polymerase chain reaction detection of bacterial 16s rrna gene in human blood

Microbiol Immunol 2008; 52: 375–382doi:10.1111/j.1348-0421.2008.00048.x

NOTE

Polymerase chain reaction detection of bacterial 16S rRNAgene in human bloodKosei Moriyama1, Chie Ando2, Kosuke Tashiro3, Satoru Kuhara3,4, Seiichi Okamura5, Shuji Nakano1,Yasumitsu Takagi6, Takeyoshi Miki6, Yoshiyuki Nakashima2 and Hideki Hirakawa4

1Nakamura Gakuen University, Faculty of Nutritional Sciences, 2Department of Medicine and 6Frontier Research Center, Fukuoka Dental College,3Graduate School of Genetic Resources Technology and 4Graduate School of Systems Life Sciences, Kyushu University and 5Department of Medicine,National Kyushu Medical Center, Fukuoka, Japan

ABSTRACTBacterial 16S ribosomal RNA genes (rDNA) were detected in blood samples from two healthy individualsby PCR under conditions involving 30 cycles that did not produce any visible products from negativecontrol saline. Even from control samples, PCR involving 35–40 cycles yielded visible bands. Major clonesdetected in the blood samples, but not in control, were the Aquabacterium subgroup, Stenotrophomonassubgroup, Budvicia subgroup, Serratia subgroup, Bacillus subgroup and Flavobacteria subgroup. Noclone was located within the bacteroides-clostridium-lactobacillus cluster, which is indigenous to gas-trointestinal flora.

Key words bacteremia, blood, polymerase chain reaction, 16S ribosomal RNA.

Diagnosis of bacterial infection and identification of theagent responsible is essential in clinical medicine, and hasbeen traditionally carried out by inoculating blood or in-fected tissues into a liquid or solid nutrient medium. Thisapproach has a limitation in that it can detect only bacte-ria and fungi that are culturable in a laboratory. Recently,PCR using species-specific primers (1–5) and nucleic acidsequence-based amplification (NASBA) (6) have also beenused as an alternative approach for detecting agents thatare responsible for infection.

These techniques can also be used for detailed analysis ofmicrobial biota in the oral cavity and gastrointestinal tract,using universal primers that anneal to conserved regions inthe 16S ribosomal RNA gene (rDNA) or gyr B gene (7–9).It is reported that half of the bacteria comprising the oraland intestinal flora have not been previously identified byin vitro culture procedures (10). Moreover, recent studiesusing PCR have raised the possibility that bacterial DNAmay be present in the human bloodstream (11–13).

CorrespondenceYoshiyuki Nakashima, Department of Medicine, Fukuoka Dental College, Tamura 2–15-1, Sawara-ku, Fukuoka 814-0193, Japan.Tel: +81 92 801 0411; fax: +81 92 865 2484; email: [email protected]

Received 29 August 2007; revised 26 March 2008; accepted 17 April 2008

List of Abbreviations: EDTA, ethylenediaminetetraacetic acid; PCR, polymerase chain reaction.

Our ultimate objective is to elucidate the role of suchsubclinically infecting unculturable or latent bacteria inthe pathogenesis of chronic vascular diseases (14–16). As afirst step, we investigated whether bacteria can translocatein some way from the oral and intestinal flora to the bloodstream in ‘healthy’ humans. In this preliminary study,blood specimen-specific bacterial sequences were detectedby PCR of rDNA. However, they were not representativeof sequences found in human intestine.

PCR was done with a REDExtract-N-Amp Blood PCRkit (Sigma-Aldrich Japan, Tokyo, Japan) using broad-range 16S ribosomal RNA gene (rDNA)-specific oligonu-cleotide primers. The PCR kit does not require any type ofpurification, organic extraction, centrifugation or alcoholprecipitation, and can be used with whole blood.

For blood sampling, the skin was first sterilized witha cotton swab moistened with popidone iodide, the an-terior brachial median vein was punctured using a ster-ile 21-gauge needle (Terumo Corporation, Tokyo, Japan),

c© 2008 The Societies and Blackwell Publishing Asia Pty Ltd 375

K. Moriyama et al.

and the blood was aspirated into an attached sterile glasstube under vacuum containing EDTA. For control sam-ples, the skin was sterilized as described above, and wipedwith an autoclaved (121 ◦C, 20 min, three times) cottonswab moistened with autoclaved saline. The saline in theswab was then squeezed directly into the EDTA-containingtube, and used as a negative control for PCR. Appendedneutralized dilution buffer with autoclaved saline was usedas another negative control.

This study was conducted in accordance with the rulesof the internal review board and the tenets of the Dec-laration of Helsinki. The Ethics Committee of FukuokaDental College, where blood sampling and laboratory ma-nipulations were carried out, approved the study, andboth of the study subjects gave informed consent toparticipate.

The forward primer was JRP-1: 5′ CTCCTACGGGAG-GCAGCAG and the reverse primer JRP-3: 5′ ACATGCTC-CACCGCTTGTG. The PCR primers were designed fromthe conserved regions of rRNA sequences of oral and in-testinal bacteria deposited in GenBank. At the presenttime, JRP-1 is completely (100%) identical to 339 766 of471 792 sequences deposited in the Ribosomal DatabaseProject II (17). Also, JRP-3 is completely identical to131 594 of 471 792 deposited sequences. Taking into ac-count one- or two-base mismatch annealing in PCR, thisprimer pair is capable of amplifying more than half of thedeposited sequences including Bacteroides and Clostrid-ium subgroups.

Either fresh whole blood or saline (10 μL; negativecontrol) was mixed with 20 μL lysis buffer, and further di-luted with 180 μL neutralization buffer. Neutralized sam-ple (2 μL) was added to 18 μL PCR mixture, and 30(or more as indicated in Results) cycles of PCR were car-ried out using TaKaRa PCR Thermal Cycler PERSONAL(TaKaRa Inc., Shiga, Japan). One cycle consisted of 1 minat 94 ◦C for denaturation, 30 s at 58 ◦C for annealing and40 s at 72 ◦C for extension. Amplicons were resolved byagarose gel electrophoresis and detected by ethidium bro-mide staining.

DNA (600–650 bp) was excised from the gel, and clonedinto the pGEM-T vector (Promega KK, Tokyo, Japan). Es-cherichia coli JM109 was transformed with the plasmidconstructs, and plated on Luria broth (LB) agar. For eachsubject, 96 white colonies were randomly detected, cul-tured in 1.5 mL LB broth, and the plasmids were extracted.The plasmids were digested with restriction enzymes PstIand EcoRI, electrophoresed in agarose gel, and the plas-mids carrying an approximately 620-bp fragment weresequenced.

Nucleotide sequence analysis was carried out usingan ABI automated DNA sequencer (model 373A) andan ABI Prism cycle sequencing kit (Perkin-Elmer Japan,

Tokyo, Japan). To confirm that the nucleotide sequenceswere 16S rDNA, homologous searches were performedagainst the non-redundant (nr) protein database in theNational Center for Biotechnology Information (NCBI)(http://www.ncbi.nlm.nih.gov/) using BLASTX (18). Se-quences that were not homologous with any proteinswere searched against 471 792 clones of 16S rDNA se-quences deposited in the Ribosomal Database Project II(RDP) (http://rdp.cme.msu.edu/) (17). For each rDNAsequence, the nearest genus and species of the type strainwas searched in the RDP, and best-hit names and se-quences were retrieved and shown as a phylogenetic tree.The alignment of the 16S rDNA sequences and the sam-pled sequences was performed using CLUSTAL W (19), andthe unrooted neighbor-joining tree was constructed usingNJPLOT (20). In phylogenic analysis, the distance betweentwo given sequences was expressed as the number of dif-ferences per 100 bases. Bacterial groups with a distanceof more than 0.02 (i.e. 12 mutations in 600 bases) weredefined as different.

To test whether rDNA of oral and intestinal bacteriais detectable in human blood, we designed PCR primersto amplify 16S ribosomal RNA genes. The amounts ofPCR products in blood samples from two healthy indi-viduals were higher than those in control samples. Using30 thermal cycles, all PCR products from blood samplesgave bands of the expected size (approximately 620 bp) byagarose gel electrophoresis and ethidium bromide stain-ing, whereas those from the negative control gave novisible bands (Fig. 1). Attempts were made to amplifyany rDNA in the negative control samples, and it was

Fig. 1. Agarose gel electrophoresis and ethidium bromide staining ofPCR products. (a) Products of 30-cycle reaction. A, whole blood of subjectA; B, whole blood of subject B; S1, saline in cotton swab used to wipethe skin of subject A; S2, saline in cotton swab used to wipe the skinof subject B. (b) Products of 40-cycle reaction. S1, saline in cotton swabused to wipe the skin of subject A; S2, saline in cotton swab used towipe the skin of subject B; C, PCR with neutralized dilution buffer withautoclaved saline.

376 c© 2008 The Societies and Blackwell Publishing Asia Pty Ltd

Bacterial rRNA gene in human blood

Table 1 PCR cycles and numbers of clones sequenced

No. colonies detected No. extracted plasmids carrying a 600–650 bpPCR cycle for small scale culture fragment for nucleotide sequence analysis No. clones identified as bacterial rRNA genes

Subject A 30 96 24 23Subject B 30 96 31 31Control† 40 96 96 86

†Control PCR (40-cycle) with intensively autoclaved saline (C in Fig. 1b).

found that 35 to 40 cycles yielded visible bands with sev-eral extra bands and smears by agarose gel electrophore-sis; amplicons obtained by 40-cycle PCR are shown inFigure 1b.

The 30-cycle PCR products from subjects A and B (Aand B in Fig. 1a), and 40-cycle PCR products from controlsaline (C in Fig. 1b) were further analyzed. The numbers ofbacterial 16S rDNAs analyzed were 23 clones from subjectA, 31 clones from subject B, and 86 clones from controlsaline (Table 1) (Appendix I).

As shown in the phylogenetic tree, members of variousbacterial groups were detected in the blood specimen-associated clone libraries (Fig. 2). Among the controlreagent-associated sequences, various subgroups in α-,β- and γ-proteobacteria were identified. Clone sequencesthat were detected in either of two human blood sam-ples, but not in the control sample, were the Aquabac-terium subgroup, Stenotrophomonas subgroup, Budvi-cia subgroup, Serratia subgroup, Bacillus subgroup andFlavobacterium subgroup. None of these sequences wasclassified as the same group as the representative sequencesfound in human intestine by Hayashi et al. (10).

PCR using 30 thermal cycles yielded positive bandsby agarose gel electrophoresis from all blood samples,whereas negative controls yielded no visible bands. How-ever, 35 to 40 cycles of PCR for the negative control salinesample gave visible rDNA bands. We used a PCR kit thatworks with raw blood specimens and requires no ma-nipulation for DNA extraction. However, rDNA can beintroduced at any stage of processing during the manu-facture of PCR enzymes and buffers, or by experimen-tal manipulations such as pipeting and opening of testtubes. The majority of the rDNA sequences in the librariesfrom the negative control samples were similar to thoseof Acinetobacter haemolyticus, Variovorax parodoxus andPseudomonas fluorescens, which are all common water-and soil-associated organisms, and have been reportedto be major contaminants of Taq polymerase and otherreagents for PCR (21–25).

Venipuncture provides an opportunity for introducingexogenous bacterial genomes into the bloodstream frombacteria resident on the skin surface. The outer diameter ofa 21-gauge needle is 0.80 mm, and therefore the punctured

area of skin is calculated to be 5 × 105 μm2. As thediameter of a bacterium is approximately 2 μm, roughly1 × 105 bacteria can theoretically adhere as a monolayerwithin this area.

Another explanation for the positive results obtainedfrom blood samples with 30 PCR cycles is that the bloodof healthy individuals contains a bacterial component.Based on the number of thermal cycles (35 cycles minus30 cycles), the copy number of rDNA in blood samples isthought to be approximately 25-fold higher than that incontrol saline.

A positive result in any PCR tube means that the tubecontains at least one copy of rDNA. Therefore, 10 μL ofstarting material (raw blood) contains at least one copy ofrDNA (100 copy/mL). Given that one bacterial cell pos-sesses 10 duplicated rDNA genes, PCR-positive blood has10 bacterial cells per 1 mL. As the one PCR used 2 μLof 105-fold diluted blood, the actual number of bacteriawould be greater. With such a degree of bacteremia, any an-imal would be critically ill. Therefore, if healthy individualsharbor a bacterial component in their blood, the rDNAmust originate from destroyed (i.e. non-viable) bacteria.Bacteria may be destroyed by physical and immunologicalbarriers, and the released rDNA may be translocated intothe bloodstream. This bacterial rDNA would be presentfreely in plasma and/or trapped in granulocytes.

The rDNA sequences found in the blood specimenswere those of Stenotrophomonas, Budvicia, Serratia, Bacil-lus, Flavobacterium and Aquabacterium subgroups. Theblood-associated rDNA sequences were not assigned toany of the major bacterial phylotypes indigenous to theperiodontal flora (7, 26–28). Furthermore, there was norDNA sequence identical to those of bacterial families in-digenous to the jejunal and ileal flora or fecal flora detectedby Hayashi et al. (10), such as Streptococcus, Lactobacil-lus, Enterococcus, Bacteroides and Clostridium subgroups(Fig. 2).

By whole-genome shotgun sequencing, Kurokawa et al.(29) reported that Bacteroides and Clostridium are ma-jor subgroups comprising the fecal flora of adult hu-mans. These taxonomic names were not found in ourblood specimen-associated subgroups. Only the Bacillussubgroup, the third population in the gut in Kurokawa’s


K. Moriyama et al.

NR_003286; Homo sapiens 18S rDNA A-3-2 S01_E02 S01_F11 S01_G09 S01_B02 A-1-4_A07 S01_H07 S01_H09 S01_F08 S01_E07 S01_C12 S01_H10 S01_D02 S01_G12 S01_B10 S01_G03 S01_C10 S01_B05 S000435815; Variovorax paradoxus IAM 12373 [β-Proteobacteria] S000619996; Variovorax paradoxus 4.2 [β-Proteobacteria] S000627894; Variovorax ginsengisoli Gsoil 3165 [β-Proteobacteria] S01_D08 S000332064; Rhodoferax antarcticus Fryx1 [β-Proteobacteria] S000804271; Acidovorax delafieldii HYY0511-SK09 [β-Proteobacteria] S01_H05 S000805102; Acidovorax avenae subsp. avenae [β-Proteobacteria] S01_A10 S01_C05 S000015591; Curvibacter lanceolatus ATCC 14669T (T) [β-Proteobacteria] S01_G08 S000428512; Curvibacter delicatus LMG 4328 (T) [β-Proteobacteria] A-1-14_G09 S000629316; Comamonas testosteroni DSM 6781 [β-Proteobacteria] S000387156; Hydrogenophaga palleronii DSM 63 (T) [β-Proteobacteria] S01_C08 S01_C07 S01_F03 A-1-9_E09 S000427920; Aquabacterium parvum B6 (T) [β-Proteobacteria] A-3-4 S000427918; Aquabacterium citratiphilum B4 (T) [β-Proteobacteria] N-2-37_H12 A-1-19_H09 A-1-1_B09 S000008887; Janthinobacterium agaricidamnosum W1r3T (T) [β-Proteobacteria] S000125062; Antarctic bacterium R-7614 S000805623; Janthinobacterium lividum H196 [β-Proteobacteria] A-3-1 A-3-10 N-1-15_D12 N-3-9 S01_A05 S01_G07 S01_E03 S01_F07 S01_D06 S01_C11 A-1-7_C09 A-3-6 A-1-8_D09 A-3-5 S000438965; Janthinobacterium agaricidamnosum SAFR-022 [β-Proteobacteria] N-1-23_G07 N-3-11 N-2-35_F12 S000824200; Herminiimonas oxalatica NS11 (T) [β-Proteobacteria] S01_A06 S01_E09 S01_B03 S01_H01 S01_G05 S000425945; Burkholderia cepacia ATCC 55487 [β-Proteobacteria] S01_E12 S01_C06 A-1-9_B07 S000824097; Stenotrophomonas maltophilia NCB0306-284 [γ-Proteobacteria] A-3-12 A-3-3 A-3-7 N-2-47_A09 N-3-6 N-3-12 S000769255; Xanthomonas axonopodis IP1-36 [γ-Proteobacteria] S01_A01 S01_G10 S01_C04 S01_H04 A-2-25_E07 A-3-11 A-3-9 S000776605; Acinetobacter junii 2R11 [γ-Proteobacteria] S01_C03 S01_F05 N-2-31_E12 N-1-1_B12 S01_E04 S01_D09 S01_F02 S01_B04 S01_H06 S01_A09 S01_H03 S01_F01 S01_B11 S01_D12 S01_G01 S01_H12 S01_B08 S01_D04 S01_A03 S01_A08 S01_B01 S01_F12 S01_B12 N-1-15_F07 N-3-1 S01_B07 S01_D05 S01_E10 N-3-10 S000434893; Acinetobacter haemolyticus AY047216 [γ-Proteobacteria] N-2-42_G08 N-3-7 S01_G02 S000559117; Pseudomonas fluorescens S16 [γ-Proteobacteria] S01_E08 S01_D10 S01_E01 S01_E11 S000824936; Pseudomonas fluorescens 29L [γ-Proteobacteria] N-3-4 N-2-38_F08 S000020702; Budvicia aquatica DSM 5075 (T) [γ-Proteobacteria] N-2-26_A08 N-3-3 N-2-34_E08 S000736666; Serratia grimesii DQ991163 [γ-Proteobacteria] N-1-2_C12 N-3-2 N-2-33_D08 N-3-8 S000381739; Enterobacter asburiae JCM6051 (T) [γ-Proteobacteria] S000436668; Aggregatibacter actinomycetemcomitans (T) [γ-Proteobacteria] S000391702; Aminobacter aminovorans Ep2-1c [α-Proteobacteria] N-2-45_H08 N-2-28_B08 N-3-5 S01_H02 S000721041; Rhizobium radiobacter MAFF 03-01224 [α-Proteobacteria] S01_F04 S000825175; Rhizobium leguminosarum BIHB 1157 [α-Proteobacteria] S01_G11 S000805621; Brevundimonas nasdae G124 [α-Proteobacteria] S01_D01 S01_H08 S01_A11 S01_D03 S000824151; Caulobacter vibrioides CB15 [α-Proteobacteria] S01_F06 S01_A07 S01_C01 A-1-21_C07 S000825335; Bacillus cereus [Firmicutes] N-2-25_H07 A-3-8 A-1-23_D07 S000375664; Enterococcus faecalis SL5 [Firmicutes] S000389017; Sporosarcina ureae DSM 2281 (T) [Firmicutes] S000128223; Streptococcus salivarius NCDO 1779 (T) [Firmicutes] S000134021; Streptococcus mutans NCTC 10449 (T) [Firmicutes] S000436427; Lactococcus lactis cremoris (T) [Firmicutes] S000414529; Lactobacillus reuteri (T) [Firmicutes] S000414606; Eubacterium cylindroides ATCC 27803 (T) [Fir micutes] S000001177; Ruminococcus gnavus ATCC 29149 (T) [Firmicutes] S000012710; Clostridium xylanolyticum ATCC 4963 (T) [Fir micutes] S000414600; Eubacterium ventriosum (T) [Firmicutes] S000436477; Ruminococcus hansenii (T) [Firmicutes] S000011936; Clostridium polysaccharolyticum DSM 1801 (T) [Firmicutes] S000437588; Butyrivibrio fibrisolvens ATCC 19171 (T) [Firmicutes] S000414263; Peptostreptococcus anaerobius ATCC 27337 (T) [Fir micutes] S000013701; Phascolarctobacterium faecium (T) [Firmicutes] S01_C02 S01_D07 N-2-29_C08 S000381050; Bifidobacterium bifidum (T) [Actinobacteria] S000484536; Rothia amarae J18 (T) [Actinobacteria] S000617880; Propionibacterium acnes mother C4 [Actinobacteria] S01_G06 S01_A12 S01_E06 S000390110; Ferrimicrobium acidiphilum T23 [Actinobacteria] S01_F09 S000006150; Desulfovibrio desulfuricans ATCC 29577 (T) [δ-Proteobacteria] S000010833; Malonomonas rubra GraMal1 (T) [δ-Proteobacteria] S000015699; Desulfuromusa kysingii Kysw2 (T) [δ-Proteobacteria] S000006649; Porphyromonas gingivalis DSM 20709 (T) [Bacteroidetes] S000414478; Tannerella forsythensis 338 (T) [Bacteroidetes] S000528932; Bacteroides fragilis NCTC 9343 [Bacteroidetes] S000135891; Flavobacteria symbiont 1 of Acromyrmex otcospinosus [Bacteroidetes] N-2-36_G12 S000436460; Clostridium leptum (T) [Firmicutes]

0.000.040.080.120.160.200.240.280.32

Fig. 2. Phylogenetic relationships inferred from bacterial 16S rDNA se-quences detected in blood specimens from two healthy individuals andfrom control clone library sequences. The sequences associated with theblood specimens (30-cycle PCR) are shown in blue (subject A) and red(subject B) (Fig. 1a), respectively, and those generated in a control (40-cycle) PCR (C in Fig. 1b) as a template are shown in green. Referencesequences showing closest similarity to the study sequences are indi-cated in black. Representative sequences found in human intestine byHayashi et al. (10) are indicated in brown. The distance between twogiven sequences was expressed as the number of differences per 100bases.

study, was included among our blood-associated taxo-nomic groups (Appendix II). Therefore, it is unlikely thatthe gastrointestinal tract, the largest reservoir of bacte-rial flora, is the main source of blood rDNA in ‘healthy’humans. If the rDNA (or bacteria) translocates from theintestinal lumen to the circulatory system, there must bestrong selective pressures for bacterial species.

The Stenotrophomonas, Budvicia, Serratia, Bacillus,Flavobacterium and Aquabacterium subgroups are soil-and water-associated bacteria. The former four sub-groups, Stenotrophomonas, Budvicia, Serratia and Bacil-lus, can be opportunistic pathogens. By using rDNA-specific PCR, Nikkari et al. have shown a possibilitythat the Stenotrophomonas, Bacillus and Riemerella sub-groups are blood sample specific (13). In our study, theStenotrophomonas and Bacillus subgroups were also de-tected as blood sample specific. This supports Nikkariet al.’s speculation that there might be a ‘normal’ popu-lation of bacterial DNA sequences in the circulatory sys-tem of ‘healthy’ humans (13). Another possibility is thatthese airborne soil bacteria are constantly aspirated, be-ing continuously destroyed in the surface mucosa of therespiratory system, and that the released rDNA may enterthe bloodstream directly or be carried by phagocytes. Thismay also explain why some of our blood-associated rDNAsequences differed from those found in studies reportedpreviously from geographically different laboratories (11–13).

ACKNOWLEDGMENT

We thank Professor Mutsuo Sekiguchi, Fukuoka DentalCollege, for pertinent advice. This work was supported bya grant-in-aid from the Ministry of Education, Science,Sports and Culture of Japan (Frontier Research Grant).

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APPENDIX I

DDBJ accession numbers

All new rDNA sequences described in the presentstudy have been registered in the DNA Data Bank ofJapan (DDBJ) as numbers AB331781 ∼ AB331834 andAB374430 ∼ AB374515.

(Control sample)

S01 A01 AB374430S01 A03 AB374431S01 A05 AB374432S01 A06 AB374433S01 A07 AB374434S01 A08 AB374435S01 A09 AB374436S01 A10 AB374437S01 A11 AB374438S01 A12 AB374439S01 B01 AB374440S01 B02 AB374441S01 B03 AB374442S01 B04 AB374443S01 B05 AB374444S01 B07 AB374445S01 B08 AB374446S01 B10 AB374447S01 B11 AB374448S01 B12 AB374449S01 C01 AB374450S01 C02 AB374451S01 C03 AB374452S01 C04 AB374453S01 C05 AB374454S01 C06 AB374455S01 C07 AB374456


K. Moriyama et al.

Continued.

(Control sample)

S01 C08 AB374457S01 C10 AB374458S01 C11 AB374459S01 C12 AB374460S01 D01 AB374461S01 D02 AB374462S01 D03 AB374463S01 D04 AB374464S01 D05 AB374465S01 D06 AB374466S01 D07 AB374467S01 D08 AB374468S01 D09 AB374469S01 D10 AB374470S01 D12 AB374471S01 E01 AB374472S01 E02 AB374473S01 E03 AB374474S01 E04 AB374475S01 E06 AB374476S01 E07 AB374477S01 E08 AB374478S01 E09 AB374479S01 E10 AB374480S01 E11 AB374481S01 E12 AB374482S01 F01 AB374483S01 F02 AB374484S01 F03 AB374485S01 F04 AB374486S01 F05 AB374487S01 F06 AB374488S01 F07 AB374489S01 F08 AB374490S01 F09 AB374491S01 F11 AB374492S01 F12 AB374493S01 G01 AB374494S01 G02 AB374495S01 G03 AB374496

Continued.

(Control sample)

S01 G05 AB374497S01 G06 AB374498S01 G07 AB374499S01 G08 AB374500S01 G09 AB374501S01 G10 AB374502S01 G11 AB374503S01 G12 AB374504S01 H01 AB374505S01 H02 AB374506S01 H03 AB374507S01 H04 AB374508S01 H05 AB374509S01 H06 AB374510S01 H07 AB374511S01 H08 AB374512S01 H09 AB374513S01 H10 AB374514S01 H12 AB374515

APPENDIX II

Referring to the metagenome dataof intestinal flora

Comparison of the blood sample-associated taxonomicnames in this study with the feces-associated taxonomicnames listed in Kurokawa’s metagenome analysis (29). Leftcolumn, bacterial sequences hit in our RDP search of bloodsample-associated sequences. Middle column, genus andspecies. Blood sample-specific taxonomic names (i.e. posi-tive in PCR of blood sample but negative in PCR of controlsaline sample) are indicated by underlines. The taxonomicnames identical to those listed in Kurokawa’s report are in-dicated in the right column. Kurokawa et al. reported thatBacteroides and Clostridium are major subgroups com-prising the fecal flora of adult humans. These taxonomicnames were not found in our blood specimen-associatedsubgroups. The Bacillus subgroup is the third populationin the gut in Kurokawa’s study.



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polymerase chain reaction detection of bacterial 16s rrna gene in human blood

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