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Journal of Microbiological Methods 42 (2000) 139147 www.elsevier.com / locate / jmicmeth

A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material
Beverley C. Millar , Xu Jiru , John E. Moore
a

a,b ,

*, John A.P. Earle b

Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7 AD, UK b The School of Biology and Biochemistry, The Queen s University of Belfast, Belfast, UK Received 2 February 2000; received in revised form 3 May 2000; accepted 25 May 2000

Abstract This study investigated the various commercially available kits and in-house methods to extract DNA from Gramnegative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads / sonication and wash / alkali / heat lysis. The results indicated that a simple wash / alkali / heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method. This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT /Alert aerobic, anaerobic and paediatric blood culture material. Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identied in all batches of BacT /Alert FAN aerobic blood culture material examined. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Bacteria; Blood-culture; Contaminant; DNA extraction; Fungi; PCR inhibitor

1. Introduction Blood culture remains the corner stone in the detection of microbiological agents responsible for infection. However, blood culture fails to yield a positive result in many instances. Due to the availability and repeated sets of blood culture material for each patient which exists in most clinical microbiology laboratories, this material may be further processed using a molecular PCR-based approach to look for the causative agent of infection. There are a number of factors which may be responsible for a
*Corresponding author. Tel.: 1 44-28-9026-3554; fax: 1 44-282589-2887. E-mail address: jemoore@niphl.dnet.co.uk (J.E. Moore). 0167-7012 / 00 / $ see front matter PII: S0167-7012( 00 )00174-3

culture-negative nding in febrile patients, namely (i) the causative organism may be fastidious in nature such as the HACEK group, Brucella spp., Neiserria spp., Legionella spp., Nocardia spp., and cell-wall decient organisms, (ii) some organisms are cell dependent such as Coxiella burnetii, Bartonella spp. and Chlamydia spp. and (iii) detection of fungi is still difcult even with improved automated blood culture methods and materials. Molecular-based diagnostic approaches are now more frequently used alongside conventional microbiological techniques in the diagnostic clinical microbiology laboratory. PCR amplication of regions of the 16S rRNA (Widjojoatmodjo et al., 1994) and 18S rRNA genes (Einsele et al., 1997) are two such molecular-based approaches widely used in

2000 Elsevier Science B.V. All rights reserved.


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the identication of bacteria and fungi, respectively. As with any clinical specimen, two factors are crucial for the successful implementation of such a universal amplication-based approach. Firstly, it is fundamental that any agent which may cause inhibition of the PCR amplication is successfully removed using a reliable, reproducible and sensitive extraction procedure. When basing identication of microbiological agents on amplication of universal conserved genes, it is essential that the clinical specimen does not contain any contaminating DNA. Such contaminating DNA may be found on the skin of the patient or indeed in the EDTA-blood vial used for collection of blood or in the virgin un-inoculated blood culture material. The aim of this study was to nd the most sensitive, reliable and cost-effective method to isolate microbial DNA from blood culture material as an important fundamental prerequisite to further downstream molecular analyses including nucleic acid amplication techniques. Various commercially available DNA extraction kits were evaluated alongside in-house methods.

2.1.1. Boiling The inoculated material (0.5 ml) was centrifuged at 13,000 3 g for 5 min and the pellet resuspended in TE buffer (10 mM TrisHCl pH 8.0 containing 1 mM EDTA, 0.1 ml) and incubated at 958C for 15 min in a heating block. The suspension was subsequently centrifuged at 13,000 3 g for 15 min and the resulting supernatant containing the extracted DNA was transferred to a sterile tube and stored at 2 208C. 2.1.2. Phenol / chloroform The inoculated material (0.5 ml) was extracted as described previously (Fredericks and Relman, 1998). 2.1.3. Commercially available DNA extraction kits The inoculated material (0.5 ml) was extracted as per the recommended instructions issued with the respective kits, i.e. QIAmp Blood kit (Qiagen, UK), Roche high PCR template preparation kit (Roche Diagnostics, UK), Puregene kit (Gentra Systems, NC, USA). 2.1.4. Wash treatments prior to extraction of microbial DNA using Roche kit The inoculated sample was mixed with either red blood cell lysis solution (Gentra Systems), Tween 20 (0.5% v / v in TE) or Triton-X-100 (1% v / v in TE) at 258C, 10 min. The resulting suspension was centrifuged at 10,000 3 g for 8 min and the pellet was resuspended in fresh wash buffer. Three washes were carried out and the nal pellet was resuspended in TE and the extraction procedure was completed as per instructions in the Roche high PCR template preparation kit. 2.1.5. Glass beads / sonication The inoculated sample was mixed with wash buffer Triton-X-100 (1% v / v in TE) at 258C, 10 min and washed three times as described above (Section 2.1.4). The nal pellet was resuspended in wash buffer (0.3 ml) and glass beads (0.2 ml). The sample was boiled at 1008C for 25 min and sonicated for a further 20 min. The suspension was centrifuged at 10,000 3 g for 20 s and the supernatant used in subsequent PCR amplications.

2. Materials and methods

2.1. Extraction of microbial DNA

All DNA isolation procedures were carried out in a Class II Biological Safety Cabinet in a room physically separate from that used to set up reaction mixes and also from the post-PCR room in order to minimise the production of false positive results. In all methods tested and where applicable, molecular grade water was employed (Biowhittaker Inc, MD, USA, LAL Grade cat no: W50-100) to reduce contamination. In addition, BacT /Alert aerobic, anaerobic and paediatric blood culture material and TE buffer (10 mM TrisHCl pH 8.0 containing 1 mM EDTA) were inoculated with either Staphylococcus aureus (10 8 colony forming units (cfu)) or Candida albicans (10 7 cfu). The organisms were prepared by harvesting two colonies from an overnight (17 h) incubation from Columbia Blood Agar (Oxoid CM331 supplemented with 5% (v / v) debrinated horse blood, Oxoid Ltd., UK) and were resuspended in the various clinical specimens and dened media, as described above.

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2.1.6. Alkali wash / lysis Bacterial DNA was extracted from blood culture material using a modication of a previously described simple alkali wash and heat lysis method (Kulski and Pryce, 1996). Briey, blood culture uid (0.5 ml) was added to 1 ml of alkali wash solution (0.5 M NaOH and 0.05 M sodium citrate) in a 1.5-ml Eppendorf tube and mixed for 10 min at room temperature by inversion using a rotary wheel. The mix was subsequently centrifuged at 13,000 3 g for 5 min and the cell pellet containing any microbial DNA was resuspended in 0.5 M TrisHCl (pH 8.0, 0.5 ml) and centrifuged at 13,000 3 g for 5 min. This latter step was repeated and the resulting pellet was resuspended in TrisEDTA (0.1 ml, 10 mM Tris HCl pH 8.0 containing 1 mM EDTA) and heated at 1008C for 1 h in a heating block after which the sample was freeze / thawed twice and subsequently centrifuged at 13,000 3 g for 15 min and the supernatant containing any extracted DNA was transferred to a clean tube and stored at 2 208C prior to PCR. The fungal extraction procedure was as described above except for the following modication, i.e. after washing with TrisHCl, the sample was incubated with TrisEDTA (0.98 ml; 10 mM TrisHCl pH 8.0 containing 1 mM EDTA) and lyticase (20 ml, 0.5 mg / ml, Roche, UK) for 1 h at 308C, prior to heating at 1008C, after which the sample was freeze / thawed twice and subsequently centrifuged at 13,000 3 g for 15 min. Extracted DNA was transferred to a clean tube and stored at 2 208C prior to PCR. 2.2. PCR Amplication of DNA

up as follows: 10 mM TrisHCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl 2 , 200 mM (each) dATP, dCTP, dGTP and dTTP; 1.25 U of Taq DNA polymerase (Amplitaq; Perkin Elmer), 0.2 mM (each) of the appropriate primers (see Table 1) and 4 ml of DNA template. The reaction mixtures following a hot start were subjected to the following thermal cycling parameters in a Perkin Elmer 2400 thermocycler: 968C for 3 min followed by 40 cycles of 968C for 1 min, 558C for 1 min, 728C for 1 min, followed by a nal extension at 728C for 10 min. During each run molecular grade water was included randomly as negative controls and appropriate DNA templates from S. aureus, and C. albicans were included as a positive control as appropriate.

2.3. Sensitivity of 16 S rRNA detection

The universal bacterial specic primer pair P11P and P13P (Widjojoatmodjo et al., 1994; Table 1) were used to amplify a portion of the 16S rRNA gene, (corresponding to the V6 region of the gene positions 1175 to 1390 of the Escherichia coli 16S rRNA gene), generating PCR products of | 216 bp. Serial dilutions were made of a tested culture-negative and PCR-negative blood culture spiked with | 10 9 cfu of S. aureus and the bacterial DNA was extracted and amplied as described above. Quantitative cultures were determined by colony counting on solid culture medium. Colony counts per millilitre of blood culture material were ascertained from the dilution containing between 30 and 100 colonies.

2.4. Sensitivity of 18 S rRNA detection

All reaction mixes were set up in a PCR hood in a room separate from that used to extract DNA and the amplication and post-PCR room in order to minimise contamination. Reaction mixes (50 ml) were set The universal fungal specic primer pair 18S (f) and 18S (r) (Einsele et al., 1997; Table 1) which bind with variable areas of the 18S rRNA gene, V7 to V9,

Table 1 Universal primers used for PCR amplication of regions of the 16S rRNA gene (bacteria) and the 18S rRNA gene (fungi) Primer name P11P(f)a P13P(r) 18S (f) 18S (r)a
a

Sequence 59 39 GAG GAA GGT GGG GAT GAC GT AGG CCC GGG AAC GTA TTC AC ATT GGA GGG CAA GTC TGG TG CCG ATC CCT AGT CGG CAT AG

Target gene 16S rRNA 16S rRNA 18S rRNA 18S rRNA

Reference Widjojoatmodjo et al., 1994 Widjojoatmodjo et al., 1994 Einsele et al., 1997 Einsele et al., 1997

Primer used for sequence analysis; f, forward; r, reverse.


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were used to amplify a portion of the gene corresponding to positions 544 to 1033 in the case of C. albicans, generating PCR products of | 490 bp. Serial dilutions were made of a tested culture-negative and PCR-negative blood culture spiked with | 10 9 cfu of C. albicans and the fungal DNA was extracted and amplied as described above. Quantitative cultures were determined by colony counting on solid culture medium. Colony counts per millilitre of blood culture material were ascertained from the dilution containing between 30 and 100 colonies.

2.5. Detection of amplicons

Following amplication, aliquots (15 ml) were removed from each reaction mixture and examined by electrophoresis (80 V, 45 min) in gels composed of 2% (w / v) agarose (Gibco, UK) in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.3), stained with ethidium bromide (5 mg / 100 ml). Gels were visualised under UV illumination using a gel image analysis system (UVP Products, UK) and all images archived as digital graphic les (*.bmp).

sequencing, particularly to remove dNTPS, polymerases, salts and primers. Appropriate primers (Table 1) were used for sequencing with the ABI PRISME Dye Terminator Cycle Sequencing Reaction with AmpliTaq DNA Polymerase , FS (PE Biosystems, Foster City, CA, USA) (968C 1 min, followed by 25 cycles of 968C for 10 s, 508C for 5 s, 608C for 4 min, followed by a 48C hold). The products were ethanol-precipitated and analysed on an ABI 373 Automatic Sequencer (PE Biosystems). The resulting sequences obtained were compared with those stored in the Genbank Data system using BLAST alignment software (http: / / www.blast. genome.ad.jp / ).

3. Results

3.1. Analysis of various DNA extraction methods to isolate microbial DNA from blood culture material
Ten different DNA extraction methods were examined in order to ascertain the most suitable method to extract microbial DNA from blood culture material (see Table 2). Inoculated TE buffer, with either S. aureus or C. albicans were used as positive controls. All of the methods successfully extracted microbial DNA as was seen by analysis on agarose gel electrophoresis (data not shown). With respect to

2.6. Sequencing of amplicons and analysis of sequence data

Amplicons chosen for sequencing were puried using a QIAquick PCR purication kit (Qiagen) eluted in TrisHCl (10 mM, pH 8.5) prior to

Table 2 Different DNA extraction methods used to isolate microbial DNA from blood culture material and TE buffer inoculated with S. aureus or C. albicans DNA extraction method Inoculated blood culture material Quantity of DNA isolated 1. Boil 2. Phenol / chloroform 3. QIAmp Blood kit 4. Roche high PCR template preparation kit 5. Red blood cell lysis treatment and 3 6. Tween 20 wash and 3 7. Triton-X-100 wash and 3 8. Puregene kit 9. Glass beads / sonication 10. Alkaline wash / lyticase / boil / freeze / thaw 1 1 1 1 1 1 1 1 1 1 Ability to amplify extracted DNA 2 2 2 2 2 2 2 2 2 111 Inoculated TE buffer Quantity of DNA isolated 1 1 1 1 1 1 1 1 2 1 Ability to amplify extracted DNA 1 2 1 1 1 1 1 1 2 1

1 11 1 1 1 1 1

1 11 1 1 1 1 1

1 11 1 1 1 1

1 , low; 1 1 , moderate; 1 1 1 , high; 2 , extracted DNA could not be amplied by PCR.

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the inoculated TE buffer, all of the extraction methods except sonication in the presence of glass beads resulted in microbial DNA which could be amplied by PCR. A possible explanation for this could be that the extracted DNA bound to the glass beads and was not present in the supernatant which was used for amplication or that some small pieces of glass remained in the extract, which may cause PCR inhibition. With respect to inoculated blood culture material (BacT /Alert anaerobic, aerobic and paediatric) although microbial DNA was extracted using all of the methods, only the alkali heat lysis method yielded DNA which could be amplied by PCR.

(10 8 cfu / ml) and the microbial DNA was extracted as per the Roche method. When the extracted DNA was used neat as a PCR template for amplication of the 16S rRNA gene, no amplied product resulted. Serial dilution of the DNA extract did however result in a PCR product (see Fig. 1, gel B).

3.3. Sensitivity of simple extraction method

The sensitivity of the simple alkali heat lysis method was ascertained for both bacteria and fungi (see Fig. 2). The sensitivity for the Gram-negative bacteria E. coli, the Gram-positive, S. aureus and the fungi, C. albicans, was 10 2 , 10 3 , 10 2 cfu / ml of blood culture material, respectively. The sensitivity was the same for BacT /Alert aerobic, anaerobic and paediatric blood culture material (data not shown).

3.2. Identication of PCR inhibitor in blood culture materials

The Roche high PCR template preparation extraction method extracted large amounts of DNA from blood culture material inoculated with S. aureus (10 8 cfu / ml), yet the DNA template containing the 16S rRNA could not be amplied by PCR. In order to ascertain if a possible explanation for this was the presence of a PCR inhibitor in the extract, 1 ml of DNA extracted from TE inoculated with S. aureus was amplied in the presence of 1 ml of LAL grade water (see Fig. 1, gel A, lane 4). However when 1 ml of LAL grade water was substituted by either 1 ml of BacT /Alert aerobic, anaerobic, or paediatric virgin blood culture material, the S. aureus DNA template could not be amplied (see Fig. 1, gel A, lanes 57). As all three BacT /Alert blood culture materials contained polyanetholesulfonic acid (SPS, 0.035% w / v), it was investigated whether SPS could be a possible PCR inhibitor; 1 ml of DNA extracted from TE inoculated with S. aureus in the presence of 1 ml SPS (0.035% w / v), could not be amplied (see Fig. 1, gel A, lane 8). An aerobic blood culture material which was culture positive for bacteria was extracted using the Roche method, however the resulting DNA extract could not be amplied by PCR (see Fig. 1, gel A, lane 10). Serial dilutions (1 / 10, 1 / 100, 1 / 1000) of this extract were made which resulted in the removal of the PCR inhibitor, allowing the bacterial DNA template to be amplied (see Fig. 1, gel A, lane 1113). SPS (0.035% w / v) was inoculated with S. aureus

3.4. Spectrum of organisms which may be extracted using the alkali lysis method
In order to ascertain whether the alkali heat lysis method could be used to extract microbial DNA from Gram-negative, Gram-positive, yeast and fungal organisms, blood culture material was inoculated with the organisms as detailed in Table 3 and the resulting PCR product sequenced to conrm the identity of the positive result.

3.5. Identication of contaminant DNA in BacT / Alert FAN aerobic material

More recently, BacT /Alert have introduced a superior culture material known as the BacT /Alert FAN Aerobic System. This material contains activated charcoal and SPS at a higher concentration (0.05% w / v). Four different paired batches of BacT / Alert anaerobic and BacT /Alert FAN Aerobic blood culture material, (batch A, (n 5 4); batch B, (n 5 4); batch C, (n 5 4); batch D, (n 5 1)) were inoculated with the culture-negative blood. All batches of BacT /Alert anaerobic blood culture material were negative by universal PCR amplication of both the 16S rRNA gene (see Fig. 3) and 18S rRNA gene (data not shown). All four batches of BacT / Alert FAN Aerobic blood culture material were negative by universal PCR amplication of the 18S


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Fig. 1. Gel A: 16S rRNA PCR analysis of DNA extracted from TE inoculated with S. aureus (10 8 cfu / ml). Lane 1, DNA molecular weight marker (AmpliSizeE DNA Size standard, 502000 bp ladder; BIO-RAD); lane 2, LAL grade water (Biowhittaker) negative control; lane 3, S. aureus DNA (2 ml); lane 4, S. aureus DNA (1 ml) plus LAL grade water (1 ml); lane 5, S. aureus DNA (1 ml) plus virgin BacT /Alert aerobic blood culture material (1 ml); lane 6, S. aureus DNA (1 ml) plus virgin BacT /Alert anaerobic blood culture material (1 ml); lane 7, S. aureus DNA (1 ml) plus virgin BacT /Alert paediatric blood culture material (1 ml); lane 8, S. aureus DNA (1 ml) plus polyanetholesulfonic acid (SPS, 0.035% w / v); lane 9, S. aureus DNA (1 ml) plus culture positive aerobic blood culture material (1 ml); lane 10, DNA isolated from culture positive aerobic blood culture material (CPA, 2 ml); lane 11, 1 / 10 dilution CPA, 2 ml; lane 12, 1 / 100 dilution CPA, 2 ml; lane 13, 1 / 1000 dilution CPA, 2 ml. Gel B: 16S rRNA PCR analysis of DNA extracted from SPS (0.035% w / v) inoculated with S. aureus (10 8 cfu / ml): lane 1, DNA molecular weight marker (AmpliSizeE DNA Size standard, 502000 bp ladder; BIO-RAD); lane 2, neat extract; lane 3, 1 / 10 dilution of extract; lane 4, 1 / 100 dilution; lane 5, 1 / 250 dilution; lane 6, 1 / 500 dilution; lane 7, 1 / 750 dilution; lane 8, 1 / 1000 dilution; lane 9, 1 / 2000 dilution; lane 10, LAL grade water (Biowhittaker) negative control.

rRNA gene (data not shown) and positive by amplication of the 16S rRNA gene. All amplicons (batch A, (n 5 4); batch B, (n 5 4); batch C, (n 5 4); batch D, (n 5 1)) were identied by sequence analysis. Sequence analysis identied the presence of DNA from Lactococcus lactis in batches A, B and D and Bacillus coagulans in batch C.

4. Discussion The molecular detection of microbiological agents of infection is now widely used, in particular, in

cases where the agent is fastidious in nature, e.g. Chlamydia spp. (Girjes et al., 1999), Bartonella spp. (Matar et al., 1999), Coxiella burnetii (Zhang et al., 1998) and Mycobacterium spp. (Ikonomopoulos et al., 1999). The wide availability of blood culture material which exists in most standard microbiology laboratories, provides the opportunity to make a molecular diagnosis alongside the conventional approach. The molecular approach would be advantageous in particular when the infectious agent is believed to be fastidious or fungal in nature, or when blood culture fails to identify the causative agent (Millar et al., 1999; Mallon et al., 2000) or where a

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Fig. 2. Sensitivity of alkali wash /(lyticase) / boil / freeze / thaw DNA extraction method. Gel A: 16S rRNA PCR analysis of DNA extracted from BacT /Alert anaerobic blood culture material inoculated with blood (10 ml, culture negative) and E. coli. Lane 1, DNA molecular weight ladder (100 bp, Gibco); lane 2, LAL grade water (Biowhittaker) negative control; lanes 310, DNA extracted from 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 cfu / ml, respectively. Gel B: 16S rRNA PCR analysis of DNA extracted from BacT /Alert anaerobic blood culture material inoculated with blood (10 ml, culture negative) and S. aureus. Lane 1, DNA molecular weight ladder (100 bp, Gibco); lane 2, LAL grade water (Biowhittaker) negative control; lanes 310, DNA extracted from 10 8 , 10 7, 10 6, 10 5, 10 4, 10 3, 10 2, 10 1 cfu / ml, respectively. Gel C: 18S rRNA PCR analysis of DNA extracted from BacT /Alert anaerobic blood culture material inoculated with blood (10 ml, culture negative) and C. albicans. Lane 1, DNA molecular weight ladder (100 bp, Gibco); lane 2, LAL grade water (Biowhittaker) negative control; lanes 310, DNA extracted from 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 cfu / ml, respectively.

quick diagnosis needs to be made. The identication of the infecting organism may thus be made earlier than the conventional approach and through ampli-

cation of antibiotic-resistance gene loci (Moore et al., 1999; Zheng et al., 1999) the most appropriate anti-microbial therapy may be initiated sooner.


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Table 3 A list of Gram-negative, Gram positive, yeast and fungal agents used to inoculate BacT /Alert blood culture material from which microbial DNA was subsequently isolated using the alkali lysis method and amplied by PCR Gram-negative Acinetobacter spp. Actinobacillus actniomycetetemcomitans Berkholderia cepacia Escherichia coli Haemophilus inuenzae Pseudomonas spp. Gram-positive Bacillus coagulans Corynebacterium spp. Lactococcus lactis Micrococcus spp. Proponibacterium acnes Staphylococcus aureus Staphylococcus epidermidis Streptococcus mitis Streptococcus oralis Yeast Candida albicans C. dublinesis C. ( Torulaspora) glabrata C. guillermondii C. kefyr C. krusei C. lustaniae C. parapsilosis C. pseudotropicalis Cryptococcus neoformans Saccharomyces cerivisiae Fungi Alternaria alternata Aspergillus niger A. avus A. fumigatus A. niduliaus A. terreus Chryosporium spp. Fusarium spp. Monolinia laxa Paecilomyces spp. Penicillium spp. Scedosporium spp.

Fig. 3. 16S rRNA PCR analysis of four different paired batches of BacT /Alert anaerobic (lanes 4, 6, 8, 10) and BacT /Alert FAN aerobic (lanes 5, 7, 9, 11) blood culture material inoculated with culture-negative blood. Lane 1, DNA molecular weight marker (AmpliSizeE DNA Size standard, 502000 bp ladder; BIO-RAD); lane 2, LAL grade water (Biowhittaker) negative control; lane 3, S. aureus -positive control.

Two inherent problems which hinder the molecular investigation of blood culture material are (i) inhibiting agents of amplication by PCR (Fredericks and Relman, 1998) and (ii) bacterial DNA which exists in virgin blood culture material (Fredericks and Relman, 1998). The ndings of our study suggest that BacT /Alert aerobic, anaerobic and paediatric materials contain potent PCR inhibitor(s). As all three blood culture materials contain the anti-coagulant and anti-complementary agent, sodium polyanetholesulfonate (SPS) and as we have shown this to be a potent PCR inhibitor, it is suggestive that this may be the main inhibitory agent in blood culture material. Indeed, recently Fredericks and Relman (1998) identied SPS by spectrophotometry, as the agent responsible for PCR inhibition in BacT /Alert anaerobic medium. A number of DNA extraction methods have been evaluated in our study in order to ascertain which method would be the most suitable to isolate DNA

which could be used further in a molecular analysis. A method should be chosen which is sensitive, reproducible, cost-effective and universal in its ability to isolate yeast, fungal, Gram-negative and Grampositive bacterial DNA. The most appropriate method was based on a simple, inexpensive heat lysis method which was originally described for the isolation solely of mycobacterial DNA from BACTEC blood culture uids (Kulski and Pryce, 1996). With respect to the BacT /Alert aerobic, anaerobic and paediatric blood culture materials, this simple method also removed any inherent bacterial DNA which existed previously in the virgin un-inoculated blood culture material. However with respect to the BacT /Alert FAN aerobic blood culture material, the inherent bacterial DNA which exists in virgin material could not be removed by this simple alkali method. A possible explanation for this is that unlike the BacT /Alert aerobic, anaerobic and paediatric materials, the FAN material contains activated char-

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coal which has the ability to bind any DNA which may exist in the blood culture material. During the centrifugation wash steps, this charcoal is pelleted and therefore it is difcult to completely remove all such inherent DNA. Such bacterial DNA could result from components in the blood culture medium which although sterile in that the microbial agents are not viable, the DNA still exists. The identity of the inherent DNA is variable but appears to be consistent within a batch. We have identied the inherent DNA to originate from mainly L. lactis (75% of blood culture batches examined) and B. coagulans (25% of blood cultures examined). It is therefore essential that when using blood culture material as a source for molecular investigation that appropriate controls are included in each stage of the molecular analysis. It is fundamental that the DNA extraction from the blood culture material is empirically optimised for each individual commercial blood culture system used, e.g. Septi-check, Oxoid, BACTEC, Biom rieux, as these may differ in terms of the cone centration of inhibitors and endogenous DNA present in un-inoculated material. In the case of broad-range amplication, the signicance of the identied microbial agent isolated from a patients blood culture material should correlate with that patients medical history, before the signicance of the result is considered.

Acknowledgements BCM and JEM are supported by a project grant from the British Heart Foundation (PG96168). XJ and JEM are supported by a project grant from the Meningitis Research Foundation.

pathogens in blood by using molecular probes. J. Clin. Microbiol. 35, 13531360. Fredericks, D.N., Relman, D.A., 1998. Improved amplication of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J. Clin. Microbiol. 36, 28102816. Girjes, A.A., Carrick, F.N., Lavin, M.F., 1999. Single DNA sequence common to all chlamydial species employed for PCR detection of these organisms. Res. Microbiol. 150, 483489. Ikonomopoulos, J.A., Gorgoulis, V.G., Zacharatos, P.V., Manolis, E.N., Kanavaros, P., Rassidakis, A. et al., 1999. Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis. Mod. Pathol. 12, 854862. Kulski, J.K., Pryce, T., 1996. Preparation of mycobacterial DNA from blood culture uids by simple alkali wash and heat lysis method for PCR detection. J. Clin. Microbiol. 34, 19851991. Mallon, P.G., Millar, B.C., Moore, J.E., Murphy, P.G., McClurg, R.B., Chew, E.W. et al., 2000. Molecular identication of Acinetobacter spp. in a patient with culture-negative endocarditis. Clin. Microbiol. Infect. 6, 277278. Matar, G.M., Koehler, J.E., Malcolm, G., Lambert-Fair, M.A., Tappero, J., Hunter, S.B. et al., 1999. Identication of Bartonella species directly in clinical specimens by PCR-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment. J. Clin. Microbiol. 37, 40454047. Millar, B.C., Moore, J.E., Mallon, P., Crowe, M.J., McClurg, R.B., Curran, M.D. et al., 1999. Molecular diagnosis of endocarditis a new Dukes criterion? Eur. Heart J. 20, 362. Moore, J.E., Millar, B.C., Yongmin, X., Crowe, M., 1999. A rapid molecular assay for the detection of antibiotic resistant organisms in infective endocarditis. Eur. Heart J. 20, 559. Widjojoatmodjo, M.N., Fluit, A.C., Verhoef, J., 1994. Rapid identication of bacteria by PCR-single-stranded conformation polymorphism. J. Clin. Microbiol. 32, 30023007. Zheng, X., Kolbert, C.P., Varga-Delmore, P., Arruda, J., Lewis, M., Kolberg, J. et al., 1999. Direct mecA detection from blood culture bottles by branched-DNA signal amplication. J. Clin. Microbiol. 37, 41924193. Zhang, G.Q., Hotta, A., Mizutani, M., Ho, T., Yamaguchi, T., Fukushi, H. et al., 1998. Direct identication of Coxiella burnetii plasmids in human sera by nested PCR. J. Clin. Microbiol. 36, 22102213.

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