Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates

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Diagnostic Microbiology and Infectious Disease 63 (2009) 403 – 408 www.elsevier.com/locate/diagmicrobio

Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates Carson D. Baldwina , Gerald B. Howea , Ranga Sampathb , Larry B. Blynb , Heather Matthewsb , Vanessa Harpinb , Thomas A. Hallb , Jared J. Draderb , Steve A. Hofstadlerb , Mark W. Eshoob , Karl Rudnickc , Karen Studarusc , David Moorec , Sharon Abbottd , J. Michael Jandad , Chris A. Whitehousea,⁎ a

U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA b Ibis Biosciences, Inc., a subsidiary of Isis Pharmaceuticals, Carlsbad, CA 92008, USA c SAIC 10260 Campus Point Drive, San Diego, CA 92121, USA d Microbial Diseases Laboratory, California Department of Public Health, Richmond, CA 94804, USA Received 26 August 2008; accepted 12 December 2008

Abstract Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified. This study shows that multilocus PCR/ESI-MS has the potential to be a useful technique for identifying a broad range of bacteria. Published by Elsevier Inc. Keywords: Broad-range PCR; Mass spectrometry; Rapid detection; Bacterial diagnostics

Classic methods for detecting and identifying pathogenic bacteria typically rely on cultivation of the organism and subsequent identification by morphology and biochemical profiles. These methods can be lengthy and are often subjective. Culture, itself, often requires 24–72 h and may be negative if the bacteria are fastidious or noncultivatable. In these cases, molecular methods, such as broad-range PCR, can be beneficial. Broad-range PCR is based on the recognition that certain genes coding for conserved proteins (e.g., heat shock proteins and RNA polymerases) are present in many different organisms and can be useful for identification within bacterial families. This method requires PCR using broad-range primers followed by sequencing of ⁎ Corresponding author. Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. Tel.: +1-301-619-2098; fax: +1-301-619-2492. E-mail address: [email protected] (C.A. Whitehouse). 0732-8893/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2008.12.012

the amplicon (Maiwald, 2004). Several broad-range PCR assays have been reported in the literature, mainly targeting either the 16S rRNA gene (Clarridge, 2004; Drancourt et al., 2000; Harris and Hartley, 2003; Rantakokko-Jalava et al., 2000; Zucol et al., 2006) or the 23S rRNA gene (Hunt et al., 2006; Miflin and Blackall, 2001; Verma et al., 1994; Yoo et al., 2006). Other common gene targets for broad-range PCR have included rpoB, coding for the β-subunit of the RNA polymerase (Adekambi et al., 2003; Adekambi et al., 2006; Drancourt and Raoult, 2002; Drancourt et al., 2001; Renesto et al., 2001). Although broad-range PCR, particularly using the 16S rRNA gene target, has proven useful in several diverse applications (Adderson et al., 2008; Whitehouse et al., 2007), the method has limitations. For example, in certain groups of bacteria, rRNA sequences might not be useful for discriminating members of that group (e.g., Bacillus cereus group) (Ash et al., 1991). Furthermore, the primary diagnostic utility of

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this method lies in testing specimens from sterile sites or culture isolates. For testing mixed cultures or microbial communities by broad-range PCR, the construction of clone libraries and the subsequent sequencing of multiple clones are needed, which is time-consuming and laborious. The Ibis T5000 biosensor (previously referred to as “TIGER”) is designed to rapidly detect and identify a variety of pathogens without prior knowledge of the pathogen's nucleic acid sequence (Ecker et al., 2008; Hofstadler et al., 2005). The T5000 uses broad-range PCR primers that target conserved regions of bacterial genomes, such as ribosomal sequences and conserved elements from essential protein-coding genes (i.e., housekeeping genes) (Table 1). The use of such broad-range priming targets across the widest possible grouping of organisms enables amplification of most species within a group. The unique aspect of the T5000 biosensor is the use of electrospray ionization-mass spectrometry to analyze the products of broad-range PCR (PCR/ESI-MS). The high mass accuracy and resolution of the PCR/ESI-MS system allows for the precise determination of the molecular mass of the PCR products (Jiang and Hofstadler, 2003; Muddiman et al., 1997). These high-precision mass measurements are used to unambiguously derive base compositions (xAxGxCxT) of the PCR products, which are then compared to a database for identifying the organism. Only well-characterized organisms (i.e., those from established culture collections or those whose whole genome has been determined) are used to populate the system database. These data allow for a multilocus identification of any bacteria in the samples with significantly less time and effort than sequencing. Previous data have also shown that the T5000 performs well with samples from a variety of

Table 1 Broad-range primers for bacteria used for PCR/ESI-MS analysis Primers pairsa

Gene target

Projected bacterial coverage

346, 347, 348, 361 349, 360 354

16S rDNA 23S rDNA rpoC

363 358

rpoC valS

359 362 367

rpoB rpoB tufB

356, 449

rplB

352 355

infB sspE

All All Bacterioidetes, fusobacteria, spirochaetes, bacilli, proteobacteria α/β/γ Proteobacteria α/β For some representatives of γ-proteobacteria: Erwinia Pantoea, Pectobacterium Proteobacteria α/β Some representatives of β - proteobacteria: Eikenella, Neisseria, Achromobacter, Bordetella, Burkholderia, Ralstonia Clostridia, fusobacteria, bacilli, and ɛ-proteobacteria (Campylobacter, Helicobacter, Wolinella) Bacilli Bacillus cereus

a

For specific primer sequences, see Hofstadler et al. (2005).

clinical and environmental matrices including blood, serum, various tissues, and even mosquito homogenates (Ecker et al., 2008). The aim of the present study was to evaluate the usefulness of PCR/ESI-MS using the T5000 biosensor for the specific identification of a diverse panel of bacterial isolates, including 124 well-characterized reference strains and 32 clinical isolates from members of the family Enterobacteriaceae. Bacterial genomic DNAs were extracted using either the Bactizol™ kit (Molecular Research Center, Inc., Cincinnati OH) or the QIAamp genomic DNA extraction kit (Qiagen, Valencia, CA) following the manufacturers' instructions or were purchased directly from the American Type Culture Collection (ATCC). The 32 clinical isolates (family Enterobacteriaceae) were extensively characterized by standard microbiological methods against a battery of more than 60 phenotypes. All of the 124 reference strains were characterized by a combination of classic microbiological methods, biochemical utilization profiling using the automated Vitek32 (bioMerieux Vitek, Hazelwood, MO), whole cell fatty acid profiling using the automated MIDI (MIDI, Newark, DE), and by 16S rRNA sequence analysis. The DNAs were blinded and coded before being analyzed to ensure no bias in the data interpretation. Each DNA sample was diluted to a final concentration of 50 pg/μL in genome dilution buffer. All PCR reactions were performed in a 50-μL reaction volume using 96-well microtiter plates. The reaction plates used 16 broad-range primers in different wells for broad bacterial detection (Table 1). These reaction plates were set up using the BioRobot 8000 (Qiagen), and an ALPS 300™ automated plate sealer (ABgene, Epsom, UK) was used to seal all PCR plates to avoid contamination and evaporation. PCR was carried out using an Eppendorf® Mastercycler® ep Thermocycler (Hamburg, Germany). The PCR reaction buffer consisted of 2.5 U of FastStart Taq (Roche, Indianapolis, IN), 1× buffer II, 2.0 mmol/L MgCl2, 0.4 mol/L betaine, 800 μmol/L dNTP mix, and 250 nmol/L propyne containing PCR primers (Table 1). All PCR reaction wells were loaded with 5 μL of DNA, resulting in a concentration of 250 pg of DNA per well. Each PCR plate contained one negative control consisting of genome dilution buffer. The following PCR conditions were used to amplify sequences: 95°C for 10 min followed by 8 cycles of 95°C for 30 s, 48°C for 30 s, and 72°C for 30 s followed by 37 cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for 20 s. After PCR was complete, the reactions were purified using magnetic beads with a weak anion exchange matrix. A Bruker Daltonics microToF (Billerica, MA) mass spectrometer (MS) was used for analyzing the purified DNA. Samples from each reaction well were individually sprayed into the MS using a LEAP autosampler (Carrboro, NC). Once the raw spectra were collected, proprietary signalprocessing software was used to deconvolute the mass/ charge (m/z) data from the MS and determine the amplicons' molecular mass. Because the assay includes 16 primers, there were multiple base counts assigned for

C.D. Baldwin et al. / Diagnostic Microbiology and Infectious Disease 63 (2009) 403–408 Table 2 Bacterial strains used in this study and results of the PCR/ESI-MS calls Organism (no. of strains/isolates)

Source/ sample no.

Isolation source

PCR/ESIMS call

Reference strains (n = 124) Acinetobacter ATCC 19606 Urine Genus baumannii Acinetobacter lwoffii ATCC 17925 NA Genus Achromobacter ATCC 27061 Ear discharge Incorrect xylosoxidans Bacillus anthracis ATCC 4728 NA Genus/species Bacillus anthracis (6) USAMRIID NA Genus/species Bacillus cereus ATCC 10987 NA Genus/species Bacillus cereus ATCC 19637 NA Genus Bacillus halodurans ATCC 21591 Soil Genus/species Bacillus licheniformis ATCC 12759 Plant Genus/species Bacillus mycoides ATCC 31101 Soil Genus Bacillus mycoides ATCC 21929 Soil Genus Bacillus sphaericus ATCC 4525 NA Genus/species Bacillus subtilis ATCC 23857 NA Genus/species Bacillus subtilis USAMRIID NA Genus/species var niger (2) Bacillus thuringiensis ATCC 35646 Sewage Genus/species Bacillus thuringiensis USAMRIID NA Genus/species Bacillus thuringiensis ATCC 33679 Insect Genus Bacteroides fragilis ATCC 25285 Appendix abscess Genus/species Bartonella henselae ATCC 49882 Human blood Genus/species Bifidobacterium ATCC 15697 Human (infant) Genus infantis intestine Bordetella ATCC 10580 Canine lung Genus/species bronchiseptica Bordetella ATCC BAA-587 Nasopharynx Genus/species parapertussis Bordetella pertussis NA NA Genus/species Borrelia burgdorferi ATCC 35210 Tick Genus/species Brevibacillus brevis ATCC 8246 NA Genus Brevibacterium ATCC 9175 Cheese Genus linens Brucella abortus (2) USAMRIID NA Genus Brucella maris (2) USAMRIID NA Genus/species Brucella USAMRIID NA Genus melitensis (2) Brucella neotame USAMRIID NA Genus Brucella ovis USAMRIID NA Genus Brucella suis (2) USAMRIID NA Genus Burkholderia cepacia ATCC 25416 Plant Genus/species Burkholderia mallei ATCC 23344 NA Genus/species Burkholderia USAMRIID NA Genus/species pseudomallei Campylobacter jejuni ATCC 33560 Bovine feces Incorrect Chryseobacterium ATCC 33958 NA Incorrect meningosepticum Citrobacter freundii ATCC 8090 NA Incorrect ATCC 824 Plant Genus/species Clostridium acetobutylicum Clostridium USAMRIID NA Genus/species bifermentans Clostridium botulinum ATCC 19397 NA Genus/species (type A) Clostridium botulinum USAMRIID NA Genus/species (type B) Clostridium botulinum ATCC 17843 NA Genus/species (type C) Clostridium botulinum ATCC 9564 NA Genus (type E) (continued on next page)

405

Table 2 (continued) Organism (no. of strains/isolates)

Source/ sample no.

Isolation source

PCR/ESIMS call

NA

Genus/species

NA

Genus/species

Lamb intestines

Genus/species

NA Soil

Genus/species Genus/species

NA

Genus/species

NA Irradiated ground meat Feces

Genus/species Genus/species

Clinical isolate

Genus/species

Spinal fluid

Incorrect

Maize

Incorrect

Human feces Human urine

Genus Genus/species

Clinical isolate Clinical isolate River water

Genus/species Genus/species Genus/species

NA

Genus/species

NA

Genus/species

NA

Genus/species

NA

Incorrect

NA

Incorrect

Sputum

Genus/species

Human

Genus/species Incorrect

ATCC 33152

Human vaginal discharge Human lung

Genus/species

ATCC 15313

Rabbit

Genus/species

NA

NA

Genus/species

ATCC 25240

NA

Genus/species

ATCC 17967

Eyeconjunctivitis Human gastric lavage NA Human with atypical pneumonia Nasopharynx

Genus/species

Clostridium botulinum USAMRIID (type F) Clostridium ATCC 13124 perfringens Clostridium ATCC 3626 perfringens Clostridium sordelli ATCC 9714 Clostridium ATCC 3584 sporogenes Corynebacterium ATCC 700971 diphtheriae Coxiella burnetii NA Deinococcus ATCC 13939 radiodurans Enterobacter ATCC 15038 aerogenes Enterobacter NA aerogenes Enterobacter cloacae ATCC 13047 subsp. cloacae Enterobacter cloacae ATCC 23373 subsp. dissolvens Enterococcus durans ATCC 6056 Enterococcus ATCC 29212 faecalis Escherichia coli ATCC 700928 Escherichia coli ATCC 25922 Francisella ATCC 25016 philomiragia Francisella USAMRIID tularensis (6) Geobacillus ATCC 7953 stearothermophilus Haemophilus ATCC 10211 influenzae Halobacterium ATCC 700922 salinarum Klebsiella pneumoniae ATCC 13883 subsp. pneumoniae Klebsiella pneumoniae ATCC 700721 subsp. pneumoniae Lactobacillus ATCC 4357 acidophilus Lactobacillus jensenii ATCC 25258 Legionella pneumophila Listeria monocytogenes Mannheimia haemolytica Moraxella cataharalis Moraxella lacunata Mycobacterium gordonae Mycobacterium bovis Mycoplasma pneumoniae Neisseria lactamica

ATCC 35760 ATCC 19015 ATCC 15531

ATCC 23970

Genus/species

Genus Genus/species Genus/species

Genus

(continued on next page)

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Table 2 (continued) Organism (no. of strains/isolates)

Table 2 (continued) Source/ sample no.

Paenibacillus ATCC 8244 macerans Paenibacillus ATCC 842 polymyxa Paenibacillus popilliae ATCC 14706 Pantoea agglomerans ATCC 29904 Pantoea ananatis ATCC 19321 Pasteurella multocida ATCC 43137 Propionibacterium ATCC 25746 acnes Proteus mirabilis ATCC 7002 Proteus vulgaris ATCC 49132 Providencia stuartii ATCC 33672 Pseudomonas ATCC 47085 aeruginosa Pseudomonas putida ATCC 47054 Rhizobium radiobacter ATCC 33970 Salmonella enterica ATCC 9150 subsp. enterica serovar Paratyphi A Salmonella enterica ATCC 700720 subsp. enterica serovar Typhimurium Serratia marcescens ATCC 13880 Serratis odorifera ATCC 33077 Shewanella oneidensis ATCC 700550 Shigella flexneri ATCC 12022 Shigella sonnei ATCC 9290 Staphylococcus ATCC 29213 aureus Staphylococcus ATCC 700699 aureus Staphylococcus ATCC 10832 aureus subsp. Aureus Staphylococcus ATCC 43809 lugdunensis Staphylococcus ATCC 43808 schleiferi Stenotrophomonas ATCC 13637 maltophilia Streptococcus ATCC 33397 anginosum Streptococcus ATCC 33400 pneumoniae Streptococcus ATCC 19615 pyogenes Streptococcus ATCC 12386 agalactiae Streptococcus ATCC 12392 sp. Group F Tannerella forsythus ATCC 43037

Ureaplasma urealyticum Vibrio cholerae Vibrio parahaemolyticus Yersinia enterocolitica

ATCC 700970 ATCC 51394 ATCC 17802 USAMRIID

Isolation source

PCR/ESIMS call

Organism (no. of strains/isolates)

NA

Genus/species

NA

Genus/species

NA NA Wheat stem rust Pig NA

Genus/species Incorrect Genus/species Genus/species Genus

Human urine Clinical isolate NA NA

Incorrect Genus/species Incorrect Genus/species

NA Cherry plant gall NA

Genus/species Genus/species

NA

Genus/species

Yersinia ATCC 33638 Human urine kristensenii Yersinia pestis USAMRIID NA (Antigua) Yersinia pestis USAMRIID NA (Nairobi) Yersinia pestis USAMRIID NA (PBM19) Yersinia pestis USAMRIID NA (Java 9) Yersinia pestis USAMRIID NA (Kim 5) Yersinia ATCC 13980 Hare pseudotuberculosis Yersinia ATCC 6902 NA pseudotuberculosis Yersinia ATCC 27802 Mink pseudotuberculosis Yersinia ATCC 907 NA pseudotuberculosis Clinical isolates (n = 32) Enterobacter CDHS 6820-10-71 Clinical isolate aerogenes Enterobacter CDHS 1311-8-72 Clinical isolate aerogenes Enterobacter CDHS 87A-02378 Clinical isolate asburiae Enterobacter CDHS 83A-00556 Clinical isolate cancerogenus Enterobacter CDHS 6217-5-70 Clinical isolate cloacae Enterobacter cloacae CDHS 0996-7-76 Clinical isolate Enterobacter CDHS 88A-04122 Clinical isolate gergoviae Enterobacter CDHS 83A-04505 Clinical isolate hormaechei Enterobacter CDHS 03X-02073 Clinical isolate sakazakii Hafnia alvei CDHS 06X-02533 Clinical isolate Hafnia alvei CDHS 06X-002534 Clinical isolate Klebsiella oxytoca CDHS 96A-14214 Clinical isolate Klebsiella oxytoca CDHS 05X-00690 Clinical isolate Klebsiella ozaenae CDHS 89A-05504 Clinical isolate Klebsiella ozaenae CDHS 6406-10-70 Clinical isolate Klebsiella CDHS 06X-03044 Clinical isolate pneumoniae Klebsiella CDHS 06X-03046 Clinical isolate pneumoniae Klebsiella CDHS 92A-02214 Clinical isolate pneumoniae Klebsiella CDHS 06X-03045 Clinical isolate pneumoniae Klebsiella CDHS 82A-00828 Clinical isolate rhinoscleromatis Klebsiella CDHS 3124-3-78 Clinical isolate rhinoscleromatis Pantoea CDHS 03X-00304 Clinical isolate agglomerans Pantoea CDHS 99A-06032 Clinical isolate agglomerans Pantoea CDHS 96A-04392 Clinical isolate agglomerans

Genus/species

Pond water Sputum Lake sediment NA NA Wound

Incorrect Genus/species Genus/species Genus/species Genus Genus/species

Surgical wound

Genus/species

NA

Genus/species

Axillary lymph node Catheter

Genus Genus

Oropharynx

Genus/species

Human throat

Genus/species

NA

Genus/species

Oropharynx

Genus/species

NA

Genus/species

Chest fluid

Genus/species†

Human periodontal abscess NA

Incorrect

Clinical isolate Shirasu food poisoning NA

Genus/species Genus/species

Genus/species

Genus/species

Source/ sample no.

Isolation source

PCR/ESIMS call Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus Genus/species Genus Genus

Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus Genus Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species Genus/species

C.D. Baldwin et al. / Diagnostic Microbiology and Infectious Disease 63 (2009) 403–408 Table 2 (continued) Organism (no. of strains/isolates)

Source/ sample no.

Isolation source

Raoultella ornithinolytica Raoultella planticola Serratia marcescens Serratia marcescens Serratia odorifera Serratia plymuthica Serratia rubidaea Serratia rubidaea

CDHS 87A-03732 Clinical isolate

Genus/species

CDHS 87A-06657 Clinical isolate

Genus/species

CDHS 04X-00054 Clinical isolate

Genus/species

CDHS 97A-6554 Clinical isolate

Genus/species

CDHS 81A-01069 Clinical isolate CDHS 87A-06656 Clinical isolate

Genus/species Genus/species

CDHS 4839-3-76 Clinical isolate CDHS 12024-11-72 Clinical isolate

Genus/species Genus/species

407

analyzed, 142 (91%) were correctly identified to the genus level and 115 (74%) were correctly identified to the genus and species level. Only 14 (9%) isolates were misidentified in this study (Table 2). Reanalyzing the data showed no clear error in data interpretation. 16S rRNA PCR and sequencing was performed on the misidentified samples, which was followed by BLAST searches to confirm their identity. Of these 14 organisms, sequence data for 10 samples confirmed the original USAMRIID panel ID. For 3 of the samples, no PCR amplicon could be produced. For Campylobacter jejuni (ATCC 33560), 16S rRNA sequencing revealed a 99% identity match to Streptococcus parasanguinis, which exactly matches the PCR/ESI-MS ID (Table 3). The T5000 biosensor using the PCR/ESI-MS bacterial surveillance assay is capable of accurately identifying both a wide range of bacterial isolates, as well as many closely related species (e.g., members of the family Enterobacteriaceae). As Table 2 demonstrates, many bacteria were identified to the species level using only this single assay, including key human and animal pathogens. Of the 27 organisms that were identified only to the genus level, many would be correctly identified at the species level with further database population. Certain genera, such as Brucella and portions of Bacillus, are classically difficult to identify because of the low genetic variability among species. However, it appears that further database population and software modifications that are currently underway will lead to a more confident separation of these and other bacterial organisms at the species level. This also holds true for the 14 samples that were misidentified. Whereas the “genus correct” calls had partial database population, leading to genus level identification, the misidentified samples were those that had little or no base composition information in the database (Table 3). Therefore, although the T5000

PCR/ESIMS call

NA = not available; Genus = correct at the genus level; /Genus/species = correct at the genus and species level; CDHS = California Department of Health Services.

each sample from various parts of the genome (i.e., multilocus). When the multiprimer data were combined, the software triangulated down to only a few, often one, probable match for pure samples. After all samples were analyzed by the system's processing software, a list of possible ID matches and correlating confidences was generated. This information was accompanied by the raw spectra, base composition, single primer matches, and other details for each sample and reaction well. The blinded panel of 156 organisms was assigned IDs using both system-generated calls and interpretation of the accompanying data. Table 2 lists all the bacterial isolates used in this study, along with the identifications that were assigned to each isolate by the PCR/ESI-MS assay. Only the genus and species assignments were made from the system data. Of the 156 isolates

Table 3 Characteristics of misidentified bacterial isolates in this study Bacterial isolate

PCR/ESI-MS ID

16S BLAST results (highest percentage match)

Number of primers for which the system had base composition data for target bacteria

Achromobacter xylosoxydans Campylobacter jejuni Chryseobacterium meningosepticum Citrobacter freundii Enterobacter cloacae Enterobacter dissolvens Halobacterium salinarum Klebsiella pneumoniae Lactobacillus jensenii Pantoea agglomerans Proteus mirabilis Providencia stuartii Serratia marcescens Tannerella forsythia

Bordetella bronchiseptica Streptococcus parasanguinis Escherichia coli Escherichia coli Escherichia coli Klebsiella pneumoniae Escherichia coli Serratia liquefaciens Tetragenococcus halophilus Escherichia coli Yersinia frederiksenii Klebsiella pneumoniae Pseudomonas agarici Shigella sonnei

Achromobacter xylosoxidans (99%) Streptococcus parasanguinis (99%)a Chryseobacterium meningosepticum (99%) Citrobacter freundii (99%) Enterobacter cloacae (99%) Enterobacter dissolvens (99%) NPb Klebsiella pneumoniae (99%) Lactobacillus jensenii (99%) Enterobacter aerogenes (98%) Proteus mirabilis (99%) NP NP Tannerella forsythia (99%)

None 7 (16S; 23S; rplB) None 4 (16S) 4 (16S) None None 11 None 11 7 None 11 None

NP = not performed. a No Campylobacter species was seen in the highest 500 BLAST hits. b No PCR amplicon was obtained.

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system is a powerful tool that uses a single assay for the broad-range amplification and detection of many bacterial isolates, the limiting factor is the completeness of the base composition database. As new strains emerge and novel system implementations arise, the database continues to mature and is readily updated. Interestingly, based on the PCR/ESI-MS data, the system identified Campylobacter jejuni (ATCC 33560) as Streptococcus parasanguinis, which was confirmed by 16S rRNA PCR and sequencing. This is consistent with other data on several isolates obtained ATCC independently, suggesting at least certain lots of the bacteria could have been mislabeled. The format and wide-range applicability of the T5000 system provide for a novel and powerful tool for identifying unknown bacteria. The cost and turnaround time of the system are comparable to other molecular methods that incorporate standard PCR. For example, the cost of analyzing a specimen using the 16-primer set bacterial surveillance assay is approximately $45–50 per sample, which equates to approximately $3 per PCR. Likewise, the turnaround time for a single specimen starting with purified DNA is generally 4 h. The system allows for the highly accurate identification of unknown bacterial isolates at the genus level. It is also capable of differentiating a wide variety of bacteria to the species level. Because the systems sample processing and database software is capable of regular updates, further database population and software modifications will increase the power of this system for separating very closely related organisms. References Adderson EE, et al. (2008) Identification of clinical coryneform bacterial isolates: comparison of biochemical methods and sequence analysis of 16S rRNA and rpoB genes. J Clin Microbiol 46:921–927. Adekambi T, Colson P, Drancourt M (2003) rpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria. J Clin Microbiol 41:5699–5708. Adekambi T, Berger P, Raoult D, Drancourt M (2006) rpoB gene sequencebased characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int J Syst Evol Microbiol 56:133–143. Ash C, Farrow JA, Dorsch M, Stackebrandt E, Collins MD (1991) Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int J Syst Bacteriol 41:343–346.

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