16S rRNA Gene Sequencing for Bacterial Pathogen Identification in ...

318 Molecular Diagnosis Vol. 6 No. 4 December 2001ported as Mycobacterium szulgai. However, bothisolates had the same 5' 500-bp sequence, and thissequence is unique from any other sequences in Gen-Bank [21]. These two isolates are likely related toeach other at the species-level and probably representa novel species, yet identification by nonsequencemethods provides no information that theseisolates are closely related to each other. Increaseduse of sequence-based identification has led to a significantincrease in newly described, medically importantMycobacterium spp. (Table 2).Sequencing was also evaluated as an identificationmethod for groups of bacteria that either arepoorly differentiated using conventional methods orrequire more than 48 hours to identify. Tang et al.[17] compared three rapid identification methodswith conventional phenotypic identification for unusualaerobic Gram-negative bacteria. The threerapid methods were based on 16S rRNA gene sequencing(MicroSeq), cellular fatty acid profiles, andcarbon source use. MicroSeq identification matchedboth the genus and species reference identity moreoften than did the identifications produced by eitherof the other methods. Additionally, their datashowed that a 5' 500-bp MicroSeq identity was comparableto a full gene identity. The full gene and500-bp 16S rRNA gene identities always agreed atthe genus level, and 93.1% of the 500-bp speciesassignments were the same as the species assignmentsby the full gene method. In another study byTang et al. [32], the MicroSeq 5' 500-bp identificationproved a useful tool for the identification ofCorynebacterium and Corynebacterium-relatedisolates. Sequence identification provided the sameTable 2. Medically Important Mycobacterium spp.Recently Recognized by 16S rRNAGene SequencingNew SpeciesReferenceM. genavense 39M. branderi 40M. interjectum 41M. conspicuum 42M. lentiflavum 43M. novocastrense 44M. triplex 45M. bohemicum 13M. heidelbergense 46M. tusciae 47M. wolinskyi 48M. heckeshornense 49genus identity as conventional and supplementedphenotypic methods for all isolates tested. Thespecies-level identity was the same for 66.7% of allisolates. Sequence identification was most reliable(100% concordance) for the two most clinically significantspecies, Corynebacterium diptheriae andCorynebacterium jeikeium. 16S rRNA gene sequencingis a particularly important method for identificationof fastidious bacteria both from culture andfrom clinical specimens. Our laboratory was able toidentify a blood culture isolate that failed to grow onconventional media as Leptotrichia sp. only after analiquot of the positive blood culture bottle was submittedfor sequence analysis [33]. Sequencing alsoindicated that this isolate is probably a novel speciesof Leptotrichia. The literature contains several similarreports. For example, the uncommon isolateAnaerobiospirillum succiniciproducens was identifiedas a cause of bacteremia in three patients bysequence identification [34]. In another report, anisolate from joint fluid of a patient with septic arthritiswas identified as a novel species of Helicobacter[35]. Sequence identification will undoubtedly beapplied to the identification of other groups of bacterialisolates. Our laboratory is currently evaluatingsequencing as a means of identifying isolates ofaerobic and anaerobic actinomycetes.More often, clinical laboratories are using sequenceidentification to detect and identify pathogensdirectly from clinical specimens that shouldotherwise be sterile. Neisseria meningitidis wasidentified in brain pus from a patient with culturenegativemeningitis [36]. The isolate presumablyfailed to grow because of prior antibiotic use. Anovel Helicobacter species was identified directlyfrom drainage of an abdominal abscess in a patientwith X-linked hypogammaglobulinemia [37]. Ourlaboratory used 16S rRNA gene sequencing todetect and identify Mycoplasma orale in the tissueof a patient with hypogammaglobulinaemia whowas suffering from a persistently culture-negativeinflammatory arthritis (M. Paessler, M. Shuster, J.B.Patel, I. Nachamkin, unpublished data). Althoughsequence-based identification directly from a clinicalspecimen is a potentially powerful technique,this technology lacks some of the advantages thatculture provides. For example, sequence-basedidentification does not easily allow for the detectionof multiple pathogens, and there is no way tomeasure the relative abundance of different organisms.Likewise, without culture there is no isolate

16S Ribosomal RNA Gene Sequencing O Patel 319for further characterization such as susceptibilitytesting. For these reasons, sequence-based identificationdirectly from a clinical specimen is primarilyused only when culture has failed or is not possible.Is Sequence-based IdentificationCost-effective?Sequencing is a relatively expensive method ofidentification. One laboratory estimated their cost atapproximately $84.25 per test, and another laboratorycalculated a cost of $40.00 to $85.00 per test[32,38]. We have estimated our cost to perform asingle identification, which includes a negativeamplification control, to be $144.00 (J.B. Patel,unpublished data). This number includes the cost ofextraction, disposables, reagents, database use, andlabor. Most of the cost is labor, so the total costdrops to $87.00 per identification if two isolates aresequenced at the same time. These figures do notinclude the cost of purchasing instrumentation.One justification for the expense of sequencebasedidentification is that the improved accuracyand speed of this method will have a positive impacton clinical care. However, there are no studies evaluatingthe impact of sequence-based identification onthe quality of patient care or the cost of treating apatient. Despite this lack of data, it may not be necessaryto look beyond a laboratory’s own budget tojustify the expense of sequencing. For example, alaboratory may not have the capability of performingmycobacterial identification in-house, but thecost of sending the specimen to a reference laboratoryfar exceeds the cost of a sequence-based identification.Such a laboratory may consider sequencing,especially in a situation in which instrumentsfor amplification and sequence analysis are alreadyavailable or can be shared.The introduction of more automated methodsundoubtedly will have the biggest impact on decreasingthe cost of sequence identification and willresult in increased use of this technology in hospitaland reference laboratories.ConclusionsSequencing of the 16S rRNA gene is a powerfulidentification method in the clinical laboratory. Thistechnique is applicable for routine identification ofseveral groups of bacteria as well as for identificationof novel isolates. As the technical resources forsequence identification become more abundant andless expensive, more clinical microbiologists willconsider using this method in their laboratories’work flow.Received June 12, 2001.Received in revised form August 8, 2001.Accepted August 10, 2001.References1. Lebrun L, Espinasse F, Poveda JD, Vincent-Levy-Frebault V: Evaluation of nonradioactive DNAprobes for identification of mycobacteria. J ClinMicrobiol 1992;30:2476–24782. Bourbeau PP, Heiter BJ, Figdore M: Use of Gen-Probe AccuProbe Group B streptococcus test todetect group B streptococci in broth cultures ofvaginal-anorectal specimens from pregnant women:Comparison with traditional culture method. J ClinMicrobiol 1997;35:144–1473. Denys GA, Carey RB: Identification of Streptococcuspneumoniae with a DNA probe. J Clin Microbiol1992;30:2725–27274. Young H, Moyes A: Comparative evaluation ofAccuProbe culture identification test for Neisseriagonorrhoeae and other rapid methods. J Clin Microbiol1993;31:1996–19995. Padhye AA, Smith G, McLaughlin D, Standard PG,Kaufman L: Comparative evaluation of a chemiluminescentDNA probe and an exoantigen test forrapid identification of Histoplasma capsulatum. JClin Microbiol 1992;30:3108–31116. Padhye AA, Smith G, Standard PG, McLaughlin D,Kaufman L: Comparative evaluation of chemiluminescentDNA probe assays and exoantigen tests forrapid identification of Blastomyces dermatitidis andCoccidioides immitis. J Clin Microbiol 1994;32:867–8707. Westin L, Miller C, Vollmer D, Canter D, RadtkeyR, Nerenberg M, O’Connell JP: Antimicrobial resistanceand bacterial identification utilizing a microelectronicchip array. J Clin Microbiol 2001;39:1097–11048. Anthony RM, Brown TJ, French GL: Rapid diagnosisof bacteremia by universal amplification of23S ribosomal DNA followed by hybridization toan oligonucleotide array. J Clin Microbiol 2000;38:781–788

320 Molecular Diagnosis Vol. 6 No. 4 December 20019. Troesch A, Nguyen H, Miyada CG, Desvarenne S,Gingeras TR, Kaplan PM, Cros P, Mabilat C:Mycobacterium species identification and rifampinresistance testing with high-density DNA probearrays. J Clin Microbiol 1999;37:49–5510. Relman DA, Loutit JS, Schmidt TM, Falkow S,Tompkins LS: The agent of bacillary angiomatosis.An approach to the identification of unculturedpathogens. N Engl J Med 1990;323:1573–158011. Relman DA, Schmidt TM, MacDermott RP, FalkowS: Identification of the uncultured bacillus of Whipple’sdisease. N Engl J Med 1992;327:293–30112. Woese CR: Bacterial evolution. Microbiol Rev1987;51:221–27113. Reischl U, Emler S, Horak Z, Kaustova J, KroppenstedtRM, Lehn N, Naumann L: Mycobacteriumbohemicum sp. nov., a new slow-growing scotochromogenicmycobacterium. Int J Syst Bacteriol1998;48 Pt 4:1349–135514. Stackebrant E, Goebel BM: Taxonomic note: Aplace for DNA-DNA reassociation and 16S rRNAsequence analysis in the present species definitionin bacteriology. Int J Syst Bacteriol 1994;44:846–84915. Kusunoki S, Ezaki T: Proposal of Mycobacteriumperegrinum sp. nov., nom. rev., and elevation ofMycobacterium chelonae subsp. abscessus (Kubicaet al.) to species status: Mycobacterium abscessuscomb. nov. Int J Syst Bacteriol 1992;42:240–24516. Springer B, Bottger EC, Kirschner P, Wallace RJ Jr.:Phylogeny of the Mycobacterium chelonae-like organismbased on partial sequencing of the 16SrRNA gene and proposal of Mycobacterium mucogenicumsp. nov. Int J Syst Bacteriol 1995;45:262–26717. Tang YW, Ellis NM, Hopkins MK, Smith DH,Dodge DE, Persing DH: Comparison of phenotypicand genotypic techniques for identification of unusualaerobic pathogenic gram-negative bacilli. JClin Microbiol 1998;36:3674–367918. Rogall T, Flohr T, Bottger EC: Differentiation ofMycobacterium species by direct sequencing ofamplified DNA. J Gen Microbiol 1990;136:1915–192019. Woese CR, Gutell R, Gupta R, Noller HF: Detailedanalysis of the higher-order structure of 16S-likeribosomal ribonucleic acids. Microbiol Rev 1983;47:621–66920. MicroSeq 16S rRNA gene kit protocol. AppliedBiosystems, Forest City, CA, 2000 (www.appliedbiosystems.com)21. Patel JB, Leonard DG, Pan X, Musser JM, BermanRE, Nachamkin I: Sequence-based identification ofMycobacterium species using the MicroSeq 50016S rDNA bacterial identification system. J ClinMicrobiol 2000;38:246–25122. Harmsen D, Rothganger J, Singer C, Albert J, FroschM: Intuitive hypertext-based molecular identificationof micro-organisms. Lancet 1999;353:29123. Liefting LW, Andersen MT, Beever RE, GardnerRC, Forster RL: Sequence heterogeneity in the two16S rRNA genes of Phormium yellow leaf phytoplasma.Appl Environ Microbiol 1996;62:3133–313924. Mylvaganam S, Dennis PP: Sequence heterogeneitybetween the two genes encoding 16S rRNA fromthe halophilic archaebacterium Haloarcula marismortui.Genetics 1992;130:399–41025. Nubel U, Engelen B, Felske A, et al.: Sequenceheterogeneities of genes encoding 16S rRNAs inPaenibacillus polymyxa detected by temperaturegradient gel electrophoresis. J Bacteriol 1996;178:5636–564326. Wang Y, Zhang Z, Ramanan N: The actinomyceteThermobispora bispora contains two distinct typesof transcriptionally active 16S rRNA genes. J Bacteriol1997;179:3270–327627. Ueda K, Seki T, Kudo T, Yoshida T, Kataoka M:Two distinct mechanisms cause heterogeneity of16S rRNA. J Bacteriol 1999;181:78–8228. Ninet B, Monod M, Emler S, et al.: Two different16S rRNA genes in a mycobacterial strain. J ClinMicrobiol 1996;34:2531–253629. Reischl U, Feldmann K, Naumann L, et al.: 16SrRNA sequence diversity in Mycobacterium celatumstrains caused by presence of two differentcopies of 16S rRNA gene. J Clin Microbiol 1998;36:1761–176430. Kirschner P, Springer B, Vogel U, et al.: Genotypicidentification of mycobacteria by nucleic acid sequencedetermination: Report of a 2-year experiencein a clinical laboratory. J Clin Microbiol1993;31:2882–288931. Springer B, Stockman L, Teschner K, Roberts GD,Bottger EC: Two-laboratory collaborative study onidentification of mycobacteria: Molecular versusphenotypic methods. J Clin Microbiol 1996;34:296–30332. Tang YW, Von Graevenitz A, Waddington MG, etal.: Identification of coryneform bacterial isolatesby ribosomal DNA sequence analysis. J Clin Microbiol2000;38:1676–167833. Patel JB, Clarridge J, Schuster MS, Waddington M,Osborne J, Nachamkin I: Bacteremia caused by anovel isolate resembling Leptotrichia species in aneutropenic patient. J Clin Microbiol 1999;37:2064–206734. Tee W, Korman TM, Waters MJ, et al.: Three casesof Anaerobiospirillum succiniciproducens bacter-

16S Ribosomal RNA Gene Sequencing O Patel 321emia confirmed by 16S rRNA gene sequencing. JClin Microbiol 1998;36:1209–121335. Husmann M, Gries C, Jehnichen P, et al.: Helicobactersp. strain Mainz isolated from an AIDSpatient with septic arthritis: Case report and nonradioactiveanalysis of 16S rRNA sequence. J ClinMicrobiol 1994;32:3037–303936. Logan JM, Orange GV, Maggs AF: Identification ofthe cause of a brain abscess by direct 16S ribosomalDNA sequencing. J Infect 1999;38:45–4737. Han S, Schindel C, Genitsariotis R, Marker-Hermann E, Bhakdi S, Maeurer MJ: Identificationof a unique Helicobacter species by 16S rRNA geneanalysis in an abdominal abscess from a patientwith X-linked hypogammaglobulinemia. J Clin Microbiol2000;38:2740–274238. Clarridge JE, Zhang Q, Heward S: 16S rDNAsequence analysis as a real-time addition to theclinical microbiology laboratory. General Meetingof the American Society for Microbiology, Orlando,FL, May 20-24, 2001 (abstr)39. Haas WH, Kirschner P, Ziesing S, Bremer HJ,Bottger EC: Cervical lymphadenitis in a childcaused by a previously unknown mycobacterium. JInfect Dis 1993;167:237–24040. Koukila-Kahkola P, Springer B, Bottger EC, PaulinL, Jantzen E, Katila ML: Mycobacterium branderisp. nov., a new potential human pathogen. Int J SystBacteriol 1995;45:549–55341. Springer B, Kirschner P, Rost-Meyer G, SchroderKH, Kroppenstedt RM, Bottger EC: Mycobacteriuminterjectum, a new species isolated from apatient with chronic lymphadenitis. J Clin Microbiol1993;31:3083–3089 (erratum J Clin Microbiol1994;32:1417)42. Springer B, Tortoli E, Richter I, et al.: Mycobacteriumconspicuum sp. nov., a new species isolatedfrom patients with disseminated infections. J ClinMicrobiol 1995;33:2805–281143. Springer B, Wu WK, Bodmer T, et al.: Isolation andcharacterization of a unique group of slowly growingmycobacteria: Description of Mycobacteriumlentiflavum sp. nov. J Clin Microbiol 1996;34:1100–110744. Shojaei H, Goodfellow M, Magee JG, Freeman R,Gould FK, Brignall CG: Mycobacterium novocastrensesp. nov., a rapidly growing photochromogenicmycobacterium. Int J Syst Bacteriol 1997;47:1205–120745. Floyd MM, Guthertz LS, Silcox VA, et al.: Characterizationof an SAV organism and proposal ofMycobacterium triplex sp. nov. J Clin Microbiol1996;34:2963–296746. Haas WH, Butler WR, Kirschner P, et al.: A newagent of mycobacterial lymphadenitis in children:Mycobacterium heidelbergense sp. nov. J Clin Microbiol1997;35:3203–320947. Tortoli E, Kroppenstedt RM, Bartoloni A, et al.:Mycobacterium tusciae sp. nov. Int J Syst Bacteriol1999;49 Pt 4:1839–184448. Brown BA, Springer B, Steingrube VA, et al.:Mycobacterium wolinskyi sp. nov. and Mycobacteriumgoodii sp. nov., two new rapidly growingspecies related to Mycobacterium smegmatis andassociated with human wound infections: A cooperativestudy from the International Working Groupon Mycobacterial Taxonomy. Int J Syst Bacteriol1999;49:1493–151149. Roth A, Reischl U, Schonfeld N, et al.: Mycobacteriumheckeshornense sp. nov., a new pathogenicslowly growing Mycobacterium sp. causing cavitarylung disease in an immunocompetent patient. J ClinMicrobiol 2000;38:4102–4107