.elegans

Helcococcus H.kunzii H.sueciensis

H.sueciensis

Ignavigranum I.ruoffiae I.ruoffiae

Rothia R.mucilaginosa

Streptococcus S.hyovaginalis S.hyovaginalis

Tetragenococcus T.solitarius

Gram-positive rods

Actinomadura A.pelletieri http://www.wendangku.net/doc/341eb6fb941ea76e58fa04ef.htmltina

Amycolatopsis A.palatopharyngis A.palatopharyngis

Arthrobacter A.scleromae A.scleromae

Bacillus B.fumarioli B.fumarioli

B.subterraneus B.subterraneus

Brevibacterium B.luteolum B.luteolum

B.sanguinis

Cellulomonas C.denverensis

Corynebacterium C.atypicum C.atypicum

C.durum C.durum

C.glucuronolyticum C.kroppenstedtii

C.imitans C.resistens

C.kroppenstedtii

C.resistens

Erysipelothrix E.inopinata E.inopinata

Janibacter J.sanguinis J.sanguinis

Leifsonia L.aquatica

Microbacterium M.resistens

Mycobacterium M.heckeshornense

Paenibacillus P.hongkongensis P.hongkongensis

Rhodococcus R.gordoniae

Streptomyces S.thermovulgaris

Zimmermannella Z.alba Z.alba

Z.bifida Z.bifida

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been overestimated.The percentage of Gram-positive bacteria estimated to be confidently identified by the MicroSeq databases in this study is low compared to the accuracies reported for selected groups of Gram-positive bacteria in the literature(Patel et al.,2000;Tang et al., 2000;Mellmann et al.,2003).This is mainly because the MicroSeq databases do not take into account bacteria with similar16S rRNA gene sequences,which can confuse the identity of the bacterium.For example,in two studies that assessed the usefulness of500-MicroSeq for identification of mycobacteria and nocardia,clinical isolates of Mycobacterium chelonae,Mycobacterium tuberculosis, Mycobacterium marinum,Nocardia asteroides,Nocardia nova and Nocardia farcinica were considered successfully identified by500-MicroSeq(Patel et al.,2000;Mellmann et al.,2003),but the present study indicated that in fact there are Mycobacterium and Nocardia species with similar 16S rRNA gene sequences.In fact,it is well known that the 16S rRNA gene is probably not the best target for identifying mycobacteria and nocardia,and alternative gene targets,such as dnaJ and secA1,as mentioned above, have been used for their identification(Yamada-Noda et al., 2007;Conville et al.,2006).Therefore,in the present study and our previous one on anaerobes(Woo et al.,2007), supplementary note is given to indicate those bacteria that cannot be confidently identified but are included in the MicroSeq databases,symbolized by?c and?f in Supplementary Table S2(a–k)(available with the online journal).In addition to this problem,the MicroSeq databases are also defective in that they do not contain a significant proportion of medically important species.To make our results more user-friendly,supplementary data is also included to indicate whether the inability of the MicroSeq databases to identify a particular bacterium is due to the species not being included in the MicroSeq databases,symbolized by6a and6d in Supplementary Table S2(a–k)(available with the online journal),or high 16S rRNA gene sequence similarity(.98%nucleotide identity)to a closely related species,symbolized by6b and 6e in Supplementary Table S2(a–k)(available with the online journal).To improve the usefulness of the MicroSeq databases,bacterial species that can be confidently identified by16S rRNA gene sequencing but are not found in the MicroSeq databases should be included(Table2). These include15medically important GPC and18 medically important GPR that should be confidently identified by full16S rRNA gene sequencing but are not included in the full-MicroSeq database,and9medically important GPC and21medically important GPR that should be confidently identified by527bp16S rRNA gene sequencing but are not included in the500-MicroSeq database(Table2).

ACKNOWLEDGEMENTS

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