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Molecular Microbiology: Diagnostic Principles and Practice, 2nd Ed.Edited by David H. Persing et al.

�2011 ASM Press, Washington, DC

31Broad-Range PCR for Detectionand Identification of Bacteria

MATTHIAS MAIWALD

BACKGROUNDBroad-range PCR is based on the recognition that thereare a number of broadly conserved molecules across a rangeof many different organisms. rRNA genes, for example, arepresent in all cellular forms of life, namely the domainsBacteria, Archaea, and Eukarya (163). rRNA genes possesshighly conserved regions that are suitable as sites for PCRprimers that recognize large, diverse groups of organisms(e.g., all members of the Bacteria) and possess variableregions that provide distinct signatures for identification atphylogenetic levels below the level initially targeted by theprimers. Commonly, broad-range PCR are aimed at mem-bers of the domain Bacteria, although broad-range PCRshave also been developed for other large relevant groupsof organisms or combinations of such groups. For example,broad-range PCRs may target Eukarya (91, 119), Archaea(1, 4, 143), Eukarya and Archaea (10), fungi (70, 130, 159),or fungi and protists (44). Fungal broad-range PCRs areoften termed panfungal PCRs in the medical literature. Be-low the level of broad groups of organisms, PCRs may beconstructed to target organisms at various other phyloge-netic levels; for example, within the Bacteria it is possibleto design phylum-, family-, or genus-specific PCRs, de-pending on the availability of phylogenetically informativesignature sequences.

The strength of broad-range PCR for diagnostic micro-biology lies in the relative absence of selectivity, assuminglittle or no prior knowledge of an infecting organism, sothat—in principle—any bacterium (in the case of bacterialassays) can be detected and identified. This is an area ofanalogy to the detection by culture and is in contrast totypical organism-specific PCR assays.

The aim of most broad-range PCRs is to amplify asbroadly as possible within the domain Bacteria and, at thesame time, to obtain sufficient portions of variable se-quence for identification. Not all broadly conserved mol-

Matthias Maiwald, Department of Pathology and Laboratory Medi-cine, KK Women’s and Children’s Hospital, Singapore 229899, Sin-gapore.

ecules, though, provide suitable priming sites as well asphylogenetically informative sequences (see chapter 9). Forexample, sequences of genes coding for conserved proteins(e.g., heat shock proteins and RNA polymerases) can bevery useful for identification within bacterial families (99)but generally do not provide sufficiently conserved sites forprimers across the domain Bacteria. rRNAs, on the otherhand, fulfill many of the necessary requirements (104).rRNA molecules have been described as the ultimate mo-lecular chronometers (163); they reflect evolutionarychanges, while functional constraints in protein translationprevent these changes from being too extensive.

In the 1980s, oligonucleotides from conserved regionswere first used for reverse transcriptase sequencing of bac-terial 16S rRNA (77). Subsequently, such conserved oli-gonucleotides were utilized as primers in PCR (23, 38).The two main molecules that are suitable for bacterialbroad-range PCR are the 16S rRNA gene, consisting ofapproximately 1,540 bp (in Escherichia coli, 1,542 bp), andthe 23S rRNA gene, consisting of approximately 2,900 bp(in E. coli, 2,904 bp). The 5S rRNA gene, the third struc-tural rRNA gene with about 120 bp, is too small for thispurpose. While the 23S rRNA gene possesses greater in-formation content and more potential priming sites due toits larger size, the 16S rRNA gene was adopted earlier, hasbecome a quasistandard, and has many more reference se-quences available for comparison. For example, the SILVAonline resource (111) contained 324,342 different small-subunit (16S rRNA-like) sequences of 1,200 bp or greater(900 bp or greater for Archaea) and 12,506 different large-subunit (23S rRNA-like) sequences of 1,900 bp or greaterin its release of 14 October 2008 (http: / /www.arb-silva.de).The numbers of small partial 16S and 23S rRNA sequencesof �300 bp are much greater.

PATHOGEN DISCOVERYBroad-range ribosomal PCR and sequence analysis haveplayed key roles in the characterization of novel bacterialpathogens, particularly in settings where detection by cul-ture has failed. The first agent discovered by this technique

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FIGURE 1 (A) Schematic drawing of the bacterial 16S rRNA gene, with conserved (open bars)and variable (shaded bars) regions. The location of broad-range primers (Table 1) is shown. Mod-ified from reference 117, with permission from the American Society for Microbiology. (B) Con-servation profile of 16S rRNA across the domain Bacteria, constructed with the software packageARB (85) and the SILVA (111) data set. The graph shows the relative frequency (y axis) of themost common nucleotide for each position (x axis) in 16S rRNA. Courtesy of Frank OliverGlockner and Elmar Pruesse (Max Planck Institute for Marine Microbiology, Bremen, Germany).

was the agent of bacillary angiomatosis in AIDS patients(118), which was microscopically visible in clinical samplesbut had not been cultivated. The 16S rRNA sequence re-vealed a relationship to Rochalimaea (subsequently namedBartonella) quintana, the agent of trench fever. The novelbacterium was subsequently cultivated, named Bartonellahenselae, and found also to be an agent of cat scratch dis-ease (13, 17, 157). Other examples of pathogen discoveriesby broad-range PCR and sequencing are Ehrlichia chaffeen-sis, an agent of human tick-borne monocytic ehrlichiosis(5), Tropheryma whipplei, the agent of Whipple’s disease(120, 160), and Mycobacterium genavense, a cause of dissem-inated infections in AIDS patients (15). Thus, broad-rangePCR joined several other molecular methods that were ca-pable of gathering insight into distinguishing features ofpathogens by obtaining molecular signatures before othercharacteristic traits were known (46). It was also recognizedthat the mere identification of a microbial molecular sig-nature in a person with a disease does not establish a causalrelationship between microbe and disease, and a set of nu-cleic acid-based criteria for causality was proposed, by anal-ogy with the historic Koch’s postulates (46). Broad-rangePCR and sequencing is not only suitable for the charac-terization of uncultivated bacteria; 16S rRNA sequencedeposition has become a mainstay of established procedureswhenever a new bacterial species is described. One reviewarticle (165) counted 215 newly described bacterial species(obtained mostly by culture) from human specimens be-tween the years 2000 and 2007, of which 29 were assignedto novel genera and 100 were found in four or more pa-tients.

PRIMER SYSTEMS FOR BROAD-RANGE PCRrRNA molecules consist of conserved as well as variableregions; a depiction of these regions and the correspondingpriming sites in the bacterial 16S rRNA gene is given in

Fig. 1A. A selection of broad-range primers for the 16Sand 23S rRNA genes is provided in Table 1. Further primerand probe sequences for 16S and 23S rRNA gene can befound in other articles (1, 4, 6, 9, 61, 63, 76, 88, 123, 133,150, 155), as well as in the European rRNA database (http:/ /bioinformatics.psb.ugent.be/webtools / rRNA) (166) andin the ProbeBase database (http: / /www.microbial-ecology.net /probebase) (84). When looking at rRNA secondarystructure, conserved regions tend to lie in loops, whilevariable regions tend to lie in stems, where nucleotides arepaired with those of the opposite strand (76). Primers fordiagnostic broad-range PCR need to amplify sufficientlylong stretches of variable sequence to be able to identifybacteria at or close to species level; generally, targets of�300 to 900 bp are used in most diagnostic broad-rangePCRs (e.g., primers 533 forward [f] plus 806 reverse [r], 27fplus 556r, 27f plus 806r, or 533f plus 1371r). Targets to-wards the shorter end of this range may be preferable whenthe extracted DNA is likely to be damaged (e.g., formalin-fixed, paraffin-embedded tissue); longer targets (e.g., prim-ers 27f /1492r) may be used when bacteria in samples areabundant. Generally, longer targets provide more variablesequence for more accurate identification. Primers that an-neal to eukaryotic nuclear or mitochondrial DNA (‘‘uni-versal’’ primers; e.g., 533f plus 1492r) should not be usedas pairs in the same assay when working with human sam-ples (117).

The distinction between conserved and variable regionsin rRNA molecules is not as sharp as the depiction in Fig.1A suggests and is instead better reflected in Fig. 1B. Withan increasing number of rRNA sequences in databases, ithas been recognized that some nucleotide positions thatare less conserved are interspersed in highly conserved se-quence stretches, and some taxa that are part of an in-tended target group of organisms have mismatches to com-monly used broad-range primers (9, 42). This is in partaccounted for by the ambiguous nucleotides in the primers

31. Broad-Range PCR for Bacterial Identification ■ 493

TABLE 1 Commonly used primers and probes for broad-range PCR with bacterial 16S and 23S rRNA genes

Name a Sequence b Location c Specificity d Reference(s) b

16S rRNA27f 5�-AGAGTTTGATYMTGGCTCAG 8–27 Most Bacteria 38355f 5�-ACTCCTACGGGAGGCAGC 338–355 Most Bacteria 3338r 5�-GCTGCCTCCCGTAGGAGT 33–355 Most Bacteria 3533f 5�-GTGCCAGCMGCCGCGGTAA 51–533 Universal 77, 120515r 5�-TTACCGCGGCKGCTGGCAC 515–533 Universal 77, 120556r 5�-CTTTACGCCCARTRAWTCCG 556–575 Most Bacteria 13806f 5�-ATTAGATACCCTGGTAGTCC 787–806 Most Bacteria 162787r 5�-GGACTACCAGGGTATCTAAT 787–806 Most Bacteria 162926f 5�-AAACTYAAAKGAATTGACGG 907–926 Most Bacteria 77907r 5�-CCGTCAATTCMTTTRAGTTT 907–926 Most Bacteria 77930f 5�-TCAAAKGAATTGACGGGGGC 911–930 Most Bacteria 120911r 5�-CCGTCAATTCMTTTRAGTTT 911–930 Most Bacteria 1201194f 5�-GAGGAAGGTGGGGATGACGT 1175–1194 Most Bacteria 231175r 5�-ACGTCATCCCCACCTTCCTC 1175–1194 Most Bacteria 231371r 5�-AGGCCCGGGAACGTATTCAC 1390–1371 Most Bacteria 231391r 5�-TGACGGGCGGTGWGTRCA 1391–1408 Universal 771492r 5�-GGTTACCTTGTTACGACTT 1510–1492 Universal 37, 1561525r 5�-AAGGAGGTGWTCCARCC 1525–1541 Most Bacteria and Archaea 38

23S rRNA1077f 5�-AGGATGTTGGCTTAGAAGCAGCCA 1054–1077 Most Bacteria 731950r 5�-CCCGACAAGGAATTTCGCTACCTTA 1950–1926 Most Bacteria 73

a Names of 16S rRNA primers are based on the 3� nucleotide position and the orientation (f, forward; r, reverse), according to the numbering system by Lane(76).

b Sequences may have been modified from the indicated references by subsequent authors. References are given for the original publications of the priming sites.c Location in E. coli 16S rRNA or 23 S rRNA.d Universal includes Bacteria, Archaea, and Eukarya.

in Table 1, but in some instances it may necessitate theredesign of commonly used broad-range primers or hybrid-ization probes to achieve more complete coverage of bac-terial phyla (30, 42). While the impact of this appears tobe somewhat smaller for diagnostic broad-range PCR incases of suspected monomicrobial infections, it can be dra-matic for polymicrobial infections, microbial flora studies,or environmental studies, where certain bacterial taxa maybecome significantly over- or underrepresented after PCRamplification (11, 39, 59, 63, 137, 142, 155).

In a general sense, the performance of PCR assays maybe affected by (i) length of the PCR product, (ii) annealingtemperature, (iii) number of cycles, (iv) template concen-tration, (v) internal composition (e.g., G�C richness) ofthe target, and (vi) primer base composition (G�C rich-ness and internal base pairing). Longer distances betweenprimer pairs generally result in less analytical sensitivity,but this may be partially offset by the finding that DNAcontaminations in PCR reagents (see below) appear to befragmented into smaller sizes (132), so that the ratio be-tween target and contaminations becomes more favorable.A large study of broad-range PCR has in fact shown quiterobust amplification of an �850-bp target from the 23SrRNA gene with diverse clinical samples (115). In additionto the possible mismatches of rRNA gene primers (seeabove), broad-range PCR has also been found to be af-fected by rRNA gene copy numbers in different bacterialgenomes and even by interference from bacterial DNA out-side the PCR target region (53, 154). In some instances,

different primer sets show different amplification efficiencydespite perfect primer-to-target matches (39).

As a consequence of performance and coverage differ-ences between broad-range PCR primers, they may haveto be redesigned and reevaluated, depending on the needsof particular diagnostic or scientific applications. There area number of tools to establish and check the matches ofprimers and probes against large 16S rRNA and 23S rRNAdata sets. The Ribosomal Database Project (RDP) website(http: / / rdp.cme.msu.edu) (27) has a probe match tool forthat purpose. A computer program called PRIMROSE canalso be used to construct primers and probes in conjunc-tion with the RDP data set (8). Other web interfacetools are the probeBase (http: / /www.microbial-ecology.net /probebase) (84) and probeCheck (http: / /www.microbial-ecology.net /probecheck) (83) resources. In ad-dition, the ARB phylogeny program package (http: / /www.arb-home.de) (85) has probe design and probe matchfunctions that can be used, for example, in conjunctionwith the SILVA 16S and 23S rRNA data sets (111).

CONTAMINATION ISSUES OFBROAD-RANGE PCRBroad-range PCR is subject to much of the same contam-ination problems and requires much of the same preventionmeasures as specific PCRs (see chapter 8). In addition, theinherent nonselective nature of broad-range PCR makes it

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TABLE 2 Methods to reduce bacterial background DNA in PCR reagents

Measure(s) Comment(s) Reference(s)

Low-DNA Taq polymerase (e.g.,AmpliTaq LD, AmpliTaq Gold LD)

Commercially available, contains 10 or fewer bacterialrRNA gene copies per 2.5 U

147

DNase I treatment of PCR mix Effective in DNA elimination; inactivation critical; minorloss of PCR sensitivity; some authors treat PCR mixwithout primers

28, 56, 57, 71, 124, 136,139, 147, 148

Restriction enzyme treatment of PCRmix

Mixed results concerning DNA elimination and retainedsensitivity; some authors treat PCR mix withoutdNTPs and primers

20, 28, 71

UV irradiation of PCR mix Taq polymerase may be sensitive to UV action; dNTPscan impede UV action; probable loss of sensitivity

28, 47, 71, 105

8-Methoxypsoralen and UV lighttreatment of PCR mix

8-Methoxypsoralen concn and UV exposure have to befine-tuned; longer exposure reduces PCR sensitivity

28, 62, 71, 92, 128

Treatment of PCR mix with ethidiummonoazide or propidium monoazideand light exposure

Concn of ethidium monoazide in PCR mix is critical;good elimination of contaminating DNA and retainedsensitivity

55, 129

Ultrafiltration of PCR mix withexclusion size of 100 kDa

Type of filter and centrifugation time and speed arecritical; good elimination of contaminating DNA andretained sensitivity

136

susceptible to minute amounts of any bacterial DNA thatmight be encountered along the various steps of testing.While specific PCR does not amplify DNA from nontargetorganisms, broad-range PCR, by its design, can amplifyDNA from almost any bacterium. Soon after broad-rangePCR was originally implemented, it was found that nega-tive controls often yielded amplified DNA of the appro-priate size. Such false-positive results would not be avoidedby common anticontamination measures. Several types ofreagents have been implicated as being sources of DNAcontamination: Taq polymerase was commonly implicated(14, 113, 132), but also other reagents, such as water andoligonucleotide solutions (49) and buffers or enzymes forsample preparation (144). The application of restrictionanalysis (113) suggested that Thermus aquaticus and E. coli(the host species for native and recombinant Taq polymer-ase) are unlikely sources of contamination in Taq poly-merase. By contrast, the columns and buffers used for theindustrial purification of Taq polymerase were thought tobe likely sources. Sequencing methods identified contami-nating DNA as originating from within the pseudomonadgroup of organisms (86, 118), which are known waterbornecontaminants. The amount of contaminating DNA was es-timated to be in the range of 100 copies of 16S rRNA geneper Taq portion needed for a single reaction (113). Taqpolymerases from different manufacturers were found tobe affected (14, 62, 132), and there appears to bemanufacturer-to-manufacturer and even batch-to-batchvariation in the amount of contaminating DNA (113, 128,132). As a consequence, the detectable limit (i.e., analyt-ical sensitivity) of PCR assays using such Taq preparationslies well above 100 copies per PCR, if one wants to distin-guish ‘‘real’’ from contaminating DNA. Shorter PCR tar-gets tend to be more susceptible to the presence of back-ground DNA, since this DNA appears to be fragmentedinto small sizes (132). There also seems to be a physio-chemical affinity between Taq polymerase and contami-nating DNA, since they appear to be strongly associatedwith each other (113).

Several protocols have been developed to avoid or re-duce contaminating DNA in PCR reagents. There are a

few commercial Taq polymerase preparations, such asAmpliTaq LD or AmpliTaq Gold LD (Applied Biosystems),where LD stands for low DNA, and these enzymes havebeen purified to contain 10 or fewer bacterial rRNA genecopies in a standard 2.5-U aliquot. Published protocols alsoinclude the treatment of PCR mixes, including Taq poly-merase, with DNase I (56, 124, 136, 139, 147, 148), re-striction enzymes (20), or UV light (47, 105). DNase I andrestriction enzymes need to be heat inactivated before theaddition of samples and commencement of cycling (20,124). Some authors pointed out that UV treatment shouldtake place prior to adding deoxynucleoside triphosphates(dNTPs), because dNTPs absorb UV light and prevent ex-posure of other PCR mix contents (47). Several others(62, 92, 128) described a combined protocol of 8-methoxypsoralen and UV treatment of PCR mixes. 8-Methoxypsoralen acts as UV-induced cross-linker of DNAin contaminated reagents. However, it was pointed out thatthe 8-methoxypsoralen concentration and time of UV ex-posure need to be carefully adjusted, because intense ex-posure damages Taq polymerase and leads to reduced PCRsensitivity. Other published approaches include the ultra-filtration of PCR reagents (136) and treatment with ethi-dium monoazide or propidium monoazide (55, 129). Ultra-filtration with a 100-kDa exclusion size (e.g., MicroconYM-100; Millipore) lets Taq polymerase (94 kDa) passthrough the filter, while typical contaminating DNA is re-tained; however, some Taq polymerase loss may be ob-served. Ethidium monoazide and propidium monoazide arephotoreactive DNA-intercalating substances that are acti-vated by visible light. Some protocols involve splittingPCR mix components for different treatments. For exam-ple, Steinman et al. (139) subjected the PCR mix withoutprimers to DNase I treatment and treated the primers sep-arately with UV light before combining the mix. Table 2contains an overview of the different anticontaminationmethods for broad-range PCR.

Several authors have examined different anticontami-nation procedures in parallel (28, 71, 136). It became clearthat most anticontamination measures provide a trade-offbetween reducing contaminating DNA and retaining PCR

31. Broad-Range PCR for Bacterial Identification ■ 495

sensitivity. Corless et al. (28) examined UV irradiation, 8-methoxypsoralen plus UV, DNase I digestion, restrictionenzyme digestion, and DNase I plus restriction enzymetreatment, using broad-range PCR in a quantitative real-time format; this format facilitates the monitoring of DNAcontamination (see below). A significant reduction in an-alytical sensitivity accompanied the reduction of contam-inating DNA in most of the treatment protocols, exceptDNase I treatment, for which the reduction in sensitivitywas relatively small, with only a 1- to 2-log reduction. Thecombination of DNase I and restriction enzyme digestionsuffered from problems with test-to-test reproducibility.The authors concluded that DNA contamination in broad-range PCR assays will continue to be a problem. Klaschiket al. (71) compared DNase I treatment, restriction enzymedigestion, UV irradiation, and 8-methoxypsoralen plus UVand found that all methods inhibited PCR. Only DNase Iwas able to eliminate contaminating DNA while retaininga useful distinction between negative controls and low lev-els of template DNA. Silkie et al. (136) compared DNaseI digestion and ultrafiltration with Microcon YM-100 (Mil-lipore) filters. It was found that both methods eliminatedcontaminating DNA and retained sensitivity, but forDNase I digestion, the concentration of DNase and thepresence of Ca2� ions in the digestion mix was critical, aswas thorough inactivation of DNase in the presence ofdithiothreitol.

Complementing the specific protocols to avoid or elim-inate DNA in PCR reagents, Millar et al. (95) proposed aset of broad, general guidelines for laboratory practice inorder to avoid contamination in the setting of broad-rangePCR assays. These included guidelines for the sampling ofclinical specimens, separation of rooms for pre- and post-PCR work, and the use of high-quality reagents.

SAMPLE PREPARATION FORBROAD-RANGE PCRIn contrast to microbial culture, where a sample in a vol-ume of up to about 10 ml can be collected and used forinoculation (e.g., into a blood culture bottle) and one sin-gle viable bacterium may lead to a positive result, the typ-ical sample volume added to a PCR tube is only about 5�l. Sample preparation for PCR is therefore extremely im-portant; its aims are to crack microbial wall structures, re-move PCR inhibitors, and purify and concentrate the tem-plate DNA, but also to avoid DNA overload (see chapter7). There are two important issues concerning broad-rangePCR: (i) sample preparation should be kept simple in orderto reduce the risk of introducing external contamination,and (ii) some reagents used for sample preparation maycontain intrinsic DNA. Proteinase K is one example of apotentially contaminated reagent; it is commonly used insample preparation and requires purification during manu-facturing, in analogy to Taq polymerase.

There are a number of in-house and commercial samplepreparation products and protocols; these include, amongothers, classical proteinase K digestion and phenol-chloroform extraction, crude lysis with proteinase K andboiling with Chelex resin, simple alkaline lysis of bloodsamples, DNA spin column kits (e.g., Qiagen kits), andfully automated sample preparation workstations, such asthe MagNA Pure LC Instrument and Kit (Roche) (35, 93,94, 114, 135, 140). There are relatively few published side-by-side evaluations of different techniques; however, theymay differ considerably in DNA recovery and the resultant

analytical sensitivity (114). It is also clear that any of theseprotocols can introduce DNA contamination. For example,some lots of Qiagen kits have been found to contain Le-gionella DNA (151). Reagents for the MagNA Pure LCworkstation have also been found to contain bacterialDNA (98, 109), and subsequently, the company (Roche)released ‘‘M-grade’’ MagNA Pure reagents that were free ofdetectable bacterial DNA.

Interesting observations were published by Tanner et al.(144); the authors conducted broad-range PCRs exposedto common sample preparation reagents and prepared aPCR product clone library from amplified DNA, presumedto be from reagents for sample preparation or PCR. Theyfound that the obtained sequences matched published se-quences from a range of ‘‘uncultured environmental bac-teria’’ well. These sequences had been amplified from verydiverse environmental samples, all with low total biomassin common, and had been deposited by various authors indatabases. This suggests that some published sequencesfrom ‘‘environmental bacteria’’ were in fact due to PCRcontamination.

There are also unusual examples of contamination in-volving sample acquisition and preparation, affecting notjust bacterial broad-range PCR. Zymolyase, an enzyme di-gesting fungal cell walls and commonly used for panfungalPCRs, was found to contain fungal DNA; it is manufac-tured by a brewery company that also handles yeast (122).Another group found an unusually high number of positiveBordetella pertussis PCRs from one particular pediatric ex-amination room; this room had previously been used foradministration of cellular pertussis vaccine, and environ-mental swabs from the room also yielded positive PCRs(146). Another study aimed at broad-range PCR diagnosisof bacterial endophthalmitis from vitreous fluid samples; all14 tested samples revealed Pseudomonas spp. sequences.The povidone-iodine antiseptic solution used for preoper-ative eye antisepsis was subsequently found to be contam-inated (149).

DETECTION OF PCR PRODUCTS ANDSEQUENCE ANALYSISThe classical way to detect and identify bacteria by broad-range PCR is via visualization of PCR products by standardgel electrophoresis, followed by sequencing, preferably forboth DNA strands of the products (26). This is the mostaccurate method. Direct sequencing of PCR products isfeasible when it can be assumed that a sample contains asingle species of microorganism. However, when direct se-quencing reveals ambiguous reads or when multiple organ-isms are present, it is generally necessary to clone PCRproducts (e.g., by using commercial plasmid vectors forPCR product cloning) and to sequence an adequate num-ber of clones (45, 116). The latter technique can also beapplied to the construction of 16S rRNA gene clone li-braries, in which a single PCR product from a polymicro-bial sample may result in a few hundred plasmid cloneswith different sequences, representing different organisms(74, 107, 108).

There are a number of tools for analysis of 16S or 23SrRNA gene sequences from broad-range PCR. The simplestmethod is to perform BLAST similarity searches in pub-lic sequence databases, such as in GenBank at the Na-tional Center for Biotechnology Information (http: / /www.ncbi.nlm.nih.gov). These searches provide a percent-age of similarity to the closest sequences in the database.

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However, this may not suffice for accurate species identi-fication; searches may reveal no identical sequences, orthey may reveal identical sequences from different species,and there is no defined similarity cutoff above which a newsequence can be assigned to the same genus or species (26,66, 165). In addition, public databases contain many se-quence errors, incorrect species and strain assignments, andrRNA gene sequence chimeras. A chimera is an artificialsequence that may be generated from a mixed microbialsample when PCR, after partial amplification of a DNAstrand of one species, continues to amplify the homologousstrand of another species, so that a phylogenetically het-erogenous sequence is generated (154). Thus, it is advisableto check sequences obtained with broad-range PCR frompolymicrobial samples; available tools are the programs Bel-lerophon (60) or Pintail (7).

More accurate rRNA gene sequence assessment is pos-sible with curated rRNA databases, web-based or commer-cial identification tools, and phylogenetic analysis. BIBI(http://pbil.univ-lyon1.fr /bibi) is a web-based identificationtool aimed at clinically relevant bacteria (33). MicroSeq(Applied Biosystems) is a commercial kit, with PCR rea-gents included, that aims at 16S rRNA-based identificationof cultivated bacterial isolates in clinical microbiology. Itcomes with its proprietary database of quality-checked se-quences from reference strains and is available in two ver-sions, covering either about 500 bp or almost the fulllength of the 16S rRNA gene (164). The Ribosomal Da-tabase Project website (http: / / rdp.cme.msu.edu) contains acurated 16S rRNA database with over 715,000 partial andfull-length sequences in its 2008 release (27). It can besearched via a web interface that generates provisional tax-onomic assignments, and a set of aligned sequences canbe downloaded and imported into a phylogeny program.Greengenes (http: / /greengenes.lbl.gov) is a taxonomicallyand chimera-checked 16S rRNA database with over290,000 sequences of �1,250 bp in length (in its 2008release) that can be interrogated via a web interface, andit has a fully aligned data set for download (31). SILVA(http: / /www.arb-silva.de) is a large database of quality-checked, aligned 16S and 23S rRNA sequences with ‘‘Ref’’and ‘‘Parc’’ data sets for each gene, where Ref stands fornear-full-length sequences and Parc for all sequences over300 bp (111). While the majority of entries in these datasets are comprised of environmental or microbial flora se-quences from uncultivated organisms, SILVA containsnearly 20,000 16S rRNA and nearly 2,000 23S rRNA se-quences from cultivated isolates (2008 release). Both thedata sets from Greengenes and SILVA can be downloadedand imported into ARB (http: / /www.arb-home.de), whichis a large phylogeny program package with a graphical userinterface (85).

Quantitative real-time PCR provides an alternative de-tection method that is rapidly replacing gel-based detectionof broad-range PCR amplicons (28, 35, 36, 68, 100, 102,127, 168). It offers several advantages. In gel-based assays,cycle numbers and other conditions need to be carefullyadjusted, so that negative controls show no bands whilesufficiently small amounts of positive control DNA becomevisible. In real-time PCR, the product is detected duringcycling, so that negative controls (e.g., water controls, sam-ple preparation controls, and known negative samples),positive controls with known copy numbers of 16S rRNAgene, and actual samples can be continuously monitored,and the quantification cycle (Cq [see chapter 4]) can beused as a criterion to compare the amount of DNA in

controls to that in samples. As a consequence, real-timePCR offers much improved distinction between ‘‘true’’ andbackground DNA. Detection of amplicons can be achievedwith fluorescent broad-range probes that simultaneouslyhybridize to the target sequence during each cycle (102),or with nonspecific nucleic acid dyes, such as SYBR Green(168). Products from real-time broad-range PCR can besequenced and analyzed using standard procedures. Real-time PCR products can be subjected to melting (i.e., ther-mal DNA dissociation) analysis, and a variant technique,high-resolution melting analysis, may be used for a provi-sional species assignment among a limited range of medi-cally relevant bacteria (24). As with gel-based assays, back-ground DNA in PCR mixes or sample preparation reagentscauses most negative controls to reach the threshold ofamplified DNA at high numbers of cycles (Fig. 2).

Pyrosequencing is an emerging alternative to standardSanger type sequencing. It is much more rapid and cheaperper base than standard sequencing, detects the incorpora-tion of nucleotides by light emission, and can be highlyautomated. Earlier generations of pyrosequencing instru-ments were limited to short sequence reads (e.g., 30 bp)and could be used in conjunction with broad-range PCRto interrogate short, informative sequence stretches thatallowed preliminary identification of bacteria, such as gramnegative or gram positive (72), Streptococcus sp., Staphylo-coccus sp., enteric gram-negative rod, etc. (67). More re-cent instruments allow for massively parallel pyrosequenc-ing with increased read lengths (up to 400 bp) and can beused for accurate characterization of broad-range PCRproducts from highly complex polymicrobial communities(64, 82).

Broad-range PCR can also be combined with amplicondetection by DNA microarrays. Several formats have beendeveloped, ranging from low-density arrays that contain aset of probes for a limited number of medically relevantpathogens (96) to high-density arrays that cover large num-bers of diverse taxa across a wide phylogenetic range (18).Such high-density arrays are commonly termed ‘‘phylo-chips’’ or ‘‘phyloarrays.’’ For example, a high-density mi-croarray has been developed on the Affymetrix platformthat contains 500,000 oligonucleotide probes coveringabout 9,000 distinct prokaryotic taxa (18). This array hasbeen used, in a research setting, to characterize bacterialpopulations in urban atmospheric aerosols (19) and inuranium-contaminated soils (18). Another type of high-density array has been used to characterize the develop-ment and diversification of intestinal flora of infants duringthe first year of life (107). High-density microarrays afterbroad-range PCR can be used for the quantitative analysisof PCR products across a wide range of taxa.

CLINICAL APPLICATIONSClinical microbiology has a constant need for alternativediagnostic methods that complement or expand on the ex-isting ones. Most diagnostic methods have limitations: mi-croscopy requires a significant number of organisms presentin samples to become positive, and identification based onmorphology is often not possible, serologic testing oftenonly provides retrospective information, and culture mayfail when fastidious or ‘‘culture-resistant’’ microorganismscause infections. For many common pathogens, the rate ofisolation is significantly reduced after the initiation of an-tibiotic therapy. Some pathogens may not be difficult tocultivate but may require special media, growth conditions,

31. Broad-Range PCR for Bacterial Identification ■ 497

FIGURE 2 Logarithmic amplification plot of a broad-range bacterial 16S rRNA gene PCR inreal-time format. This amplification pattern has arisen in a study in which blood from healthysubjects was compared with negative controls (102). Samples were analyzed in triplicate and for40 cycles. The Cq value is derived from the number of cycles needed for the amplified DNA toreach the quantification cycle (threshold). �Rn, relative fluorescence. Reprinted from reference102, with permission from the American Society for Microbiology.

or lengthy incubation times; thus, they normally need tobe considered in order to become detected (45). Broad-range PCR offers two potential benefits: it lacks selectivityfor particular groups of bacteria, and it can detect as wellas identify culture-resistant, fastidious, damaged, and slow-growing microorganisms.

Since the inception of broad-range PCR in the late1980s, many clinical microbiology laboratories have imple-mented this technique for research and clinical applica-tions. In diagnostic settings, broad-range testing usuallytakes place in selected cases, with selected specimen types,on specific request by physicians, and by using in-houseassays (115). When implemented, broad-range PCR testingis usually done with samples from primary ‘‘sterile’’ sites,such as blood, cerebrospinal fluid, synovial fluid, and tissue.Clinical applications of broad-range PCR have been pub-lished in multiple case reports, small case series, and anumber of large prospective and retrospective evaluationstudies.

Published case reports include instances where the di-agnosis was missed because all standard tests using conven-tional diagnostic methods (culture, serology, and micros-copy) were unrevealing. Examples are the detection of B.quintana in culture-negative endocarditis (65), T. whippleiin spondylodiscitis (2) and endocarditis (51), Mycoplasmapneumoniae in osteomyelitis (78), and Granulicatella elegansin endocarditis (22).

A number of clinical scenarios and specimen types haveattracted particular interest and more thorough evaluationstudies for broad-range PCR. These include excised heartvalves in suspected bacterial endocarditis (16, 50, 89, 153),bloodstream infections in neonates or hematology-oncology patients (69, 80, 103, 110, 138), cerebrospinalfluid in suspected meningitis or shunt infections (32, 73,134, 158, 167), bone samples or synovial fluid samples inosteomyelitis or septic arthritis (40, 41, 127), vitreous or

aqueous fluid in intraocular infections (152), tissue or pusfrom brain or spinal abscesses (75, 115), amniotic fluid inchorioamnionitis or preterm labor (34, 58), and variousother scenarios.

In one of the earlier studies, Goldenberger et al. (50)applied broad-range PCR to resected heart valves in a se-ries of 18 endocarditis cases. Cultures of the valves werepositive in only 4 cases, blood cultures were positive in 15cases, and PCR was positive in 15 cases. Tentative bacterialidentification based on 16S rRNA sequences was possiblein 13 cases. Two PCR-positive cases were missed by cul-tures: DNA sequences of a Streptococcus sp. and of T. whip-plei were detected in these valves. Similar findings weremade in other studies of heart valve PCR: at the time ofoperation, most patients were treated with antibiotics andvalve cultures were often negative, and among the casesdetected by PCR but not by culture were pretreated casesand infections by fastidious bacteria (16, 89, 153).

Ley et al. (80) published findings of broad-range PCRdirectly from EDTA-anticoagulated blood samples from111 episodes of fever and neutropenia in cancer patientsand compared the results with those of conventional bloodcultures. Broad-range PCR was positive in 9 of 11 culture-positive episodes but also in 20 culture-negative episodes,18 of which occurred under antibiotic treatment. It wasconcluded that broad-range PCR can augment culture inpatients under antibiotic treatment. Another study (69)examined 1,233 blood samples from neonates being eval-uated for early-onset sepsis. Whole blood samples taken forcell count analysis were preincubated for 5 h in broth andexamined by PCR, and the results were compared to con-ventional blood cultures. Seven samples were concordantlypositive. PCR failed to detect 10 of 17 culture-positivesamples, some of which yielded skin organisms, and waspositive in 30 culture-negative cases. The authors pointedout that due to ethical reasons for study design, samples for

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PCR were often taken by heel pricks, which may haveintroduced skin organisms that were then multiplied bypreincubation. Bloodstream infections have also been ad-dressed by the design of a commercial kit, the SeptiFasttest (Roche). This kit is based on real-time PCR with dif-ferent sets of primers and probes for a limited range of 25gram-positive, gram-negative, and fungal organisms com-monly involved in bloodstream infections and requiresabout 6 h to obtain results (21).

Kotilainen et al. (73) examined 56 cerebrospinal fluidsamples from patients with suspected meningitis and foundthat PCR detected Neisseria meningitidis sequences in 5samples, 4 of which were also culture positive. One PCR-positive sample from a patient under antibiotic treatmentwas negative by microscopy and culture, but autopsy find-ings were consistent with meningococcal disease. One ad-ditional case of culture-positive Listeria monocytogenes men-ingitis was missed by broad-range PCR. Similar findingswere made in larger studies evaluating broad-range PCRfor the diagnosis of meningitis: PCR failed in some casesthat were positive by culture but was often successful whenpatients were pretreated with antibiotics (134, 158).

A large study of 525 bone and joint samples, comprisingtissues and fluid aspirates, was published by Fenollar et al.(41). Concordantly negative results between culture andPCR were obtained in 386 cases, concordantly positive re-sults in 89 cases, and discrepant results in 50 cases. Cultureand PCR each had similar rates of false-positive and false-negative results. Among 21 culture-negative, PCR-positivecases were 7 cases with antibiotic treatment and 2 caseswith fastidious organisms. Seven cases revealed mixed se-quences by PCR; some of the sequences in these cases be-longed to established bone and joint pathogens, others toenvironmental organisms rarely involved in bone and jointinfections.

A large study from Finland (115) applied broad-rangePCR to 536 diverse clinical samples from 459 hospitalizedpatients and compared PCR with culture. Samples con-sisted of body fluids and tissues and were collected over a4-year period in a routine diagnostic setting. Results of cul-ture and PCR were concordant in 447 (83%) specimens.PCR-positive but culture-negative results occurred in 19samples, which included specimens taken during antibiotictreatment or infections caused by bacteria with unusualgrowth requirements. For 11 patients in the study, broad-range PCR was the only method that provided evidence ofthe etiologic agent of the infection, and for 10 of thesepatients, the agent identified by sequencing was consideredsignificant in the clinical context. The tendency of broad-range PCR to be positive after the initiation of antibiotictherapy, as opposed to culture, was statistically significant.

A large, population-based study of unexplained deathsand critical illnesses due to possible infectious causes wasinitiated by the Centers for Disease Control and Preven-tion (CDC) and conducted in four states of the UnitedStates (52, 101). Included were cases in which all conven-tional diagnostic methods had yielded negative results, al-though the patients demonstrated clinical signs and symp-toms suggestive of infection. A total of 137 cases wereidentified, and broad-range PCR was performed in 46 cases.The PCR results were positive for eight cases, and bacterialsequences (e.g., N. meningitidis and Streptococcus pneumon-iae) recovered from six patients were consistent with theclinical symptoms. Seven of the eight patients had beenpretreated with antibiotics.

Bacterial broad-range PCR has also attracted interest intransfusion medicine, in order to assess blood products forbacterial contamination (35, 36, 97, 126). Platelet concen-trates are of particular concern, because they require stor-age and agitation at room temperature, thus making themprone to bacterial growth from very small inocula. Culturein blood culture bottles requires several days, and slow-growing bacteria such as Propionibacterium spp. may require4 to 5 days, so that cultures may become positive at a timewhen platelets have been transfused. Several broad-rangereal-time PCR assays have been designed and evaluated forthat purpose (35, 36, 97, 126). One study evaluated 2,146platelet concentrate samples in parallel with culture andPCR (97). The test had a turnaround time of 4 h andshowed concordant results between the two methods, thusshowing the feasibility of PCR testing for platelet concen-trates.

ANALYSIS OF POLYMICROBIAL INFECTIONSAND MICROBIAL COMMUNITIESBroad-range PCR can also be applied to mixed infectionsor to determine the composition of complex polymicrobialcommunities, such as in samples from the environment orsamples containing microbial flora of humans and animals.This type of analysis has originally been established in en-vironmental microbiology (48). Such studies as well as insitu hybridization experiments (4) have provided estimatesthat �99% of microorganisms in environmental commu-nities are not detectable by standard culture techniques.PCR-based community studies usually involve the con-struction and phylogenetic analysis of 16S rRNA geneclone libraries and commonly reveal many sequences cor-responding to new species (‘‘phylotypes’’ or ‘‘operationaltaxonomic units’’) that are not detected by culture.

Analysis of human microbial flora has been performedon specimens such as feces (141, 161) and subgingivalplaque material (74, 108). These studies revealed a consid-erable fraction (40 to 75%) of sequences that did not be-long to known bacterial species. The diversity of sequenceswas greater for PCR-amplified clone libraries than for bac-terial colonies from cultures that were set up in parallel(74, 161), but larger numbers of cycles (35 cycles versus 9)in the initial PCRs reduced diversity, probably due to pref-erential amplification (161). Based on clone library data,it was estimated that the number of different bacterial spe-cies in subgingival plaque communities exceeds 400 (108).Another study, combining broad-range PCR with bothclone libraries and microarray analysis, examined the stoolflora development of 14 infants during the first year of life(107). Each of the babies had and retained a distinct com-munity profile, and by the end of the first year, the profileshad diversified into patterns that were similar to those ofthe adult gastrointestinal tract.

Other studies have focused on the association betweenhuman microbial flora disturbances and conditions of im-paired health. One study examined samples from womenwith bacterial vaginosis and women with normal vaginalflora by broad-range PCR followed by clone libraries, aswell as by in situ hybridization (43). Bacterial vaginosissamples contained much greater microbial diversity, in-cluding 16 presumptive new species, among which threespecies were very strongly associated with bacterial vagi-nosis and were only distantly related to any other knownbacteria. Another set of interesting findings was reportedby Ley et al. (81). The authors examined the stool flora of

31. Broad-Range PCR for Bacterial Identification ■ 499

obese and lean people, as well as obese people during thecourse of diet. For the two dominant phyla in stool, theFirmicutes and the Bacteroidetes, they found that the num-ber of Bacteroidetes was smaller in obese people than in leanpeople and that obese people under diet underwent a shifttowards fewer Firmicutes and more Bacteroidetes, approach-ing the ratios of lean people. It was pointed out that thesechanges constitute a major shift of bacterial phyla that isassociated with health disturbance.

Broad-range PCR has also been applied to polymicrobialinfections, such as destructive periodontitis (25), prostatitis(145), dental root canal infections (125), and destructivekeratitis (131). These studies also revealed the unexpecteddiversity of bacteria associated with polymicrobial infec-tions, as well as sequences corresponding to unknown spe-cies.

UNANSWERED QUESTIONSInteresting questions were raised in a study by Nikkari etal. (102). By use of a real-time broad-range PCR assay,blood samples from healthy volunteers were compared withnegative controls (i.e., water treated as for sample prepa-ration), human tissue culture cells, and known amounts ofextracted bacterial DNA (Fig. 2). Negative controls con-sistently gave rise to amplification products, reaching thequantification cycle around cycle 38. The quantificationcycles of healthy blood samples were four to six cycles ear-lier, indicating a higher load of bacterial DNA. Clone li-braries were prepared from both a negative control and ablood sample and revealed sequences that belonged to phy-logenetic groups similar to those of sequences previouslyreported from negative controls (144), but blood samplesyielded additional sequences from phylogenetic groups thatwere not represented in negative controls. The origin andsignificance of the blood-derived sequences remained un-clear, but the possibility was raised that supposedly sterilebody compartments may harbor a ‘‘normal’’ burden of bac-terial DNA. This appears consistent with previous findingsof culture-positive bacteremia after toothbrushing (12) andwith pneumococcal DNA found in the blood of healthychildren (29).

Apparently similar findings were made by another groupafter the incidental observation of pleomorphic bacteria bydark-field microscopy in the blood of one healthy subject(90). The findings were confirmed by in situ hybridizationand broad-range PCR in several more healthy subjects; 16SrRNA gene and gyrB gene sequences were similar but notidentical to those of Stenotrophomonas maltophilia, but cul-tures on standard media remained negative. The signifi-cance of these findings remained unclear. Interesting ob-servations were also made in two other studies, examiningcoronary arteries after death from various causes (79) andatherosclerotic lesions from abdominal aortas (121) bybroad-range PCR. In the second study (121), the authorstook particular care to avoid background DNA from rea-gents by cloning and subtracting sequences obtained afterextensive cycling of negative controls, but despite this, se-quences from typical oral commensals and low-level patho-gens were detected in both studies. The significanceremained unclear, but the authors speculated that com-mensals may become embedded in vascular tissues duringtransient bacteremias. Again, these studies point towardsthe possibility that human blood and tissue may have anormal background of bacterial DNA, and this may have

enormous implications on the analysis of samples from sup-posedly sterile sites by broad-range PCR.

Among the clinical evaluation studies of broad-rangePCR, there were some that reported largely consistent re-sults between PCR and culture, with moderate numbers ofsamples positive by PCR but negative by culture, and viceversa (50, 73, 89, 115, 134). Others reported a greater pro-portion of samples that were positive by PCR and negativeby culture (54, 80, 138, 167). As pointed out by Peters etal. (110), the interpretation of such studies is difficult, withculture being the reference. A positive PCR may be theresult of greater sensitivity of PCR, or it may be a false-positive result. It may be the result of true pathogen DNAor of presumed normal background DNA in human bloodor tissue, or it may be due to bacterial DNA in reagents.Interpretation in the context of clinical findings and otherlaboratory results is necessary. This is feasible in thoroughscientific evaluation studies, but it may not be feasible ina routine diagnostic setting.

CONCLUSIONSConducting broad-range PCR analysis at a level of highanalytical and clinical sensitivity is a complex task andremains one of the most difficult and challenging PCR ap-plications. Implementation of broad-range PCR in a diag-nostic setting requires appropriate laboratory facilities andstaff, familiarity with the theoretical underpinnings andpractical aspects, extensive precautions to avoid contami-nation, and thorough in-house evaluations of analytical aswell as clinical sensitivity and specificity. The presence ofubiquitous bacterial background DNA and the problemsassociated with its elimination suggest that dealing with acertain, low level of interfering DNA will be unavoidable,at least in the foreseeable future. Some of the decontami-nation methods in Table 2 describe the reduction of con-taminating DNA to levels of about 10 copies of rRNA geneper PCR test, which means that the amount of real targetDNA that can be distinguished will have to be significantlyabove that level, and broad-range PCRs will not achievethe same level of sensitivity as organism-specific PCRs.

Careful implementation of positive and negative con-trols is necessary. The presence of bacterial DNA in PCRmixes, in sample preparation reagents, and presumably insome normal human tissues mandates that negative con-trols should include DNA-free controls in the PCR mix,sample preparation controls, and control samples from pa-tients without the disease in question, in order to detectand be aware of the levels of background DNA in all ofthese settings. Positive controls must contain serial dilu-tions of known copy numbers of bacterial DNA or bacterialcells, both in the PCR mix and spiked into negative sam-ples, to determine the threshold of detectable bacterialDNA that is clearly above the background level.

Sequence-based identification of positive diagnosticbroad-range PCR products is generally advisable, evenwhen real-time technology is used. Some authors have pro-posed using a second, independent, broad-range primer sys-tem in order to confirm results (73, 112, 115). Additionalcontrols may include PCRs using a human gene (e.g., thebeta-globin gene) in order to assess the presence of am-plifiable DNA in a sample or inhibition of PCR. SomePCR tests use internal, coamplified DNA controls (126).

In addition to the specific measures to eliminate back-ground DNA (Table 2), a wide range of general anticon-tamination precautions is necessary, including separate

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rooms for pre- and post-PCR work, UV decontaminationof surface areas, use of high-quality reagents, and adequatesampling techniques and vials for clinical specimens (95).It is important to understand that the traditional conceptsof sterile laboratory techniques from culture-based micro-biology do not apply in the same way in the setting ofmolecular tests, as shown by experiments in which exten-sive autoclaving failed to eliminate amplifiable DNA (87).

Clinical studies have shown a number of situations inwhich bacterial broad-range PCR can provide considerablebenefits. Clear benefits exist when bacteria are seen by mi-croscopy but fail to be recovered in culture, when infec-tions may be caused by fastidious or slow-growing organ-isms, and when patients have been pretreated withantibiotics. The examination of resected heart valves ex-emplifies these benefits very well: endocarditis can becaused by various organisms, including fastidious ones,valve resection is a highly invasive sampling procedure thatwarrants considerable diagnostic efforts, the examinationby blood cultures may fail to detect the pathogen, and pa-tients have often been pretreated with antibiotics, so thatheart valves at the time of operation are often culture-negative (50). However, diagnostic broad-range PCR, withfull amplicon sequencing, is expensive and time-consumingand requires appropriate laboratory facilities (115).

The primary diagnostic utility of broad-range PCR liesin the testing of specimens from primary sterile sites, es-pecially if they are difficult to obtain, and in clinical situ-ations in which other methods are known to pose difficul-ties. The analysis of polymicrobial infections and complexcommunities becomes feasible with the aid of clone librar-ies and the emerging techniques of pyrosequencing andhigh-density microarrays, but it still requires considerablymore effort and resources and is currently done almost ex-clusively in research laboratory settings.

Other emerging technologies may take the current placeof broad-range PCR as a nonprescient tool for the exami-nation of unknown infections. This has been exemplifiedby the discovery of a novel transplant-associated arenavirusthat caused fatal infection in three organ recipients (106).The virus was discovered by random amplification of RNAtranscripts from infected organs, followed by unbiased high-throughput pyrosequencing. The resulting sequences werethen subjected to bioinformatic subtraction of human se-quences, and the novel arenavirus sequence was found.This was subsequently confirmed by virus culture, specificPCRs, and antibody testing.

I thank David A. Relman, Paul Eckburg, and Elisabeth M. Bik(Stanford University, Palo Alto, CA) for providing helpful commentson this chapter in the earlier edition; Linda L. Blackall and Tim Beck-enham (University of Queensland, Brisbane St Lucia, QLD, Austra-lia) for help with selecting primers for Table 1; and Frank Oliver Glock-ner and Elmar Pruesse (Max Planck Institute for Marine Microbiology,Bremen, Germany) for providing Fig. 1B.

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