1. Introduction
 Fecal contamination of surface waters is a serious environmental
 problem that limits the use of water for various purposes, such as
 agriculture, aquaculture, source of drinking water and recreation
 (Simpson et al., 2002). The most widely used method for measuring
 fecal pollution of recreational water is to count fecal indicator
 bacteria, such as total coliforms, fecal coliforms, fecal enterococci
 and Escherichia coli. However, there are several limitations associated
 with traditional application of E. coli or fecal coliforms as indicators.
 First, standard methods involve the lengthy culturing time of at least
 18–24 h. Second, major microfloral populations of the human gut are
 not E. coli or fecal coliforms, but are obligate anaerobes, such as the
 classes Clostridia and Bacteroidetes in the phyla Firmicutes and
 Bacteroidetes/Chlorobi, respectively (Victor et al., 1996; Hayashi et
 ☆ Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank
 databases under the accession numbers GU130198 to GU130203.
 ⁎ Corresponding author. College of Public Health, Division of Environmental Health
 Sciences, The Ohio State University, Columbus, Ohio 43210, USA. Tel.: +1 614 292
 5546; fax: <PRESIDIO_ANONYMIZED_PHONE_NUMBER>.
 E-mail address: <PRESIDIO_ANONYMIZED_EMAIL_ADDRESS> (J. Lee).
 0167-7012/$– see front matter © 2010 Elsevier B.V. All rights reserved.
 doi:10.1016/j.mimet.2010.07.012
 al., 2002; Hold et al., 2002).These are not easily cultivated (Eckburg et
 al., 2005).
 Recently, in order to overcome this hindrance, rapid detection
 methods, such as real-time PCR (Seurinck et al., 2005; Layton et al.,
 2006) or FISH (Collado and Sanz, 2007; Goh et al., 2009), have been
 developed by targeting 16S rRNA genes of significant fecal bacteria
 (e.g. genus Bacteroides (Okabe and Shimazu,2007). Oneadvantagefor
 using Bacteroides is that the genus hasa highdegreeofhost-specificity
 attributable to differences in source animal digestive systems, which
 enables Bacteroides to be used as an effective tool for microbial source
 tracking (MST) in water when molecular techniques are employed
 (Bernhard and Field, 2000; Simpson et al., 2004; Dick et al., 2005;
 Seurinck et al., 2005; Layton et al., 2006; Reischer et al., 2007).
 Even though many field tests have confirmed the efficacy of 16S
 rRNA gene-based molecular markers as human-specific or human
associated, false-positive and cross-amplification from other fecal
 sources, such as pig (Layton et al., 2006), dog (Ahmed et al., 2008),
 and cat (Kildare et al., 2007), have been reported. While some
 discrimination could be possible between homeothermic animal- and
 human-derived fecal contamination by 16S rRNA markers, it has been
 reported that 16S rRNA markers may not meet the standards as a
 useful tracking tool (McLain et al., 2009). The underlying problem is
312
 C.S. Lee, J. Lee / Journal of Microbiological Methods 82 (2010) 311–318
 the inherent limitation of the 16S rRNA gene as a marker for MST. 16S
 rRNA genes have a slow rate of evolutionary change and are thereby
 relatively conserved across related species which has resulted in few
 interspecies polymorphisms. Multiple ribosomal RNA (rrn) operon
 copies reside in a single bacterial genome (Acinas et al., 2004)
 contributing to the limited amount of interspecies polymorphism
 observed in some taxonomic groups (Boyer et al., 2001; Clarridge,
 2004).
 Furthermore, the genera Bacteroides and Prevotella are closely
 related to each other. The genus Prevotella is a moderately
 saccharolytic and bile-sensitive species, which once belonged to the
 genus Bacteroides (Shah and Collins, 1990). Many species of Prevotella
 have been isolated from human sources associated with the oral
 cavity (Sakamoto et al., 2004; Downes et al., 2005; Sakamoto et al.,
 2005a,b) and from tissues of the pelvic region, including the
 genitourinary tract, vagina and cervix (Brook and Frazier, 1997;
 Puapermpoonsiri et al., 1997; Mikamo et al., 1999; Fredricks et al.,
 2005; Hyman et al., 2005). All of these limitations of 16S rRNA-based
 methods make it difficult to discriminate target bacteria particularly
 when Bacteroides and Prevotella coexist in the contamination source.
 Recently, several housekeeping genes, such as rpoB, gyrB, elonga
tion factor Tu (EF-Tu), phosphoglycerate kinase (pgk), and heat shock
 protein (dnaK) genes, have been reported to evolve much faster than
 16S rRNA genes. Consequently they are used as alternative genetic
 markers providing better discrimination among closely related
 species (Volokhov et al., 2007). Eubacteria have two indispensable
 type II DNA topoisomerases; DNA gyrase encoded by gyrB and gyrA,
 and topoisomerase IV encoded by parE and parC (Huang, 1996).
 Amongthese genes, gyrB is a single-copy gene, present in all bacteria,
 whichencodestheB-subunitofDNAgyrase(topoisomerasetypeII).It
 maybeapotentiallyuseful geneforthedetectionandidentification of
 bacteria (Bhattacharya et al., 2003; Kawamura et al., 1999; Liu et al.,
 2003; Victor et al., 1996).
 Based on these advantages, this study focused on the gyrB gene
 marker in order to achieve higher specificity when targeting B. fragilis
 and to differentiate B. fragilis from other Bacteroides and Prevotella
 species. The performance of the real-time PCR system as a sensitive,
 specific and rapid tool for detection of recreational water contami
nation was investigated using human and cow fecal samples.
 2. Materials and methods
 2.1. Bacterial strains
 The type species and environmental isolates used in this study are
 listed on Table 1. The type strains were purchased from the American
 Type Culture Collection (Bacteroides fragilis ATCC 25285T, Bacteroides
 ovatus ATCC 8483T and Prevotella melaninogenica ATCC 25845T). B.
 fragilis ATCC 25285T and B. ovatus ATCC 8483T were propagated on B.
 fragilis bile-esculin agar (Oxyrase, Inc., Mansfield, OH) in GasPak jars
 (BBL Microbiology Systems, Cockeysville, MD) containing an Anaero
Pack system(Mitsubishi GasChemical, Tokyo,Japan) for 72 h at 37 °C.
 P. melaninogenica ATCC 25845T was grown at 37°C on Eggerth
 Gagnon (EG) agar (Eiken Chemical Co. Ltd., Tokyo, Japan) supple
Table 1
 Type strains used in this study.
 Strain
 environmental isolate, B. fragilis strains 1AL
 environmental isolate, B. fragilis strains 1B2
 environmental isolate, B. fragilis strains 1BL
 B. fragilis ATCC 25285T
 B. ovatus ATCC 8434T
 Accession no.
 gyrB
 GU130198
 GU130199
 GU130200
 CR626927
 AAXF02000049
 P. melaninogenica ATCC 25845T
 ACSI01000031
 16S rRNA
 GU130201
 GU130202
 GU130203
 CR626927
 AB050108
 AY323525
 mented with 5% (v/v) horse blood, 5 mg/l of haemin, and 0.1 mg/l of
 vitamin K1 in GasPak jars for 72 h at 37 °C. We also used three B.
 fragilis strains, 1AL, 1B2 and 1BL, which were isolated from
 environmental waterandidentifiedasB.fragilis with100%similarities
 of the 16S rRNA sequence of B. fragilis ATCC 25285T.
 2.2. Primer design based on gyrB gene
 Atotal of 322 gyrB sequences were retrieved from NCBI's GenBank
 database and aligned using Clustal X (Larkin et al., 2007). After the
 alignment, the sequences were manually searched for homologous
 regions specific for Bacteroides fragilis by using Bioedit (Hall, 1999).
 The selected regions were analyzed further to search homologous
 nucleotide sequences in the GenBank database by using the
 BLAST database search program (http://www.ncbi.nlm.nih.gov/
 tools/primer-blast/) and Probe Match application at the Ribosomal
 Database Project II (http://rdp.cme.msu.edu/probematch/). Several
 regions were selected for designing of primers and probe and each
 candidate was double-checked using the search programs, Primer
Blast and Probe Match, in order to minimize false cross-amplification
 with other bacteria. The Primer-Blast results were chosen because the
 NCBI database had more sequence information from environmental
 isolates and clones. Finally, one sequence subset was selected as a
 primer/probe set which showed the satisfying specificity for the B.
 fragilis. After obtaining the optimal sequence region of B. fragilis,
 forward andreverseprimers,and aminorgroovebinder(MGB)probe
 were determined by Primer Express® software (Applied Biosystems,
 Foster City, CA).
 2.3. Primer specificity tests with PCR
 In order to compare the specificity of the new gyrB-based primers,
 we conducted PCR amplification with a gyrB-based primer and two
 16S rRNA-based primer sets (qHS601F/qBac725R (Okabe et al., 2007)
 andg-Bfra-F/g-Bfra-R (Matsuki et al., 2002) with genomic DNAfrom3
 environmental isolates (1AL, 1B2, 1BL) and 3 type strains(B. fragilis, B.
 ovatus and P. melaninogenica). DNA templates were extracted from
 these 6 strains with a DNeasy Tissue Kit (Qiagen, Valencia, CA)
 following the manufacturer's instructions. Their concentrations were
 measuredwithNanoDrop(NanoDropTechnologies,Wilmington,DE).
 The DNA amount was adjusted as 10ng for template DNA per 1 PCR
 tubebecauseanamplificationreactionwouldbeconsiderablyaffected
 by the initial DNA concentration. PCR was conducted using a
 MultiGene Thermal Cycler (Labnet International, Inc., Edison, NJ) to
 generate a different optimal annealing point at the same time.
 Weused two reference 16S rRNA-based primers. For convenience
 of throughout this study, they will be referred to as Human-Bac1
 primer set for qHS601F/qBac725R and B.fragilis group-specific primer
 set for g-Bfra-F/g-Bfra-R, respectively. We compared our newly
 designed gyrB-based primers with these two references. The
 Human-Bac1 primer set had an optimal annealing temperature of
 62 °C and the B. fragilis group-specific primers had a low annealing
 temperature of 50 °C. The optimal annealing temperature for the gyrB
 primers was 58 °C and the amplification condition was the same with
 the exception of the annealing temperature. PCR was carried out by
 incubation for 3 min at 94 °C, 30 cycles of denaturation at 94 °C for
 1 min, annealing at each appropriate temperature for 30 s, extension
 at 72 °Cfor1 min,finalextensionat72 °Cfor10 minwitheachprimer
 set. After PCR amplification, aliquots (5 μl) of the PCR products were
 analyzed by electrophoresis on a 1.0% agarose gel stained with 0.1 μg/
 ml ethidium bromide in TBE buffer (89 mM Tris–borate, 2 mM EDTA)
 at 100 V for 40min. The bands were documented with a Bio-Rad
 Quantity One Gel Doc system (Bio-Rad, Hercules, CA) with a 1 kb
 (Invitrogen, Carlsbad, CA) DNA ladder as molecular weight marker.
C.S. Lee, J. Lee / Journal of Microbiological Methods 82 (2010) 311–318
 313
 2.4. Sequencing and phylogenetic analysis
 Excess primers and dNTPs were removed from the remaining PCR
 products using a QIAquick PCR purification kit (Qiagen, Valencia, CA).
 The amplified DNA products were sequenced at the Plant-Microbe
 Genomics Facility at The Ohio State University (http://pmgf.biosci.
 ohio-state.edu/) by an ABI Prism 3730 DNA analyzer (Applied
 Biosystems). Multiple alignments of the DNA sequences allowed
 phylogenetic comparisons between the Bacteroides and Prevotella
 species with the gyrB and 16s rRNA genes. Reference sequences of
 Bacteroides and Prevotella were retrieved from GenBank database,
 aligned by Clustal X and trimmed by Bioedit which allowed the
 construction of two phylogenetic trees based on 16S rRNA and gyrB
 genesusingMEGA(Tamuraetal.,2007).Theneighborjoiningmethod
 with Kimura 2 parameter model (Saitou and Nei, 1987), Maximum
likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge and
 Farris, 1969) were used to draw trees. A bootstrap analysis was
 performed to assess the confidence limits of the branching with 1000
 replicates. Only values greater than 50 were included in the results.
 2.5. Standard curves and comparison of gyrB-based and 16S rRNA-based
 primers by TaqMan real-time PCR
 With the newly designed gyrB-based primers, real-time PCR was
 carried out to obtain standard curves of B. fragilis. The Human Bac1
 primerset wastestedasa16SrRNA-basedreferenceprimerset.TheB.
 fragilis group-specific primer set, which is another 16S rRNA-based
 reference, was not originally designed for the TaqMan system, but for
 the SYBR probe system, so it could not be directly comparable with
 our primer set. Serial dilutions of B. fragilis were prepared by spiking
 cultured cells into 1 ml of sterile phosphate buffered saline (PBS;
 0.14 M NaCl, 2.7 mM KCl, 10.1mM Na2HPO4, 1.8mM KH2PO4 [pH
 7.6]) (Sigma-Aldrich, St. Louis, MO). Ten μl of the well mixed solution
 of B. fragilis was used to count colony forming units (CFU) after
 anaerobic culture on BBE plates and the 990 μl of the remaining
 solution was used for DNA extraction using a DNeasy Tissue kit
 (Qiagen, Valencia, CA). The extracted DNA was used for real-time PCR
 to quantify B. fragilis. Real-time PCR was performed and a standard
 curve was derived by plotting threshold cycles (CT) against the log
transformed cell counts (CFU/PCR).
 An ABI 48-well StepOneTM Real-Time System (Applied Biosys
tems) was used with a total 25 μl mixture which consisted of 5 μlof
 template DNA, 12.5 μl of TaqMan universal PCR master mix (PCR
 buffer, deoxynucleoside triphosphates, AmpliTaq Gold polymerase,
 internal reference signal 6-carboxy-x-rhodamine [ROX], Amp Erase
 uracil N-glycosylase [UNG], MgCl2; Applied Biosystems), 500 nM of
 each primer, and 250nM of each probe labeled with 6-carboxy
 f
 luorescein (FAM). A mixture of all PCR regents without any template
 DNA was used as a negative control for each PCR reaction. Thermal
 cycling consisted of an initial cycle of 50 °C for 2 min (activation of the
 UNG) and 95°C for 10min (activation of the AmpliTaq Gold DNA
 polymerase), followed by 40 cycles of denaturation at 95 °C for 15 s
 and annealing and extension at 60 °C for 1 min. For the Bacteroides
Prevotella-specific primer set, PCR reactions were carried out by
 incubation for 2 min at 50 °C and for 10 min at 95°C, followed by
 40 cycles of denaturation at 95 °C for 15 s and annealing and
 extension at 62 °C for 1 min.
 2.6. End-point and real-time PCR assays for cow, pig, dog, and human
 fecal samples
 In order to check the host-specificity of the newly designed primer
 set, we testedthe specificity of all three primersets withfecal samples
 from cow, pig, dog, and human. Individual fresh fecal samples were
 collected from apparently healthy human and animal sources. Cow
 and pig feces were obtained from the Waterman farm (Dairy Science
 Center, TheOhioStateUniversity,Columbus,Ohio).Dogsampleswere
 obtained from local pet owners and represented several different
 breeds. All fecal samples were collected using sterile swabs (BBL
 CultureSwab; Becton Dickinson, Sparks, MD) and immediately
 transported to the laboratory on ice. For DNA extraction, 10 ml of
 sterile PBS was added directly into the swabs and vortexted. The fecal
 suspension (1 ml) was transferred into a sterile microcentrifugal tube
 and its weight was measured by subtracting the post-centrifugation
 weight from the pre-centrifugation weight. The centrifugation was
 performed at 10,000 g for 1 min at 4 °C. With the known concentra
tion of fecal suspensions, all the fecal samples were adjusted to the
 same amount of 5mg fecal pellet by transferring appropriate volume
 into the tube. DNA was extracted using the QIAmp DNAStool Mini Kit
 (Qiagen) for end-point and real-time PCR assays with Human Bac1
 and gyrB-based primers. The concentration of the DNA was deter
mined from the final elute of 200 μl from the DNA extraction with a
 NanoDrop system (NanoDrop Technologies).
 3. Result
 3.1. Designing of gyrB-based primers for B. fragilis
 Analysis of the 322 gyrB sequences of Bacteroides and Prevotella in
 the GenBank database allowed the identification of B. fragilis-specific
 regions and the design of the primer set (Fig. 1 and Table 2). One
 primer set covering only 54-bp size was composed of a forward
 primer (Bf904F), a reverse primer (Bf958R), and the TaqMan probe
 (Bf923MGB). An additional reverse primer was designed (Bf1272R)
 because it could generate enough size of sequences (368-bp) for
 phylogenetic analysis.
 Multiple alignment analysis showed that our reverse primer,
 Bf958R, supported higher specificity for B. fragilis than Bf904F. We
 evaluated the specificity of the newly designed gyrB-based primer
 (Bf904F/Bf958R), 16S rRNA-basedHumanBac1(qHS601F/qBac725R),
 and B. fragilis group-specific (g-Bfra-F/g-Bfra-R) primer sets using B.
 fragilis, other Bacteroides (B. ovatus) and Prevotella (P. melaninogenica)
 by end-point PCR. The results showed a distinction in the amplicons
 bythethreeprimersetsintargetingB. fragilis (Fig. 2). As expected, we
 obtained 54-bp size gyrB gene products from both environmental B.
 fragilis isolates and type strain of B. fragilis (ATCC 25285T), and
 negative results from B. ovatus and P. melaninogenica when using
 Bf904F/Bf958R (Fig. 2A). The 150-bp size 16S rRNA gene regions by
 qHS601F/qBac725R were amplified in all of environmental B. fragilis
 isolates and type strains of B. fragilis, B. ovatus and P. melaninogenica
 (Fig. 2B). The 495-bp size 16S rRNA gene region by g-Bfra-F/g-Bfra-R
 was amplified only in environmental B. fragilis isolates and type
 strains of B. fragilis and B. ovatus, but not from P. melaninogenica
 (Fig. 2C). The PCR results confirm that our gyrB-based primer, Bf904F/
 Bf958R, can discriminate between B. fragilis and other closely related
 species, such as B. ovatus and P. melaninogenica.
 3.2. Sequencing and phylogenetic analysis
 The PCR products produced by our gyrB-based primer set and one
 16S rRNA-based Human Bac1 primer set (qHS601F/qBac725R) were
 further investigated by sequence analysis. The other 16S rRNA-based
 B. fragilis group-specific primer (g-Bfra-F/g-Bfra-R) was not compared
 for this purpose because it had a significantly different amplicon size
 (495-bp) from the amplicons produced by the gyrB-based (54-bp)
 and HumanBac1(150-bp) primers. The sequences (Fig. 3) confirmed
 again that new proposed primer set could amplify only the gyrB gene
 of B. fragilis. Although we obtained the PCR products from B. fragilis by
 using the Bf904F/Bf958R, we could not obtain sequencing data
 because their sequences were too short to analyze sequencing. In
 order to overcome this limitation, another reverse primer (BF1272R)
 was used to amplify N100 bp of PCR amplicons for sequence analysis.
C.S.Lee, J.Lee/ JournalofMicrobiologicalMethods82(2010)311
Fig.1.MultiplealignmentsoftheB. fragilis-specificprimers,Bf904F,Bf958R,Bf1275R,andtheTaqManprobeBf923MGBfortheB. fragilisgyrBsequenceincomparisonwiththe
 homologousgyrBsequencesofotherBacteroidesandPrevotella
 Analyzed sequences were added up with other 16S rRNA and gyrB
 gene sequences from GenBank and EMBL databases to construct
 phylogenetic dendrograms.
 Bacteroides 16S rRNA sequences are as follows: B. acidifaciens
 (AB021164), B. caccae (X83951), B. capillosus (AY136666), B. cellulosi
lyticus (AJ583243), B. dorei (AB242142), B. eggerthii (AB050107), B.
 f
 inegoldii (AB222699), B. fragilis (CR626927), B. gallinarum (AB253732),
 B. graminisolvens (AB363973), B. helcogenes (AB200227), B. intestinalis
 (AB214328), B.massiliensis (AY126616), B. nordii (AY608697), B.ovatus
 (AB050108), B. pectinophilus (DQ497993), B. plebeius (AB200217), B.
 pyogenes(AB200229),B.salyersiae(AY608696),B.stercoris(X83953),B.
 tectus (AB200228), B. thetaiotaomicron (AE015928), B. uniformis
 (AB050110), B. vulgatus (CP000139), B. xylanisolvens (AM230650).
 Prevotella 16S rRNA sequences are as follows: P. bryantii (AJ006457), P.
 buccae (L16477), P. copri (AB064923), P. heparinolytica (L16487), P.
 micans (AF481228), P. nanceiensis (AY957555), P. oralis (AY323522),
 P. oris (L16474), P. paludivivens (AB078827), P. ruminicola (L16482), P.
 shahii (AB108825).
 Bacteroides gyrBsequencesareasfollows:B.caccae(AAVM02000009),
 B. capillosus (AAXG02000004), B. cellulosilyticus (ACCH01000128),
 B. dorei (DS995534), B. eggerthii (ABVO01000051), B. finegoldii
 (ABXI02000076), B. fragilis (CR626927), B. intestinalis (ABJL02000007),
 B. ovatus (AAXF02000049), B. pectinophilus (ABVQ01000037), B. plebeius
 (ABQC02000004), B. stercoris (ABFZ02000020), B. thetaiotaomicron
 (AE015928), B. uniformis (AAYH02000048), B. vulgatus (CP000139),
 Prevotella gyrB sequences are as follows: P. copri (ACBX01000202), P.
 melaninogenica (ACSI01000031), P. oralis (AB048191), P. tannerae
 (ACIJ02000017).
 Twophylogenetic trees were created with the available sequences
 of 16S rRNA and gyrB gene from GenBank and EMBL database, so not
 all the bacterial species could be equally entered in each tree. Thus, a
 simple and direct comparison of the genetic distances of the two
 phylogenetic trees was not feasible. However, the following observa
tions were made. First, the rate of genetic divergence of the sequence
 amplified by the gyrB-based primer set differed greatly from that of
 the 16S rRNA-based primer set. For example, compared with the
 sequence amplified by the 16S rRNA-based primers, the sequence
 amplifiedbythegyrBprimersetshoweda4-to10-foldincreaseinthe
 length of branches between the closely related species of Bacteroides
 and Prevotella. Moreover, the topology of the phylogenetic tree based
 on the 16S rRNA sequence was quite different from the tree based on
 the gyrB sequence. It demonstrated that 8 Bacteroides species (B.
 acidifaciens, B. nordii, B. fragilis, B. salyersiae, B. thetaiotaomicron, B.
 xylanisolvens, B. finegoldii and B. ovatus) were clustered into one group
 in the 16S rRNA-based phylogenetic tree (Fig. 3A), but they were
 totally separated into each cluster in the gyrB gene-base tree (Fig. 3B).
 Secondly, there was a difference in the classification of the target
 bacteria between the sequences amplified by the 2 primers. For
 instance, even though the 16S rRNA sequences of 3 environmental
 isolates, 1AL, 1B2 and 1BL, were 100% identical to the type strain of B.
 fragilis, their intrageneric relatedness with other species of Bacteroides
 was too close to be positioned solely with the B. fragilis species
 (Fig. 3A). In contrast, with the amplification by the gyrB gene, these 3
 isolates were clearly clustered only with the B. fragilis species (Fig. 3B).
 3.3. Performance of real-time PCR with new primers and probe for
 B. fragilis
 Aseries of experiments were conducted with B. fragilis to compare
 the results obtained using gyrB-based (Bf904F/Bf923MGB/Bf958R)
 and the 16S rRNA-based primers and probe (qHS601F/qHS624MGB/
 qBac725R)(Fig. 4). For this, 10-fold serial dilutions of known B. fragilis
 cells were prepared and CT values were checked with the 48-well
 StepOne™Real-TimeSystem (Applied Biosystems) using each primer
 set. The TaqMan PCR reagent contains a passive reference dye as an
 internal reference to which the reporter dye was normalized during
 data analysis. This allows correction of fluctuations in fluorescence
 due to changes in concentration and volume of the reaction mixture.
 Table2
 Nucleotidesequencesandtargetsofprimersandprobesusedforreal-timePCRassayinthisstudy.
 Name Sequence Gene Target Reference
 Bf904F 5′-GGCGGTCTTCCGGGTAAA-3′ gyrB B. fragilis Thisstudy
 Bf923MGB 5′-TGGCCGACTGCTC-3′ gyrB B. fragilis Thisstudy
 Bf1272R 5′-TGGCATATAGCGGAAGAAAAAAG-3′ gyrB B. fragilis Thisstudy
 Bf958R 5′-CACACTTCTGCGGGTCTTTGT-3′ gyrB B. fragilis Thisstudy
 qHS601F 5′-GTTGTGAAAGTTTGCGGCTCA-3′ 16SrRNA B. fragiliscluster Okabeetal.(2007)
 qHS624MGB 5′-CGTAAAATTGCAGTTGA-3′ 16SrRNA B. fragiliscluster Okabeetal.(2007)
 qBac725R 5′-CAATCGGAGTTCTTCGTGATATCTA-3′ 16SrRNA Bacteroides–Prevotella BernardandField(2000)
 g-Bfra-F 5′-ATAGCCTTTCGAAAGRAAGAT-3′ 16SrRNA B. fragilisgroup Matsukietal.(2002)
 g-Bfra-R 5′-CCAGTATCAACTGCAATTTTA-3′ 16SrRNA B. fragilisgroup
TheCTvalueisthecycleatwhichastatisticallysignificantincreasein
 ΔRnwasfirstdetectedassociatedwithexponential increaseofPCR
 product.Linearregressionanalysesofthereal-timePCRresultsofthe
 gyrBandthe16SrRNAampliconsproducedstandardcurveswith
 similar slopes (−3.367,−3.332)andgoodcorrelationcoefficients
 (r2=0.998, 0.997), butdifferenty-intercepts(41.89, 38.99) inthe
 rageof1.1×102to1.1×107(CFU/PCR).
 3.4.End-pointandreal-timePCRassays forcow,pig,dog,andhuman
 fecalsamples
 Host-specificitywas testedwithhumanandotheranimal fecal
 samplesusingend-pointPCR(Table3).TheextractedDNAconcen
trations were 2.8±0.6ng/μl (cow, n=10), 4.0±0.9ng/μl (pig,
 n=10), 3.4±0.9ng/μl (dog, n=10), and3.6±0.5ng/μl (human,
 n=10).Allthethreeprimersetsshowed100%positivePCRreaction
 with thehumansamples. However, somecross-amplificationwas
 observed fromnon-human fecal samples.With theHumanBac1
 (qHS601F/qBac725R)primerset,positivereactionswereobservedin
 70%pig,40%cow,and30%dogsamples.WiththeB. fragilisgroup
specificprimer(g-Bfra-F/g-Bfra-R),positivereactionswereobtained
 from50%pig,30%cow,and10%dogsamples.WiththegyrB-based
 primerset(Bf904F/BF958R),positivereactionswereobservedinonly
 10%pigsamplesandnocross-amplificationwasobtainedfromallthe
 cowand dog samples. This result indicates that the gyrB-based
 B. fragilis-specific primer has superior specificity against human
originatedfecalcontamination.
 Thesamesetoffecalsampleswastestedwithreal-timePCRassayfor
 validatinghuman-specificfecalcontamination.WiththeuseofHuman
 Bac1, theaverageCtvalueswere18.61±1.23(human),21.33±2.00
 (pig), 24.75±1.40 (cow), and 28.07±4.89 (dog) and theywere
 equivalentto1.4±0.8×1010,3.8±4.6×109,2.2±1.7×108,and2.1±
 3.2×108CFU/goffeces.Thisconversionwasbaseduponthestandard
 curve(Fig.4B)andthedilutionfactors.Thesameprocedurewasused
 withthegyrB-basedprimerset.TheaverageCtvalueswere27.86±0.66
 (human),35.64±0.68(pig),35.91±0.80(cow)and35.85±0.56(dog)
 and theywere equivalent to 1.3±0.7×108, 4.6±4.5×105, 4.0±
 4.0×105,2.1±2.8×105CFUB.fragilis/goffeces,respectively.
 4.Discussion
 The B. fragilis-specific detection by real-time PCR allows the
 quantificationofB. fragilis,whichisasignificantandrepresentative
 human-specificfecal indicator.Thisisthefirststudy1)todevelopa
 primersetthattargetsthegyrBgeneofB.fragilisasanewfecalsource
 marker;and2)optimizetheTaqManreal-timePCRsystemforrapid
 detectionofahuman-specificfecalsourceinrecreationalwater.We
 also investigatedthespecificityof ournewlydesignedprimersby
 comparingpreviouslyknown16SrRNA-basedprimersets.Byshifting
 thetargetmarker from16SrRNAtothegyrBgene,weareableto
 proposeanalternativeprimer systemwhich isveryspecific toB.
 fragilisasarepresentativespecies inBacteroides.Variouscandidate
 markersweredeterminedaftermultiplealignmentsof gyrBgenes
 fromtheavailabledatabaseand twoprimer setswere chosen. A
 primerset,Bf904F/Bf958R,wasusedtoamplifythe904–958regionof
 thegyrBgeneinareal-timePCRandcomparedwiththe16SrRNA
based referenceprimer set (HumanBac1). Theother primer set,
 Bf904F/Bf1272R,wasusedtoamplifythe904–1272regionofthegyrB
 geneinordertoanalyzesequencesforphylogeneticinvestigation.The
 newgyrB-basedprimer showedhigh specificity toB. fragilis and
 negativereactiontoB. ovatusandP.melaninogenica,whicharethe
 mostcloselyrelatedtoB. fragilis.
 TheothergyrB-basedprimerset(368-bpcoveringBf904F/Bf1272R
 primerset)wasmoreusefulforanalyzingsequencesofPCRamplicons
 andconstructingphylogenetictreesbecausetheampliconsproduced
 bytheBf904F/Bf958Rprimer setwasnot longenoughtoprovide
 sufficientinformationonsequences.PCRampliconsweresequenced
 andanalyzedwith16SrRNAfragmentsandthegyrB-basedphyloge
netic tree showed that it had greater resolution enabling the
 generationofamoredivergeddendrogram.Eachphylogenetictree
 wasconstructedwithpreviouslyknownsequences intheGenBank
 database. The twoconstructeddendrogramsdemonstrate that the
 gyrB-basedprimersetdifferentiatedB. fragilisfromotherBacteroides
 andPrevotellaspecies, but the16SrRNA-basedprimersetdidnot.
 OnlyB. fragiliswasamplifiedby thegyrB-basedprimer, but all B.
 fragilis, B. ovatusandP.melaninogenicawereamplifiedbythe16S
 rRNA-basedprimers.Thismayserveasapossibleexplanationtowhy
 manyfieldstudiesofMSTtargetingBacteroidesperformedinconsis
tentlyinthepast(Laytonetal.,2006;Kildareetal.,2007;Ahmedetal.,
 2008;McLainetal.,2009).
 ThenewlydevelopedTaqManprimer/probesystemforreal-time
 PCRwasoptimizedtoquantifyB. fragilisinwaterbyspikingaknown
 numberofbacterialcells.Thereal-timePCRresultsdemonstratethat
 thegyrB-basedprimersetcanbeusedtoquantifyB. fragiliswiththe
 detectionlimitof110CFU/reaction.ThegyrB-basedreal-timePCRhad
 aslightlylesssensitivitythantheoneusing16SrRNA-basedprimer
 set. The difference in y-intercepts of the calibration curveswas
 approximately3cyclesbetweenthegyrB-basedandthe16SrRNA
basedsystem,whichindicatesthat3morecyclesareneededtoyield
 thesameCTvalues.Itisequivalenttoapproximatelya1logdifference
 in colony forming unit (CFU). The differences in real-time PCR
 conditionsofthegyrB-basedandthe16SrRNA-basedprimers,suchas
 themelting point, mayhave affected the sensitivity of the PCR
 amplification,whichisexpressedasCTvalues.Additionally,16SrRNA
 genesaremorelikelytobeamplifiedthangyrBgenesbecauseatleast
 6 copies of 16S rRNAgene are contained in one B. fragilis cell
 (Klappenbachet al., 2001), butonlyonegyrBgeneexistsper cell
 (Huang,1996).Thus, fromthesamenumberofB. fragilis, inherently
 differentamountofgyrBand16SrRNAgeneswouldbeobtainedin
 theextractedDNA.Thereby, it isexpectedtoobservedifferencesin
 they-interceptsof thestandardcurvesaswellasdifferencesinthe
 sensitivityofdetectionbyreal-timePCR.
 Duringour preliminary study,we investigated the gyrB-based
 assaysystemwithrecreationalwaterinordertocheckwhetherthe
 detectionisfeasibleandwhetherthedetectionlimitisaffectedbyPCR
 inhibitorspossiblypresent inrecreationalwater.Thereal-timePCR
 Fig.2.Specificityofthenewlydesignedprimers.PCRproductsbynewgyrB-basedprimer(A)andtwo16SrRNA-basedprimers(B,C).
 315 C.S.Lee, J.Lee/ JournalofMicrobiologicalMethods82(2010)311
testforB. fragilispresent intherecreationalwaterandPBSresulted
 very similar CT values (data not shown). Statistically significant
 differencebetweenthePBSandtherecreationalwatersamplewas
 notobservedinthereal-timePCRdetectionofB. fragilis(datanot
 shown).Thisindicatedthattheenvironmentalwatermatrixdidnot
 affectthereal-timePCR.
 Anessential traitof ageneticmarker toserveasastableand
 library-independent MST tool is its temporal and geographical
 Fig.3.Comparisonofphylogenetictreesbasedon16SrRNAgeneandthegyrBgeneforthegenusBacteroidesandPrevotella.
 316 C.S.Lee, J.Lee/ JournalofMicrobiologicalMethods82(2010)311
humanandthe16SrRNA-basedhuman-specificmarkersmayhave
 thepossibilityofcross-amplificationswithswinefeces.
 Wehavedescribedarapidmonitoringsystemforahuman-specific
 fecal indicator, B. fragilis, based on the gyrB genemarker for
 indispensable type II DNA topoisomerases inbacteria. The gyrB
basedprimerhadsufficientresolutionpowertoamplifyonlyB.fragilis
 species and gyrB genemarker canbe used to designate and/or
 differentiatebacterial speciesorsubspecieswhicharespecific toa
 host.Duringthisstudy,wetestedourprimersetwithfecalsamples
 fromanimal andhumansources. ThegyrB-basedprimer set could
 differentiatehuman fecal samples fromanimal fecal samples and
 producepositiveresultsforB. fragilis.16SrRNAgeneisstillusefulfor
 groupingandclusteringinphylogeneticstudy.However, switching
 thetargetgenefrom16SrRNAtogyrBmaybeveryuseful formore
 accurate identification, classification, and phylogenetic sorting of
 bacterial lineages (YamamotoandHarayama, 1995; LaDucet al.,
 2004). Thismethod could serve as a complementary tool for
 overcoming the limitation of 16S rRNA-basedMSTmethods in
 delineatingabilityamongclosely relatedbacteriaoriginated from
 humansources.Inaddition,itcanbeappliedtoothermoleculartools
 that involveprimers andprobes, suchasmicroarray, T-RFLP, and
 othernucleotide-basedmethods


//

 Abstract Salmonella causes the majority of infections in
 humans and homeothermic animals. This article describes a
 specific polymerase chain reaction (PCR) method devel
oped for a rapid identification of Salmonella.AgyrB-tar
geted species-specific primer pair, S-P-for (50-GGT GGT
 TTC CGT AAA AGT A-30) and S-P-rev (50-GAA TCG
 CCT GGT TCT TGC-30), was successfully designed. PCR
 with all the Salmonella strains produced a 366- bp DNA
 fragment that was absent from all the non-Salmonella
 strains tested. The detection limit of the PCR was 0.01 ng
 with genomic DNA or 3.2 cells per assay. Good specificity
 was also demonstrated by fecal samples, from which only
 the gyrB gene of Salmonella was amplified. Using the
 culture-PCR method, 27 isolates on Salmonella-Shigella
 X. Ye Y. Wang X. Lin
 State Key Laboratory of Soil and Sustainable Agriculture,
 Institute of Soil Science, Chinese Academy of Sciences,
 Beijing East Road, 71, Nanjing 210008,
 People’s Republic of China
 X. Ye Y. Wang X. Lin
 Joint Open Laboratory of Soil and the Environment,
 Hongkong Baptist University & Institute of Soil Science,
 Chinese Academy of Sciences, Nanjing 210008,
 People’s Republic of China
 X. Ye
 Graduate University of Chinese Academy of Sciences,
 Beijing 100049, People’s Republic of China
 X. Lin (&)
 Department of Biology and Biochemistry, Institute of Soil
 Science, Chinese Academy of Sciences, Beijing East Road, 71,
 Nanjing 210008, People’s Republic of China
 e-mail: <PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>
 (SS) medium were rapidly identified as Salmonella, which
 was confirmed by the sequencing of the gyrB gene.
 Introduction
 Salmonella is a facultative intracellular pathogen [7]. They
 cause a wide range of human diseases, such as enteric
 fever, gastroenteritis, and bacteremia. Gastroenteritis
 associated with food-borne outbreaks is probably the most
 common clinical manifestation of the infection [3, 5, 10,
 15, 22]. Annually pathogens account for more than 25
 million infections worldwide, resulting in approximately
 200,000 deaths [9]. A study on the prevalence of Salmo
nella in broiler flocks in Europe revealed that on an average
 more than 20% of the flocks were Salmonella positive with
 a range from 0% to nearly 70% depending on the country
 [11, 12]. In China, surveillance of foodborne pathogens in
 Chinese Center for Disease Control and Prevention
 (CCDC) results showed that food of the detection of
 Salmonella had reached 7.76% [8].
 Awidely accepted standard for detecting Salmonella is a
 traditional microbiological method based on a non-selec
tive pre-enrichment, selective enrichment, subsequent
 plating on selective media and biochemical identification
 [4]. However, culture-based methods are typically time
consuming, and it may take several days before the results
 are obtained. Nucleic acid techniques offer faster detection
 systems, a number of nucleic acid-based assays for Sal
monella have been developed [18]. Numerous targeted
 probe specific for Salmonella have been described. Targets
 include antigen determinants, outer membrane proteins,
 DNA-binding proteins, and virulence-associated genes [20,
 24]. The invasion A (invA) and sefA gene, which encode
 the main subunit of the SEF14 fimbrial protein have been
 123
478
 Species-specific Primers Design
 X. Ye et al.: A gyrB-targeted PCR for Rapid Identification
 particular popular and useful targets [6, 21, 25]. Probes
 specific for Salmonella enteritidis and other group D sal
monellae have also been reported [1, 14, 19]. Otherwise,
 the GENE-TRAK and GeneQuence assays target 16 s
 rRNA. This has been a favored approach in probe assay
 development [18]. In this assay, it presents the target gene
 gyrB gene encoding the B-subunit of DNA gyrase. The
 gyrB gene has been used as the target sequence for the
 detection and identification of several species of bacteria
 by PCR-based assays [17, 27], and Salmonella has also
 been identified by microarrays method with the specific
 gyrB gene probe [16]. However, the higher cost and
 equipment requirements of the microarrays method make it
 difficult to be widely used, despite high efficiency of
 identification.
 This study describes a specific PCR detection technique
 based on the gyrB gene which can be used successfully for
 rapid identification of Salmonella from others, and first
 present a culture-PCR method to reduce time in preparation
 of samples tested.
 Materials and Methods
 Bacterial Strains
 Strains used in this study were obtained from China’s
 National Institute for the Control of Pharmaceutical and
 Biological Products. The strains representing six genera
 (Table 1) were used for primer development and PCR
 specificity assays. Bacteria were cultured on Luria–Bertani
 (LB) agar plates at 37C for 24–48 h.
 Table 1 Strains used in the study
 Name
 Strain
 Accession number
 for partial gyrB
 gene seqence
 Salmonella sub-genus II
 CMCC50215
 Salmonella sub-genus III b CMCC50381
 Salmonella enteritidis
 Salmonella enteritidis var.
 danysz
 Salmonella gallinarum
 Salmonella manchester
 Salmonella senftenberg
 Escherichia coli
 Streptococcus fecalis
 Enterobacter cloacae
 Enterobacter aerogenes
 CMCC50335
 CMCC50100
 CMCC50770
 CMCC50380
 CMCC 50210
 ATCC 25922
 CMCC 32223
 CMCC 45301
 AB598750
 AB598754
 AB598753
 AB598752
 AB598751
 AB598748
 AB598747
 AB598755
 AB598756
 AB598757
 CMCC (B) 45103 AB598749
 Citro Bacter freumdii
 Shigella flexneri
 CMCC48001
 CMCC 51229
 AB598758
 AB598759
 All the strain gyrB gene sequences (Table 1) were analyzed
 using Clustal_X, version 1.83 [28]. The sequence regions
 unique to Salmonella were used to design a series of for
ward and reverse primers. The specificity of the primers
 was then analyzed by BLASTN search of the GenBank
 database (Altschul et al. 1990), and the resulting a pair of
 primers S-P-for (50-GGTGGTTTCCGTAAAAGTA-30)
 and S-P-rev (50-GAATCGCCTGGTTCTTGC-30) with the
 best specificity to Salmonella were selected and synthe
sized by Sangon Biotech (Shanghai, China).
 PCR Reaction Volumes and Conditions
 Genomic DNA strains (Table 1) from pure cultures were
 purified using the E.Z.N.A. TM Bacterial DNA Kit(Omega
 Bio-Tek, USA), following the manufacturer’s instructions.
 The PCR reaction volumes for species detection were
 carried out in 25 ll consisting 2.5 ll buffer (100 mM
 Tris-HCl, 15 mM MgCl2, 500 mM KCl), 0.2 ll 2.5 U
 Taq polymerase (Takara Bio, China), 0.2 ll 10mM
 dNTP, and 0.5 ll of each specific primer and 1 ll purified
 DNA as template. Reaction conditions were carried out
 with profile of pre-denaturation (94C, 5 min), 35 cycles
 of denaturation (94C, 1 min), annealing (58C, 1 min),
 and extension (72C, 40 s) with final extension (72C,
 4 min). After amplification, 5 ll of each PCR product
 was analyzed through electrophoresis in 2% agarose gel
 in 19 Tris-acetate-EDTA (TAE) buffer, stained with
 ethidium bromide and visualized using an ultraviolet
 transilluminator.
 To simplify the specific PCR, a culture-PCR was
 developed on the basis of the PCR conditions. Pure cultures
 were 10-fold diluted with sterile distilled water, and then
 the dilution was used directly as PCR template. Only
 before carrying out the PCR reaction, the volumes were
 paralyzed at 99C for 10 min to release the DNA.
 Sensitivity Determined With S. enteritidis
 The sensitivity of the designed species-specific primers was
 detected by S. enteritidis CMCC50335. Liquid cultures of
 S. enteritidis at 37C for 24–48 h were used to extract the
 genomic DNA. Then, the genomic DNA was 10-fold
 serially diluted with sterile distilled water. One microliter
 of each dilution was used as a PCR template. The number
 of PCR cycles was 30. Sensitivity was also determined by
 culture-PCR. The liquid culture was 10-fold serially diluted
 with sterile distilled water. One microliter of each dilution
 was used directly as PCR template. Three independent
 123
X. Ye et al.: A gyrB-targeted PCR for Rapid Identification
 Results and Discussion
 479
 PCR assays were conducted. Each dilution was also plated
 onto LB medium to determine the cell concentration by a
 standard plate count method in triplicate, and the sensi
tivity was then calculated.
 Specificity Determination
 The specificity of PCR was validated using fresh pigs’
 fecal samples as the representative. The fecal samples were
 collected from a farm in Wuxi China. The total fecal
 genomic DNA was extracted using the E.Z.N.A. TM Stool
 DNAKit (Omega Bio-Tek, USA). The extracted DNA was
 used as a template for the specific PCR. The amplified
 product was recovered using the AxyPrepTM DNA Gel
 Extraction Kit (Takara Bio, China), cloned into a pGEM-T
 easy vector (Promega, Madison, WI, USA), and 22 clones
 were randomly chosen, sequenced by Invitrogen Life Sci
ence (Shanghai, China).
 Identification of Salmonella Isolates
 The developed culture-PCR was applied to identify the
 isolates from fecal samples. The above fresh fecal samples
 were 10-fold serially diluted with sterile distilled water and
 each 100 ll dilution was spread onto the Salmonella
Shigella (SS) media [2] and incubated for 12 h at 37C.
 The 27 isolates morphologically similar to Salmonella
 were selected and incubated in LB media for 12 h at 37C.
 The culture was used for PCR detection, and then the
 amplified products were sequenced as above.
 Phylogeny Analysis
 The gyrB sequences of the 22 clones and the 27 isolates
 were analyzed to find related sequences in the GenBank
 database by performing a BLASTN search. The similari
ties of the sequences were analyzed using the Meg Align
 program, version 7.1.0 (DNAStar, Madison, WI, USA).
 Phylogenetic tree was constructed with the MEGA pro
gram, version 4.0 [26], using the Neighbor Joining
 method [23]. A bootstrap resampling analysis was per
formed to estimate the confidence of the tree topologies.
 Nucleotide Sequence Accession Numbers
 The 22 clones from the total fecal DNA were deposited in
 the DDBJ database under accession numbers AB607965
AB607986. The gyrB sequences of the 27 isolates have
 been assigned with the following DDBJ accession numbers
 AB621372–AB621398.
 The last 20 years have seen a revolution in the methodol
ogy for the detection of Salmonella. The era of ‘‘rapid
 methods’’ began with the introduction of immunological
 methods, such as fluorescent antibody procedures and
 enzyme-linked immunosorbent assay (ELISA), followed
 soon after by DNA probe-based methods [11, 13]. With a
 variety of genetic methods for Salmonella to choose from,
 what are the advantages of our methods? There are several
 factors as follows.
 Amplification With Specific Primers
 The gyrB genes of the Salmonella species from test sam
ples were amplified and analyzed by 2% agarose gel
 electrophoresis to confirm that the primers were specific for
 all Salmonella species (Fig. 1 lanes 1–7). It obtained spe
cific amplification of a 366-bp DNA fragment by PCR with
 primers S-P-for and S-P-rev. No amplification products
 were observed from E. coli, S. fecalis, E. cloacae,
 E. aerogenes, C. Bacter freumdii, and S. flexneri genomic
 DNAs (Fig. 1, lanes 8–13), indicating that there is no
 similar or homologous region in these samples’ DNAs.
 These results showed that only a single band was amplified
 from every Salmonella samples, and so the specific primers
 used were appropriate for amplification of the gyrB region
 of Salmonella at the genus level.
 Two of the principal advantages of genetic methods are
 the superior inclusivity and specificity offered by well
designed DNA probes. This assay presents the target gene
 gyrB gene owing to three benefits. First, gyrB gene
 Fig. 1 Amplification of Salmonella DNAs with primers S-P-for and
 S-P-rev. Lanes 1–13, amplification of gyrB fragments from samples:
 1 S. sub-genus II, 2 S. sub-genus III b, 3 S. enteritidis, 4 S. enteritidis
 var. danysz, 5 S. gallinarum, 6 S. manchester, 7 S. senftenberg,
 8 E. coli, 9 S. fecalis, 10 E. cloacae, 11 E. aerogenes, 12 C. freumdii,
 13 S. flexneri, M molecular size markers (DL2000). The length of
 each amplified fragment was 366 bp. 215 9 121 mm (72 9 72 DPI)
 123
480
 X. Ye et al.: A gyrB-targeted PCR for Rapid Identification
 sequences are highly conserved and, the average base
 substitution rate of change of 0.7% per 100 million years
 to 0.8%, faster than 16S rDNA for each 1% change in 5000
 years. Second, the gyrB gene sequences permit design of
 probes with an impressive array of specificities. Finally,
 because of its encoded protein as protein, the genetic code
 inherent in children’s acquisition makes more DNA
 sequence, variations can occur without changing the amino
 acid sequence, especially in the third codon base, which
 makes the gyrB gene with high resolution in the identifi
cation of areas [29]. It was shown that gyrB gene had
 relatively higher discrimination ability, which was well
 proven by the present study.
 Among the 13 tested samples including seven species of
 Salmonella, primer pair S-P-for and S-P-rev gave the best
 discriminative of Salmonella with a unique amplification
 product of 366-bp in size. Therefore, primer pair S-P-for
 and S-P-rev, was used to develop a rapid culture-PCR
 method for identification of Salmonella. Although the
 number of the pure culture samples tested is relatively low,
 fortunately, the specificity determination results shown that
 the sequences of amplicon of the 366-bp-length gyrB gene
 from fecal sample proved that all to belong to Salmonella.
 The culture-PCR method developed was also successfully
 used in the identification of Salmonella isolates from fecal
 sample. 100% of the isolates morphologically similar to
 Salmonella on the SS medium were identified as Salmo
nella, which was confirmed through sequencing of the
 366-bp-length gyrB gene.
 Sensitivity Determined With S. enteritidis
 The detection limit of the PCR was determined by DNA
PCR and culture-PCR using 30 cycles of amplification.
 Serial 10-fold dilutions DNA of S. enteritidis were used as
 templates, and the dilutions that contained more than
 0.01 ng per microliter gave the expected amplification
 band (Fig. 2), which showed the DNA-PCR limit was
 calculated to be 0.01 ng per assay. At the same time, serial
 10-fold diluted cultures of S. enteritidis were also used as
 templates and the dilutions that contained more than 3.2
 per microliter gave the expected amplification band
 (Fig. 3) in three replicates. The PCR detection limit was
 calculated to be 4–10 cells per assay, indicating the sen
sitivity of the rapid culture-PCR method.
 The concentration of Salmonella in the environment is
 usually lower, and so whether the method could detect it
 with high sensitivity is crucial. The gyrB gene is a single
copy gene, but a sensitivity of 0.01 ng with genomic DNA
 or 3.2 cells per PCR was obtained in the assay. It is obvious
 that the PCR detection limit was little influenced by the
 single copy of the target gene, which is ascribed to the high
 efficiency of PCR. This detection sensitivity ensured that
 Fig. 2 The representative image of amplification products of serial
 DNA dilutions of S. enteritidis CMCC50335. Lanes 1–6: 1, 0.1,
 0.1 9 10-1, 0.1 9 10-2 and 0.1 9 10-3, lane 7, negative control,
 M molecular size markers (DL2000). The length of each amplified
 fragment was 366 bp. 133 9 101 mm (72 9 72 DPI)
 Fig. 3 The representative image of amplification products of serial
 culture dilutions of S. enteritidis CMCC50335. Lanes 1–6: 3.2 9 104,
 3.2 9 103, 3.2 9 102, 3.2 9 101, 3.2 9 100 and 3.2 9 10-1 cells
 per PCR mixture, lane 7 negative control; M 100 bp ladder marker.
 The length of each amplified fragment was 366 bp. 148 9 106 mm
 (72 9 72 DPI)
 even if the Salmonella in environment samples is very low,
 it could still be detected.
 Specificity Determination
 To check the specificity of the PCR method, total DNA
 from fresh fecal sample was used as a template to detect
 Salmonella. A single product of the predicted size (366 bp)
 was successfully amplified. The amplicon was cloned, and
 22 of the clones were randomly selected for sequencing
 (DDBJ accession no. AB607965–AB607986). The phylo
genetic tree indicated that the 22 clones were clustered
 with the species of Salmonella (Fig. 4). The gyrB sequence
 of only Salmonella was amplified from the total fecal
 123
X. Ye et al.: A gyrB-targeted PCR for Rapid Identification
 481
 Fig. 4 Phylogenetic tree based
 on gyrB sequences of the clones
 and the isolates from fecal
 samples. It was constructed
 using the Neighbor-Joining
 method (MEGA version 4.0).
 The number at each node
 indicates bootstrap values
 ([50%) of 1,000 repetitions.
 22 clones and 27 isolates shared
 100% similarity with
 Salmonella. 337 9 668 mm
 (300 9 300 DPI)
 123
482
 X. Ye et al.: A gyrB-targeted PCR for Rapid Identification
 DNA,demonstrating the specificity and validity of the PCR
 method.
 Identification of Salmonella Isolates
 The culture-PCR method was applied to identify the iso
lates of Salmonella from fecal sample. Twenty-seven iso
lates morphologically similar to Salmonella were obtained
 from the fecal sample. The 366-bp gyrB genes of the 27
 isolates were amplified and sequenced. The sequences of
 the 27 isolates with positive amplifications in the culture
PCR shared 100% similarity with that of Salmonella. The
 phylogenetic tree indicated that the 27 isolates were clus
tered with the species of Salmonella (Fig. 4).
 Consideration of Ease of use
 The culture-PCR developed method developed is a time
saving one with good sensitivity. In comparison to the time
 consumed in classic method (2 days) and the method of
 sequencing (3 days) or DNA–DNA hybridization (2 days),
 only 4–5 h duration is required in the culture-PCR method.
 Typically, the template of PCR was always DNA, but the
 time consumed in extracting DNA was at least 2 h. In our
 assay, on the lyses condition at 99C, 10 min, the bacterial
 DNAcould be completely free, so the DNA extraction was
 omitted when pyrolysis bacterial was used as a template. In
 addition, our previous experiments results have shown that
 LB medium did not affect the PCR reaction results (data
 not shown). Therefore, this method could be utilized for
 handling a large numbers of new isolates, such as in a
 biodiversity study or a large isolate-screening program.
 In conclusion, a species-specific PCR method was suc
cessfully developed for the rapid identification of Salmo
nella from pure isolates and detection of the species from a
 complex fecal sample. More studies should be carried out
 to test the potential of the special PCR in applications
 involving large sample throughput, such as in food,
 wastewater, soil, and in the identification of a large number
 of novel isolates.
 Acknowledgments This research was supported by the National
 Natural Science Foundation of China (20607024), The Knowledge
 Innovation Program of Chinese Academy of Sciences (KSCX2-EW
N-08), Special Fund for Agro-Scientific Research in the Public
 Interest (200903011) and the Knowledge Innovation Program of the
 Chinese Academy of Sciences (ISSASIP0721)

//

 a 
b 
s 
t 
r 
a 
c 
t
 The 
bacterial 
genus 
Clavibacter 
has 
only 
one 
species, 
Clavibacter 
michiganensis, 
containing 
five 
subspecies.
 All 
five 
are 
plant 
pathogens, 
among 
which 
three 
are 
recognized 
as 
quarantine 
pests 
(mentioned 
on 
the
 EPPO 
A2 
list). 
Prevention 
of 
their 
introduction 
and 
epidemic 
outbreaks 
requires 
a 
reliable 
and 
accurate
 identification. 
Currently, 
identification 
of 
these 
bacteria 
is 
time 
consuming 
and 
often 
problematic, 
mainly
 because 
of 
cross-reactions 
with 
other 
plant-associated 
bacteria 
in 
immunological 
tests 
and 
false-negative
 results 
in 
PCR 
detection 
methods. 
Furthermore, 
distinguishing 
closely 
related 
subspecies 
is 
not 
straight
forward. 
This 
study 
aimed 
at 
evaluating 
the 
use 
of 
matrix-assisted 
laser 
desorption 
ionization-time 
of
 flight 
mass 
spectrometry 
(MALDI-TOF 
MS) 
and 
a 
fragment 
of 
the 
gyrB 
sequence 
for 
the 
reliable 
and 
fast
 identification 
of 
the 
Clavibacter 
subspecies. 
Amplification 
and 
sequencing 
of 
gyrB 
using 
a 
single 
primer 
set
 had 
sufficient 
resolution 
and 
specificity 
to 
identify 
each 
subspecies 
based 
on 
both 
sequence 
similarities 
in
 cluster 
analyses 
and 
specific 
signatures 
within 
the 
sequences. 
All 
five 
subspecies 
also 
generated 
distinct
 and 
reproducible 
MALDI-TOF 
MS 
profiles, 
with 
unique 
and 
specific 
ion 
peaks 
for 
each 
subspecies, 
which
 could 
be 
used 
as 
biomarkers 
for 
identification. 
Results 
from 
both 
methods 
were 
in 
agreement 
and 
were
 able 
to 
distinguish 
the 
five 
Clavibacter 
subspecies 
from 
each 
other 
and 
from 
representatives 
of 
closely
 related 
Rathayibacter, 
Leifsonia 
or 
Curtobacterium 
species. 
Our 
study 
suggests 
that 
proteomic 
analysis
 using 
MALDI-TOF 
MS 
and 
gyrB 
sequence 
are 
powerful 
diagnostic 
tools 
for 
the 
accurate 
identification 
of
 Clavibacter 
plant 
pathogens.
 © 2011 Elsevier GmbH. All rights reserved.
 Introduction
 Clavibacter 
michiganensis 
harbours 
aerobic, 
non-sporulating
 rods 
that 
belong 
to 
the 
GC 
rich 
subgroup 
of 
Gram-positive 
bac
teria. 
It 
constitutes 
the 
only 
species 
within 
the 
genus 
Clavibacter
 and 
currently 
consists 
of 
five 
subspecies, 
all 
of 
which 
are 
plant
 pathogens: 
C. 
m. 
subsp. 
insidiosus 
(Cmi), 
C. 
m. 
subsp. 
michiganensis
 (Cmm), 
C. 
m. 
subsp. 
nebraskensis 
(Cmn), 
C. 
m. 
subsp. 
sepedonicus
 (Cms) 
and 
C. 
m. 
subsp. 
tesselarius 
(Cmt) 
[12]. 
They 
are 
harmful 
on
 important 
agricultural 
crops 
such 
as 
alfalfa, 
tomato, 
corn, 
potato
 and 
wheat. 
The 
host 
range 
of 
each 
subspecies 
is 
limited, 
mostly
 restricted 
to 
one 
plant 
species, 
and 
most 
of 
them 
are 
pathogens 
of
 vascular 
plants, 
causing 
wilt 
as 
main 
symptom. 
Based 
on 
cell 
wall
 composition, 
menaquinones 
and 
additional 
phenotypic 
markers,
 former 
Clavibacter 
species 
were 
reclassified 
to 
the 
genus 
Curtobac
terium, 
Leifsonia 
and 
Rathayibacter 
[4,13,45]. 
Cmm, 
Cms 
and 
Cmi
 ∗Corresponding 
author. 
Tel.: 
+32 
92645101; 
fax: 
+32 
92645092.
 E-mail 
address: 
<PRESIDIO_ANONYMIZED_EMAIL_ADDRESS> 
(J. 
Zaluga).
 have 
quarantine 
status 
in 
plant 
health 
legislation 
in 
Europe 
[2].
 Every 
year 
they 
cause 
serious 
economic 
damage 
due 
to 
actual 
yield
 reduction 
[7] 
but 
also 
due 
to 
statutory 
measures 
taken 
to 
eliminate
 the 
pathogen 
from 
seed 
stocks 
[44]. 
Transport 
of 
certain 
plants 
and
 plant 
products 
within 
the 
EU 
is 
regulated, 
as 
well 
as 
introduction 
of
 certain 
commodities 
from 
third 
countries 
into 
the 
EU. 
Plant 
health
 and 
seed 
certifications 
are 
mandatory 
to 
prevent 
the 
introduction,
 spread 
and 
establishment 
of 
specific 
plant 
pests 
and, 
if 
detected, 
to
 implement 
control 
strategies 
with 
the 
aim 
of 
eradication 
[2].
 Diagnostic 
procedures 
for 
phytopathogenic 
C. 
michiganensis
 subspecies 
are 
available 
from 
either 
the 
European 
Union 
or 
Euro
pean 
Plant 
Protection 
Organization 
(EPPO) 
[9–11]. 
Fundamental 
in
 the 
diagnostic 
process 
is 
the 
rapid, 
reliable 
and 
accurate 
identifi
cation 
of 
the 
phytopathogen 
to 
confirm 
its 
presence 
and 
identity,
 which 
for 
C. 
michiganensis 
subspecies 
can 
be 
done 
by 
pheno
typic 
properties, 
serological 
tests, 
fatty 
acid 
or 
protein 
profiles 
and
 molecular 
assays. 
Final 
confirmation 
is 
obtained 
in 
a 
pathogenic
ity 
test. 
Usually, 
a 
serological 
test 
or 
fatty 
acid 
profile 
is 
combined
 with 
a 
molecular 
test 
such 
as 
PCR, 
real-time 
PCR 
or 
a 
fingerprint
 PCR, 
e.g., 
BOX-PCR. 
PCR 
identification 
of 
the 
Clavibacter 
michiga
0723-2020/$ – 
see 
front 
matter© 
 2011 Elsevier GmbH. All rights reserved.
 doi:10.1016/j.syapm.2011.05.001
J. 
Zaluga 
et 
al. 
/ 
Systematic 
and 
Applied 
Microbiology 
34 (2011) 400– 
407 401
 nensis 
subspecies 
targets 
the 
16S–23S 
rRNA 
spacer 
region 
[29],
 other 
chromosomal 
regions 
[27] 
or 
plasmid 
encoded 
genes 
[21,28].
 This 
diversity 
of 
tests 
and 
targets, 
each 
with 
its 
intrinsic 
specificity,
 can 
trigger 
inconsistent 
or 
conflicting 
results 
in 
identification 
of 
C.
 michiganensis 
subspecies. 
PCR 
using 
a 
uniform 
target 
region 
present
 in 
all 
C. 
michiganensis 
subspecies 
will 
allow 
complete 
differenti
ation 
within 
the 
whole 
species, 
greatly 
simplifying 
identification
 of 
these 
important 
plant 
pathogens. 
Sequencing 
of 
the 
16S 
rRNA
 gene 
is 
the 
most 
common 
identification 
tool 
in 
bacterial 
taxonomy
 [17], 
but 
its 
resolution 
is 
not 
sufficient 
for 
accurate 
delineation 
at
 subspecies 
level. 
Other 
targets, 
such 
as 
housekeeping 
genes 
atpA,
 rpoB, 
gyrB 
and 
others, 
have 
already 
proven 
to 
be 
useful 
for 
reliable
 identification 
in 
genera 
such 
as 
Ensifer 
[25], 
Microbacterium 
[36]
 and 
others. 
Sequence 
analysis 
of 
the 
gyrase 
B 
gene 
(gyrB), 
deserves
 attention 
as 
potential 
identification 
tool 
for 
the 
genus 
Clavibacter
 as 
it 
was 
already 
proposed 
as 
a 
possible 
useful 
phylogenetic 
marker
 for 
members 
of 
the 
family 
Microbacteriacea, 
of 
which 
Clavibacter
 is 
a 
member 
[35].
 Recently, 
a 
number 
of 
studies 
demonstrated 
the 
usefulness 
of
 MALDI-TOF 
MS 
(matrix 
assisted 
laser 
desorption/ionization 
time
 of 
flight 
mass 
spectrometry) 
for 
rapid 
identification 
of 
clinical
 pathogens 
[3,14], 
lactic 
acid 
bacteria 
[41], 
nonfermenting 
bacteria
 [26], 
environmental 
bacteria 
[37], 
etc. 
In 
this 
type 
of 
mass 
spec
trometry, 
samples 
are 
prepared 
by 
embedding 
analyte 
molecules
 in 
a 
crystal 
matrix 
of 
small 
acidic 
molecules. 
A 
brief 
laser 
pulse
 irradiates 
the 
sample 
and 
the 
matrix 
absorbs 
the 
laser 
energy
 resulting 
in 
ablation 
of 
a 
small 
volume 
of 
matrix 
and 
desorption 
of
 the 
embedded 
analyte 
molecules 
which 
are 
ionized. 
Subsequently,
 predominantly 
single 
charged 
analyte 
ions 
can 
be 
detected 
and 
ana
lyzed 
[23]. 
Characterization 
and/or 
identification 
using 
MALDI-TOF
 MS 
is 
based 
on 
differences 
in 
mass 
to 
charge 
ratio 
(m/z) 
fingerprints
 of 
whole 
cell 
proteins, 
mainly 
representing 
ribosomal 
proteins
 which 
are 
most 
abundantly 
expressed 
under 
all 
growth 
conditions
 [38].
 The 
objective 
of 
this 
study 
was 
to 
develop 
a 
robust 
identifica
tion 
method 
applicable 
to 
all 
C. 
michiganensis 
subspecies. 
GyrB 
and
 MALDI-TOF 
MS 
profiling 
were 
evaluated 
as 
identification 
tools 
on 
a
 large 
set 
of 
strains 
of 
all 
C. 
michiganensis 
subspecies 
retrieved 
from
 outbreaks 
worldwide, 
complemented 
with 
a 
collection 
of 
reference
 strains 
and 
representatives 
of 
phylogenetically 
closely 
related 
taxa.
 Applicability 
of 
both 
techniques 
for 
correct 
subspecies 
assignment
 was 
evaluated 
in 
two 
ways: 
by 
cluster 
analysis 
and 
use 
of 
specific
 signature 
positions 
in 
case 
of 
sequence 
data 
and 
by 
cluster 
analysis
 together 
with 
biomarkers 
for 
spectral 
data.
 Materials 
and 
methods
 Bacterial 
strains 
and 
growth 
conditions
 A 
total 
of 
173 
strains 
were 
received 
from 
the 
BCCM/LMG 
Bac
teria 
Collection 
(Ghent, 
Belgium), 
PD 
collection 
(Wageningen, 
The
 Netherlands) 
GBBC 
collection 
(ILVO, 
Merelbeke, 
Belgium) 
and 
Insti
tuto 
Canario 
de 
Investigaciones 
Agrarias 
(Tenerife, 
Spain). 
This
 Clavibacter 
strain 
subset 
consisted 
of 
67 
Cmm, 
65 
Cms, 
13 
Cmi, 
9
 Cmn, 
4 
Cmt 
and 
7 
unidentified 
Clavibacter 
sp. 
of 
which 
four 
strains
 were 
described 
as 
Cmm 
look-alikes. 
Cmm 
look-alikes 
were 
isolated
 from 
infected 
tomato 
seeds, 
positively 
identified 
as 
belonging 
to 
the
 subspecies 
Cmm 
using 
the 
current 
diagnostic 
protocols 
[9], 
though
 failed 
the 
pathogenicity 
test 
in 
a 
bioassay 
(personal 
communica
tion 
PD 
collection). 
The 
following 
outgroup 
strains 
were 
included,
 Rathayibacter 
rathayi 
LMG 
3717, 
Rathayibacter 
tritici 
LMG 
3726,
 Rathayibacter 
iranicus 
LMG 
3677T, 
Leifsonia 
aquatica 
LMG 
18699,
 and 
four 
Curtobacterium 
strains. 
All 
strains 
are 
listed 
in 
Table 
S1. 
The
 bacteria 
were 
grown 
aerobically 
at 
25◦C 
for 
24–48 
h. 
Stock 
cultures
 were 
stored 
at 
−20◦C 
in 
MicrobankTMbeads 
(Pro-Lab 
Diagnostics,
 Canada) 
until 
analysis. 
For 
MALDI-TOF 
MS 
analysis, 
bacteria 
were
 grown 
on 
MTNA 
medium 
without 
antibiotics 
added 
[18]. 
To 
assess
 the 
influence 
of 
different 
growth 
media 
on 
species 
recognition
 using 
MALDI-TOF 
MS, 
two 
additional 
media 
recommended 
by 
the
 BCCM/LMG 
bacteria 
collection 
(http://bccm.belspo.be/index.php),
 M6 
(glucose 
10 
g, 
yeast 
extract 
5 
g, 
peptone 
5 
g, 
agar 
15 
g, 
distilled
 water 
1 
L, 
pH 
7.0) 
and 
M39 
(medium 
M6 
supplemented 
with 
0.1 
g
 casein 
hydrolysate 
per 
liter) 
were 
used. 
These 
two 
media 
were 
also
 used 
to 
obtain 
cultures 
for 
DNA 
extraction.
 DNA 
extraction 
and 
sequencing
 Total 
genomic 
DNA 
was 
extracted 
according 
to 
the 
guanidium
thiocyanate–EDTA–sarkosyl 
method 
described 
by 
Pitcher 
[31]
 which 
was 
adapted 
for 
Gram-positive 
bacteria 
with 
an 
additional
 lysozyme 
step 
as 
described 
before 
16S 
rRNA 
gene 
amplification
 was 
performed 
as 
described 
previously 
with 
primers 
pA-forward
 and 
pH-reverse 
[16]. 
Subsequently, 
PCR 
products 
were 
purified
 and 
partially 
sequenced 
as 
described 
below 
using 
the 
primers
 BKL1-reverse 
(5-ACTGCTGCCTCCCGTAGGAG-3, 
position 
536–516
 in 
the 
Escherichia 
coli 
numbering 
system) 
and 
gamma-reverse 
(5
ACTGCTGCCTCCCGTAGGAG-3, 
position 
358–339) 
[16].
 A 
fragment 
of 
the 
gyrB 
was 
amplified 
by 
PCR 
with 
the 
previously
 described 
primers 
gyrB-2F 
and 
gyrB-4R 
[35]. 
The 
PCR 
mixture 
had 
a
 total 
volume 
of 
25 
l, 
containing 
1× 
PCR 
buffer 
(100 
mM 
Tris–HCl,
 15 
mM 
MgCl2, 
500 
mM 
KCl 
[pH 
8.3]), 
dNTP’s 
0.2 
mM 
each, 
0.6 
M 
of
 each 
primer, 
0.5 
U 
AmpliTaq 
DNA 
polymerase, 
and 
50–60 
ng 
tem
plate 
DNA. 
The 
PCR 
conditions 
were 
as 
described 
previously 
[35].
 The 
expected 
amplicon 
size 
was 
about 
550 
bp. 
Resulting 
amplicons
 were 
purified 
using 
the 
Nucleofast®96 
PCR 
clean 
up 
membrane
 system 
(Macherey-Nagel, 
Germany). 
Sequencing 
reactions 
were
 performed 
in 
a 
total 
volume 
of 
10 
l 
with 
3 
l 
of 
purified 
ampli
con, 
0.286 
l 
of 
BigDyeTMmixture 
(Terminator 
Cycle 
Sequencing
 Kit 
version 
3.1, 
Applied 
Biosystems), 
1× 
sequencing 
buffer 
and
 1.2 
M 
of 
each 
of 
the 
amplification 
primers 
(gyrB 
2F 
and 
gyrB 
4R).
 The 
thermal 
program 
consisted 
of 
30 
cycles 
(96◦C 
for 
15 
s, 
35◦C 
for
 1 
s, 
60◦C 
for 
4 
min). 
Subsequently, 
the 
sequencing 
products 
were
 purified 
using 
the 
BigDye 
XTerminator 
Kit 
(Applied 
Biosystems)
 and 
analyzed 
on 
a 
3130xl 
Genetic 
Analyzer 
(Applied 
Biosystems).
 Sequence 
analysis
 The 
16S 
rRNA 
gene 
and 
gyrB 
sequences 
were 
assembled 
with
 BioNumerics 
version 
5.1 
(Applied 
Maths, 
Belgium) 
and 
aligned
 using 
ClustalW 
[42]. 
The 
identity 
assigned 
to 
each 
strain 
by 
the
 three 
culture 
collections 
was 
verified 
with 
an 
nBLAST 
of 
the 
16S
 rRNA 
gene 
sequence 
of 
each 
strain 
and 
a 
neighbor-joining 
cluster
 analysis 
with 
all 
type 
strains 
of 
all 
species 
of 
all 
genera 
mentioned 
in
 the 
twenty 
highest 
BLAST 
hits. 
The 
strains 
were 
assigned 
to 
a 
genus
 based 
on 
the 
obtained 
pairwise 
16S 
rRNA 
gene 
sequence 
similarity
 (>98% 
gene 
sequence 
similarity).
 GyrB 
sequences 
were 
checked 
by 
amino 
acid 
translation 
with
 Transseq 
(http://www.ebi.ac.uk/Tools/emboss/transeq/) 
and 
pres
ence 
of 
the 
gyrB 
protein 
domain 
was 
confirmed 
with 
pBlast 
[1].
 Molecular 
Evolutionary 
Genetics 
Analysis 
software 
(Mega 
4.1) 
[40]
 was 
used 
to 
calculate 
evolutionary 
distances 
based 
on 
a 
500 
bp 
(this
 equal 
length 
was 
used 
for 
all 
strains) 
gyrB 
fragment 
of 
the 
gyrB
 amplicon, 
and 
to 
infer 
Maximum 
Parsimony 
(MP), 
Maximum 
Like
lihood 
(ML) 
and 
Neighbor-Joining 
(NJ) 
trees 
using 
the 
maximum
 composite 
likelihood 
(MCL) 
model.
 TaxonGap 
software, 
version 
2.4.1 
[39], 
was 
used 
to 
define 
the
 discriminatory 
power 
of 
both 
sequence 
markers, 
16S 
rRNA 
and
 gyrB, 
and 
to 
identify 
sequence 
characters 
using 
strains 
of 
Clavibac
ter 
subspecies 
with 
established 
name 
and 
confirmed 
pathogenicity.
 To 
establish 
the 
positions 
within 
the 
gyrB 
and 
16S 
rRNA 
gene, 
the
402 J. 
Zaluga 
et 
al. 
/ 
Systematic 
and 
Applied 
Microbiology 
34 (2011) 400– 
407
 whole 
Cmm 
genome 
sequence 
(AM711867) 
was 
used 
as 
a 
refer
ence.
 MALDI-TOF 
MS
 Preparation 
of 
cell 
extracts. 
The 
formic 
acid–acetonitrile 
extrac
tion 
as 
developed 
by 
Bruker 
Daltonics 
[24] 
was 
optimized 
to 
retain
 mostly 
ribosomal 
proteins. 
For 
this, 
a 
small 
amount 
of 
bacterial 
cells
 (yellow 
loop) 
was 
suspended 
in 
300 
l 
of 
distilled 
water 
after 
which
 900 
l 
of 
absolute 
ethanol 
was 
added. 
This 
mixture 
was 
centrifuged
 at 
18,000 
× 
g 
for 
3 
min 
and 
the 
supernatant 
was 
discarded. 
Subse
quently, 
the 
pellet 
was 
resuspended 
in 
50 
l 
of 
formic 
acid 
(70%
 [v/v]) 
after 
which 
50 
l 
of 
acetonitrile 
(ACN) 
was 
added. 
Following
 centrifugation 
at 
18,000 
× 
g 
for 
3 
min, 
one 
microliter 
of 
the 
super
natant 
was 
spotted 
on 
a 
steel 
target 
plate 
(AB 
Sciex, 
Belgium) 
and
 air 
dried 
at 
room 
temperature. 
Finally, 
the 
sample 
spot 
was 
overlaid
 with 
1 
l 
of 
a 
0.5% 
(w/v) -cyano-4-hydroxy 
cinnamic 
acid 
(
CHCA) 
solution 
in 
ACN-MilliQ 
water-trifluoroacetic 
(50:48:2 
v/v/v)
 and 
air 
dried.
 MALDI-TOF 
MS 
analysis. 
Measurements 
were 
performed 
on 
a
 4800 
Plus 
MALDI 
TOF/TOFTMAnalyzer 
(AB 
Sciex, 
Belgium) 
in 
lin
ear, 
positive-ion 
mode 
with 
a 
200-Hz 
frequency 
tripled 
UV 
Nd:YAG
 laser 
operating 
at 
a 
wavelength 
of 
355 
nm. 
Generated 
ions 
were
 accelerated 
at 
20 
kV 
through 
a 
grid 
at 
19.2 
kV 
and 
separated 
accord
ing 
to 
their 
m/z 
ratio 
in 
a 
1.5 
m 
long 
linear, 
field-free 
drift 
region.
 Each 
generated 
spectrum 
resulted 
from 
40 
laser 
shots 
at 
50 
random
 positions 
within 
the 
measuring 
spot 
(2000 
spectra/spot). 
MALDI
TOF 
mass 
spectra 
were 
generated 
in 
the 
range 
2–20 
kDa. 
Calibration
 was 
performed 
using 
an 
external 
protein 
calibration 
mix 
composed
 of 
the 
Protein 
Calibration 
Standard 
I 
developed 
by 
Bruker 
Dalton
ics 
(Bruker) 
[composition: 
insulin 
([M+H]+, 
m/z 
5734.6), 
ubiquitin
 I 
([M+H]+, 
m/z 
8565.9), 
cytochrome 
C 
([M+H]+, 
m/z 
12361.5), 
myo
globin 
([M+H]+, 
m/z 
16952.3)] 
and 
an 
ACTH 
clip 
18–39 
([M+H]+,
 m/z 
2466.7) 
(Sigma–Aldrich), 
resulting 
in 
five 
peaks 
spread 
over 
the
 whole 
fingerprint 
spectrum. 
The 
error 
allowed 
to 
match 
the 
peak
 of 
the 
proteins 
was 
set 
at 
200 
ppm. 
Bruker 
Bacterial 
Test 
Standard
 (Bruker) 
of 
E. 
coli 
DH5 
was 
included 
with 
every 
set 
of 
measure
ments 
as 
a 
positive 
control. 
Each 
sample 
was 
spotted 
at 
least 
in
 duplicate 
to 
check 
the 
reproducibility.
 Analysis 
of 
the 
spectral 
data. 
Mass 
spectra 
were 
generated 
in
 t2d 
format 
and 
converted 
to 
txt 
files 
using 
ABI 
Data 
Explorer 
4.0
 software 
(AB 
Sciex), 
after 
which 
they 
were 
imported 
into 
BioNu
merics 
5.1 
software 
(Applied 
Maths, 
Belgium). 
To 
obtain 
reliable
 data 
analysis, 
the 
spectra 
with 
extensive 
noise 
and/or 
insufficient
 signal 
intensity 
were 
excluded 
from 
further 
analysis. 
High 
levels
 of 
noise 
signals, 
possibly 
due 
to 
interference 
of 
remaining 
polysac
charides 
or 
salts 
in 
the 
cell 
extracts 
at 
the 
beginning 
of 
the 
profile
 were 
left 
out 
of 
the 
analysis 
as 
excluding 
the 
first 
5% 
of 
the 
profile
 (shifting 
the 
m/z 
range 
from 
2 
kDa 
to 
3 
kDa) 
was 
enough 
to 
obtain
 more 
reliable 
profiles.
 The 
similarity 
between 
the 
spectra 
was 
expressed 
using 
Pear
son’s 
product 
moment 
correlation 
coefficient, 
a 
curve 
based
 analysis, 
and 
the 
spectra 
were 
clustered 
using 
the 
UPGMA
 (unweighted 
pair 
group 
method 
with 
arithmetic 
mean) 
clustering
 algorithm. 
Next 
to 
cluster 
analysis, 
spectral 
data 
were 
investigated
 for 
the 
presence 
of 
biomarkers 
characteristic 
for 
each 
Clavibac
ter 
subspecies. 
After 
visual 
inspection 
and 
comparison, 
the 
most
 intensive 
and 
predominantly 
present 
protein 
peaks 
were 
selected
 and 
screened 
in 
representatives 
of 
each 
subspecies. 
Because
 the 
in-house 
cell 
extraction 
protocol 
was 
designed 
to 
retain
 mainly 
ribosomal 
proteins, 
selected 
biomarkers 
could 
be 
assigned
 to 
specific 
ribosomal 
proteins 
by 
comparing 
protein 
masses
 to 
the 
Rapid 
Microorganism 
Identification 
Database 
(RMIDb)
 http://www.rmidb.org/cgi-bin/index.pl. 
This 
database 
was 
created
 to 
identify 
bacteria 
and 
viruses 
using 
existing 
mass 
spectrom
etry 
protocols. 
RMIDb 
contains 
all 
bacterial, 
virus, 
plasmid 
and
 environmental 
protein 
sequences 
from 
the 
Ventor 
Institute’s 
Com
prehensive 
Microbial 
Resource 
(CMR), 
UniProt’s 
Swiss-Prot 
and
 TrEMBL, 
Genbank’s 
Protein, 
and 
RefSeq’s 
Protein 
and 
Genome 
[30].
 Nucleotide 
accession 
numbers
 The 
Clavibacter 
sequences 
have 
been 
deposited 
in 
the 
EMBL
 Database 
with 
accession 
numbers 
FR728257–FR728381 
and
 FR727975–FR728147 
for 
the 
16S 
rRNA 
gene 
and 
gyrB 
sequences
 respectively.
 Results
 Strain 
set
 A 
total 
of 
165 
Clavibacter 
strains 
and 
eight 
outgroup 
strains
 were 
included 
in 
this 
study, 
covering 
a 
wide 
geographical 
and
 temporal 
spread 
(Table 
S1). 
Information 
received 
from 
the 
cul
ture 
collections 
on 
subspecies 
allocation 
of 
strains 
was 
initially
 regarded 
as 
correct. 
However, 
partial 
16S 
rRNA 
gene 
sequence 
anal
ysis 
demonstrated 
that 
not 
all 
strains 
belonged 
to 
Clavibacter 
(data
 not 
shown). 
Neighbor 
joining 
analysis 
of 
16S 
rRNA 
gene 
sequences
 distinguished 
two 
separate 
groups 
of 
Clavibacter, 
one 
containing
 the 
majority 
of 
clavibacters 
and 
the 
second 
containing 
four 
strains,
 three 
of 
which 
were 
originally 
assigned 
to 
the 
Cmm 
and 
one 
to
 the 
subspecies 
sepedonicus. 
However, 
BLAST 
analysis 
of 
members
 of 
this 
second 
group 
showed 
100% 
16S 
rRNA 
gene 
sequence 
sim
ilarity 
to 
members 
of 
the 
genus 
Curtobacterium 
(Curtobacterium
 flaccumfaciens, 
accession 
number 
GU586309.1, 
Curtobacterium 
sp.,
 accession 
number 
GU120656.1 
and 
Curtobacterium 
flaccumfaciens
 pv. 
flaccumfaciens, 
accession 
number 
AM410688.1). 
These 
results
 indicated 
that 
these 
strains 
had 
been 
incorrectly 
identified 
as
 Clavibacter, 
which 
was 
confirmed 
by 
pairwise 
sequence 
similar
ity 
analysis 
with 
type 
strains 
of 
genus 
Curtobacterium. 
The 
separate
 position 
of 
these 
four 
Curtobacterium 
strains 
was 
later 
also 
observed
 in 
gyrB 
sequence 
and 
MALDI-TOF 
MS 
analyses. 
Because 
of 
the
 relevance 
to 
diagnostic 
purposes, 
Cmm 
look-alikes 
from 
the 
PD
 collection 
were 
also 
included 
in 
the 
strain 
set. 
These 
bacteria 
were
 isolated 
from 
infected 
tomato 
seeds 
and 
had 
been 
positively 
iden
tified 
as 
Cmm 
although 
they 
could 
not 
be 
confirmed 
as 
pathogenic
 with 
the 
bioassay 
test. 
Both 
MALDI-TOF 
MS 
and 
gyrB 
sequence 
anal
ysis 
grouped 
these 
Cmm 
look-alikes 
in 
separate 
clusters. 
Moreover,
 both 
gyrB 
sequence 
analysis 
and 
MALDI-TOF 
MS 
were 
able 
to 
assign
 three 
unidentified 
Clavibacter 
spp., 
with 
strain 
numbers 
PD 
5715,
 PD 
5716 
and 
PD 
5714, 
to 
subsp. 
nebraskensis, 
subsp. 
michiganen
sis 
and 
subsp. 
tesselarius, 
respectively. 
In 
addition, 
the 
outgroup
 strains 
Rathayibacter; 
Curtobacterium 
and 
Leifsonia 
were 
included
 to 
evaluate 
the 
sensitivity 
and 
robustness 
of 
MALDI-TOF 
MS 
and
 gyrB 
sequence 
analysis.
 GyrB 
sequence 
analysis
 GyrB 
amplicons 
were 
retrieved 
from 
all 
strains 
and 
sequences
 were 
translated 
into 
amino 
acids 
as 
a 
quality 
check. 
Sequence
 data 
were 
subsequently 
analyzed 
with 
two 
different 
approaches,
 namely 
by 
phylogenetic 
analysis 
and 
by 
identification 
of 
signature
 nucleotides 
specific 
for 
each 
subspecies. 
Cluster 
analysis 
clearly
 grouped 
all 
gyrB 
sequences 
from 
Clavibacter 
spp. 
separately 
from
 outgroups 
and 
Curtobacterium 
spp. 
Also, 
sequences 
from 
each 
sub
species 
formed 
distinct 
clusters, 
supported 
with 
high 
bootstrap
 values 
(Fig. 
1). 
In 
Cmn 
and 
Cmi 
the 
sequences 
were 
identical,
 whereas 
in 
Cms 
sequences 
similarity 
ranged 
from 
97.1 
to 
100%,
 in 
Cmm 
98.2% 
to 
100% 
and 
in 
Cmt 
96.2 
to 
99.3%. 
Interestingly,
 the 
gyrB 
sequences 
from 
the 
Cmm 
look-alikes 
grouped 
outside
 the 
Cmm 
cluster, 
although 
these 
strains 
were 
positively 
identi
fied 
as 
Cmm 
using 
the 
current 
diagnostic 
procedure. 
As 
indicated
J. 
Zaluga 
et 
al. 
/ 
Systematic 
and 
Applied 
Microbiology 
34 (2011) 400– 
407 403
 subsp . michiganensis C. michiganensis 
 Cmm look-alikes
 . tesselarius C. michiganensis subsp
 subsp . sepedonicus C. michiganensis 
 nebraskensis C. michiganensis subsp.
 insidiosus C. michiganensis subsp.
 r sp.  Rathayibacte
 LMG18699Leifsonia aquatica
 sp. Curtobacterium
 100
 99 70
 100
 99
 91
 95
 82
 100
 98
 100
 0.02
 Number of base substitutions per site
 Fig. 
1. 
Phylogenetic 
analysis 
of 
gyrB 
sequences. 
Rooted 
neighbor-joining 
tree 
was 
based 
on 
partial 
gyrB 
sequence 
(position 
646–1143 
of 
Cmm 
NCPPB 
382 
accession 
number
 AM711867) 
of 
173 
Clavibacter 
strains. 
Bootstrap 
values 
were 
generated 
from 
1000 
replicates. 
Rathayibacter 
sp. 
and 
Leifsonia 
aquatica 
were 
included 
as 
outgroups. 
NJ 
tree 
is
 in 
accordance 
with 
maximum 
parsimony 
and 
maximum 
likelihood 
analysis. 
GyrB 
sequences 
had 
minimum 
length 
of 
500 
bp.
 by 
branch 
lengths 
in 
the 
phylogenetic 
tree 
(Fig. 
1), 
gyrB 
sequence
 variation 
between 
strains 
of 
the 
same 
subspecies 
was 
very 
limited
 and 
even 
absent 
within 
Cms, 
Cmi 
and 
Cmn. 
This 
limited 
diver
gence 
in 
gyrB 
sequence 
was 
translated 
in 
only 
a 
few 
changes 
in
 amino 
acid 
composition. 
Therefore, 
the 
suitability 
of 
the 
gyrB 
and
 16S 
rRNA 
sequences 
as 
phylogenetic 
markers 
was 
investigated 
fur
ther 
using 
the 
TaxonGap 
2.4.1 
software 
(Fig. 
S2) 
for 
visualization 
of
 the 
intra- 
and 
intersubspecies 
sequence 
heterogeneity 
and 
com
parison 
of 
the 
discriminative 
power 
of 
sequence 
markers 
for 
a
 set 
of 
taxonomic 
units. 
Analysis 
was 
performed 
on 
both 
gyrB 
and
 16S 
rRNA 
gene 
sequences 
of 
strains 
that 
were 
assigned 
to 
a 
cer
tain 
subspecies 
(only 
strains 
with 
an 
established 
name 
were 
used.
 All 
Cmm 
look-alike 
strains 
and 
strains 
received 
as 
Clavibacter 
spp.
 were 
excluded 
from 
this 
analysis). 
For 
all 
subspecies, 
gyrB 
clearly
 allowed 
distinct 
higher 
sequence 
divergence 
between 
sequences 
of
 different 
subspecies 
than 
within 
the 
subspecies. 
For 
Cmt, 
the 
gyrB
 sequence 
heterogeneity 
within 
the 
subspecies 
was 
higher 
than 
in
 other 
subspecies.
 In 
addition 
to 
the 
standard 
cluster 
analysis 
approach, 
aligned
 gyrB 
sequences 
were 
manually 
searched 
for 
the 
presence 
of 
sig
nature 
positions 
specific 
for 
each 
subspecies. 
In 
total, 
39 
positions
 differed 
among 
subspecies 
throughout 
the 
gyrB 
fragment 
of 
500 
bp.
 Based 
on 
these 
positions; 
unique 
signatures 
could 
be 
selected 
for
 each 
subspecies 
(Fig. 
S3). 
Numbers 
of 
unique 
nucleotides 
ranged
 from 
two 
for 
Cmm 
to 
nine 
for 
Cms 
(Table 
1). 
Almost 
all 
sequence
 divergences 
at 
these 
positions 
were 
the 
result 
of 
silent 
mutations
 whereas 
only 
four 
changes 
in 
amino 
acid 
sequence 
were 
found
 throughout 
all 
strains 
of 
C. 
michiganensis.
 MALDI-TOF 
MS
 All 
strains 
were 
subjected 
to 
MALDI-TOF 
MS 
analysis. 
Fifteen
 percent 
of 
the 
strains 
were 
analyzed 
in 
duplicate 
to 
assess 
the
 reproducibility 
(mean 
± 
standard 
deviation; 
91.98% 
± 
3.91) 
of 
the
 whole 
MALDI-TOF 
MS 
procedure, 
starting 
from 
growth 
and 
cell
 extraction 
over 
spectrum 
generation 
to 
data 
analysis. 
In 
addition,
 extracts 
were 
always 
spotted 
twice 
on 
the 
target 
plate, 
to 
evaluate
 variation 
(mean 
± 
SD; 
99.21 
± 
0.95) 
introduced 
by 
the 
MALDI-TOF
 MS 
method. 
The 
standard 
protocol 
included 
MTNA 
growth 
medium
 for 
all 
strains 
of 
Clavibacter. 
However, 
in 
diagnostics, 
different
 growth 
media 
are 
applied 
for 
isolation 
of 
different 
Clavibacter
 subspecies. 
Therefore, 
the 
influence 
of 
alternative 
growth 
media,
 namely 
M6 
and 
M39, 
recommended 
by 
BCCM/LMG 
collection, 
on
 MALDI-TOF 
MS 
spectra 
was 
investigated 
for 
54 
strains 
(31% 
of 
all
 strains). 
The 
choice 
of 
growth 
medium 
had 
no 
influence 
on 
the 
cor
rect 
subspecies 
grouping 
(Fig. 
S1). 
The 
low 
variations 
introduced 
by
 the 
technique 
itself 
and 
through 
the 
use 
of 
different 
growth 
condi
tions 
together 
with 
its 
high 
reproducibility 
provide 
evidence 
for 
the
 robustness 
of 
the 
MALDI-TOF 
MS 
for 
analysis 
of 
Clavibacter 
strains.
 Table 
1
 Overview 
of 
signatures 
in 
gyrB 
gene 
sequences, 
which 
are 
unique 
combinations
 of 
characters 
for 
individual 
subspecies 
of 
Clavibacter. 
Specific 
nucleotide 
present
 at 
each 
discriminative 
position 
(position 
in 
reference 
gyrB 
sequence 
of 
Cmm 
NCPPB
 382 
accession 
nr 
AM711867) 
is 
given. 
A, 
adenine; 
G, 
guanine; 
C, 
cytosine; 
T, 
thymine.
 Name 
(# 
strains) 
Positions 
of 
specific
 nucleotide
 Number 
of 
unique
 signatures
 Cmm 
(67) 
702G, 
1119G 
2
 Cms 
(65) 
687T, 
728A, 
786G, 
1035T,
 1056G, 
1059T, 
1080C,
 1089T, 
1113G
 9
 Cmn 
(9) 
1041G, 
1053C, 
1077G 
3
 Cmi 
(13) 
725G, 
726C, 
789T, 
969T,
 981G, 
1092G
 6
 Cmt 
(4) 
798T, 
855G, 
957C 
3
 Cmm 
look-alikes 
(4) 654G, 
743C, 
825C 
3
404 J. 
Zaluga 
et 
al. 
/ 
Systematic 
and 
Applied 
Microbiology 
34 (2011) 400– 
407
 Table 
2
 Characteristic 
masses 
(in 
daltons) 
selected 
as 
possible 
biomarkers 
for 
identification 
of 
Clavibacter. 
Masses 
observed 
in 
4 
or 
all 
subspecies 
are 
marked 
in 
bold. 
Assigned 
proteins
 calculated 
using 
RMIDb.
 Cmm Cms Cmt 
Cmi 
Cmn 
Assigned 
ribosomal 
protein 
(mass 
in 
Da,
 protein 
subunit/name, 
origin 
organism)
 3224a
 3238 3239 
3237 
3237
 3353 
3353 
3353 
3350 
3350
 3525 
3529 
3529 
3524
 4343 
4341 
4343 
4346-50S/L36 
Cmm
 4358a 4360-50S/L36 
Cms
 4613a 4613-50S/L36 
Cmm/Cms
 4772 4772 
4773 
4770 
4771
 5078 
5078 
5078 
5076 
5076 
5078-50S/L34 
Cmm/Cms
 5724 
5724 
5724
 5738a
 5752 5754 5755 5751 5752
 5823a
 6044a
 6068a
 6145a
 6454a 6456-50S/L33 
Cms
 6482 6482 
6479 
6480 
6483-50S/L33 
Cmm
 6713 
6713 
6713 
6710 
6712 
6714-50S/L30 
Cmm/Cms
 7013a 7012-50S/L35 
Cmm
 7058 
7059 
7060 
7057
 7070a 7069-50S/L35 
Cms
 7500 7501 7501-50S/L32 
Cms
 7998a
 8489a
 8510a
 8526 
8525 
8523 
8525-50S/L28 
Cmm/Cms
 8560 8560 8563 8559 8564-50S/L27 
Cmm/Cms
 9004 
9004 
9005 
9002 
9002-30S/S20 
Cmm
 9302 9300 9300 
9300 
9302-30S/S18 
Cmm
 9360a 9360-30S/S18 
Cms
 9549 9549 
9550 
9544 
9547
 a Subspecies 
unique 
mass 
values 
marked 
in 
Fig. 
2.
 The 
MALDI-TOF 
MS 
data 
analyzed 
in 
BioNumerics 
5.1 
and 
Data
Explorer 
4.0 
software 
were 
in 
agreement 
with 
the 
gyrB 
sequence
 analysis. 
Indeed, 
cluster 
analysis 
of 
the 
whole 
spectral 
profile
 showed 
distinct 
clusters 
for 
each 
subspecies, 
Cmm 
look-alikes 
and
 outgroups 
(data 
not 
shown). 
However, 
Cmm 
strains 
formed 
two
 separate 
groups, 
one 
with 
only 
a 
few 
strains 
that 
closely 
clustered
 to 
Cmn 
strains 
and 
the 
other 
with 
the 
majority 
of 
the 
Cmm 
strains.
 Next 
to 
cluster 
analyses, 
the 
obtained 
spectral 
profiles 
were 
also
 screened 
for 
the 
presence 
of 
recurring 
peaks 
or 
biomarker 
ions 
spe
cific 
for 
the 
whole 
species 
or 
a 
subspecies. 
A 
selection 
of 
20 
profiles
 of 
representatives 
of 
all 
Clavibacter 
subspecies, 
including 
the 
type
 strains, 
was 
visually 
screened 
for 
the 
most 
informative 
and 
inten
sive 
peaks. 
A 
typical 
MALDI-TOF 
MS 
spectrum 
of 
C. 
michiganensis
 contains 
about 
50 
ion 
peaks 
between 
2000 
and 
20,000 
Da, 
with 
the
 highest 
intensity 
peaks 
between 
4000 
and 
10,000 
Da. 
Table 
2 
sum
marizes 
32 
selected 
m/z 
values. 
Six 
m/z 
values 
were 
detected 
in
 all 
subspecies, 
making 
them 
characteristic 
for 
the 
genus 
Clavibac
ter. 
Unique 
peaks 
for 
each 
subspecies 
ranged 
from 
one 
in 
Cmt 
to
 five 
in 
Cms 
(Fig. 
2). 
In 
order 
to 
validate 
these 
peaks 
as 
biomark
ers, 
analysis 
of 
another 
25 
randomly 
selected 
Clavibacter 
profiles
 was 
performed 
and 
the 
assignment 
to 
the 
expected 
subspecies 
was
 obtained.
 The 
applied 
cell 
extraction 
protocol 
favors 
isolation 
of 
riboso
mal 
subunit 
proteins. 
Therefore, 
we 
tried 
to 
assign 
the 
selected
 biomarkers 
by 
comparing 
the 
experimentally 
obtained 
protein
 masses 
with 
those 
of 
the 
ribosomal 
subunit 
proteins 
deduced 
from
 two 
fully 
sequenced 
Clavibacter 
genomes 
(Cmm 
NCPPB382 
and
 Cms 
ATCC 
33113T) 
using 
the 
RMIDb 
database. 
In 
this 
way, 
eleven
 biomarkers 
could 
be 
identified 
as 
specific 
ribosomal 
proteins
 (Table 
2). 
However, 
our 
assignment 
procedure 
left 
several 
masses
 unidentified, 
probably 
because 
of 
slight 
differences 
in 
protein 
mass
 between 
different 
subspecies 
(only 
genomes 
of 
representatives 
of
 two 
subspecies 
were 
available), 
inclusion 
of 
nonribosomal 
proteins
 in 
the 
analyzed 
cell 
extracts, 
and 
possible 
occurrence 
of 
2+ 
ions.
 Discussion
 This 
study 
demonstrated 
the 
suitability 
of 
gyrB 
sequences
 and 
MALDI-TOF 
MS 
protein 
profiles 
for 
identification 
of 
plant
 pathogenic 
bacteria 
of 
the 
genus 
Clavibacter. 
The 
identification
 can 
be 
based 
on 
the 
generated 
data 
as 
a 
whole, 
or 
on 
signa
tures 
within 
these 
data 
that 
have 
enough 
discriminatory 
power
 to 
distinguish 
between 
the 
different 
subspecies 
of 
C. 
michiganen
sis. 
Moreover, 
both 
the 
sequence 
and 
the 
protein 
method 
suggested
 that 
diversity 
within 
this 
Clavibacter 
species 
is 
higher 
than 
currently
 accepted. 
This 
study 
reports 
for 
the 
first 
time 
on 
the 
use 
of 
gyrB 
as
 a 
phylogenetic 
marker 
in 
the 
genus 
Clavibacter. 
Molecular 
mark
ers 
for 
identification 
purposes 
must 
exhibit 
the 
smallest 
amount
 of 
heterogeneity 
within 
a 
taxonomic 
unit 
and 
should 
maximally
 discriminate 
between 
different 
species/subspecies 
[25], 
which 
was
 demonstrated 
in 
this 
study 
for 
gyrB 
sequences 
in 
the 
genus 
Clav
ibacter. 
In 
addition 
to 
cluster 
analysis, 
a 
selection 
of 
positions 
in
 the 
gyrB 
contained 
nucleotide 
signatures 
characteristic 
for 
each
 subspecies 
and 
were 
shown 
to 
be 
decisive 
for 
subspecies 
assign
ment. 
This 
character-based 
identification 
approach 
was 
previously
 proposed 
as 
an 
alternative 
for 
tree 
building 
methods 
[33]. 
How
ever, 
in 
bacterial 
taxonomy, 
this 
approach 
is 
not 
as 
popular 
as 
in
 human 
medicine 
where 
SNPs 
(single 
nucleotide 
polymorphisms)
 are 
commonly 
used 
in 
identification 
of 
pathogens 
or 
loci 
associ
ated 
with 
complex 
diseases 
[5,22]. 
These 
SNPs 
have 
also 
been 
used
 to 
design 
highly 
specific 
molecular 
tools 
for 
identification 
[43]. 
Fur
thermore, 
the 
fact 
that 
the 
gyrB 
fragment 
can 
be 
amplified 
and
 sequenced 
with 
the 
same 
protocol 
in 
all 
Clavibacter 
and 
closely
 related 
strains 
tested, 
creates 
great 
potential 
for 
future 
application
J. 
Zaluga 
et 
al. 
/ 
Systematic 
and 
Applied 
Microbiology 
34 (2011) 400– 
407 405
 Fig. 
2. 
MALDI-TOF 
MS 
protein 
mass 
fingerprints 
of 
type 
strains 
of 
genus 
Clavibacter. 
Similar 
and 
different 
marker 
masses 
for 
the 
identification 
of 
Clavibacter 
subspecies 
are
 listed 
in 
Table 
2. 
Relative 
intensities 
of 
ions 
(percentages) 
are 
shown 
on 
the 
y 
axis 
and 
the 
masses 
(in 
Daltons) 
of 
the 
ions 
are 
shown 
on 
the 
x 
axis. 
The 
m/z 
values 
represent
 mass-to-charge 
ratios. 
*Unique 
peaks 
positions 
for 
each 
of 
subspecies.
 as 
a 
new 
diagnostic 
tool. 
Based 
on 
the 
signature 
nucleotide 
posi
tions, 
new 
methods 
for 
specific 
PCR 
and 
Q-PCR 
detection 
in 
plant
 material 
may 
also 
be 
developed, 
which 
is 
especially 
relevant 
for 
the
 EU-quarantine 
pathogens 
Cmm, 
Cms 
and 
Cmi.
 Because 
of 
its 
simplicity 
and 
applicability, 
MALDI-TOF 
MS
 is 
already 
widely 
used 
for 
identification 
and 
characterization
 of 
diverse 
microorganisms 
[15,32,34]. 
Currently, 
spectral 
data
 are 
commonly 
analyzed 
using 
two 
different 
approaches. 
Specific
 biomarker 
ions 
are 
identified 
based 
on 
additional 
information 
on
 theoretical 
protein 
masses 
(from 
e.g., 
genome 
projects). 
New 
pro
files 
of 
unknown 
strains 
are 
then 
screened 
for 
presence/absence
 of 
these 
unique 
biomarker 
ions 
to 
subsequently 
assign 
to 
a 
certain
 taxonomic 
group 
[30]. 
This 
approach 
is 
more 
suitable 
for 
discrim
ination 
between 
closely 
related 
species 
as 
well 
as 
for 
accurate
 identification 
[6]. 
Another 
workflow 
uses 
a 
database 
with 
reference
 fingerprinting 
profiles 
obtained 
as 
a 
consensus 
profile 
from 
a 
set 
of
 type 
strains. 
New 
spectra 
are 
then 
compared 
with 
database 
entries
 and 
the 
closest 
spectrum 
is 
found 
[19]. 
Widely 
used 
commercial
 systems 
like 
SARAMIS 
(Spectral 
ARchiving 
and 
Microbial 
Identifi
cation 
System, 
AnagnosTec) 
and 
Maldi 
Biotyper 
(Bruker 
Daltonics)
 contain 
average 
spectra 
together 
with 
peak 
lists 
of 
the 
most 
dis
criminating 
biomarker 
ions 
which 
are 
scored 
according 
to 
their
 occurrence 
and 
specificity. 
Because 
we 
wanted 
to 
use 
both 
the
 whole 
spectral 
profile 
and 
biomarker 
ions, 
we 
used 
BioNumer
ics 
and 
DataExplorer 
4.0 
(AB 
Sciex) 
respectively, 
as 
an 
alternative
 to 
commercially 
available 
database 
systems. 
Both 
software 
appli
cations 
allowed 
us 
to 
look 
at 
the 
spectra 
in 
detail, 
perform 
our
 own 
quality 
check 
and 
analyze 
the 
data 
in 
different 
ways. 
More
over, 
BioNumerics 
software 
allowed 
the 
coupling 
of 
additional 
data
 from 
more 
than 
one 
experiment 
type 
(e.g., 
sequencing 
information)
 making 
the 
analysis 
easier, 
more 
comprehensive 
and 
straightfor
ward. 
However, 
the 
use 
of 
non-specialized 
software 
also 
had 
the
 disadvantages 
that 
(i) 
no 
specific 
experiment 
type 
was 
available 
for
 spectral 
profiles, 
and 
(ii) 
biomarker 
ions 
detection 
had 
to 
be 
per
formed 
manually 
because 
appropriate 
tools 
were 
lacking. 
There 
is
 still 
debate 
on 
the 
discriminatory 
level 
of 
MALDI-TOF 
MS, 
which 
is
 said 
to 
be 
accurate 
at 
genus, 
species 
[34] 
or 
even 
below 
species 
level
 [3]. 
Nevertheless, 
a 
very 
good 
discrimination 
between 
Clavibacter
 subspecies 
was 
consistently 
obtained. 
As 
MALDI-TOF 
MS 
is 
gaining
 more 
popularity 
as 
a 
quick 
and 
robust 
identification 
tool, 
required
 software 
tools 
will 
probably 
be 
available 
in 
the 
near 
future.
 To 
evaluate 
the 
robustness 
and 
discriminatory 
power 
of 
gyrB
 sequencing 
and 
MALDI-TOF 
MS 
analysis, 
the 
closely 
related 
Clav
ibacter 
strains 
were 
analyzed 
together 
with 
Cmm 
look-alikes.
 Discrimination 
of 
these 
closely 
related 
non-pathogenic 
Clavibac
ter 
by 
MALDI-TOF 
MS 
and 
gyrB 
sequence 
analysis 
indicates 
that
 these 
Cmm 
look-alikes 
may 
be 
used 
to 
study 
specific 
mechanisms
 of 
plant 
disease 
development 
caused 
by 
pathogenic 
Cmm. 
Cmm
 look-alikes 
are 
an 
especially 
interesting 
group 
of 
organisms 
that
 possibly 
form 
a 
new 
group 
of 
Clavibacter 
since 
there 
is 
almost 
no
 evidence 
of 
wild-type 
non-pathogenic 
bacteria 
within 
this 
genus
 [46]. 
Both 
techniques 
applied 
in 
this 
study 
were 
discriminative
406 J. 
Zaluga 
et 
al. 
/ 
Systematic 
and 
Applied 
Microbiology 
34 (2011) 400– 
407
 enough 
to 
differentiate 
strains 
of 
the 
closely 
related 
genus 
Curto
bacterium. 
Misidentification 
of 
Curtobacterium 
as 
Clavibacter 
could
 be 
attributed 
to 
their 
high 
morphological 
similarities 
with 
Clavibac
ter 
or 
contamination 
during 
the 
handling. 
Most 
of 
the 
members
 of 
the 
genus 
Curtobacterium 
have 
been 
isolated 
from 
plants 
but
 only 
one 
species, 
Curtobacterium 
flaccumfaciens 
pv. 
flaccumfaciens,
 is 
regarded 
as 
a 
plant 
pathogen. 
Because 
of 
its 
importance, 
this
 species 
is 
also 
classified 
as 
a 
quarantine 
organism 
on 
the 
A2 
EPPO
 list 
and 
in 
the 
Council 
Directive 
2000/29/EC 
[2,8]. 
Problems 
in 
the
 discrimination 
of 
Curtobacterium 
from 
Clavibacter 
were 
encoun
tered 
previously 
in 
a 
numerical 
classification 
study 
of 
coryneforms
 [20], 
where 
members 
of 
both 
groups 
were 
allocated 
in 
the 
same
 cluster.
 The 
results 
obtained 
with 
the 
genetic- 
and 
protein-based
 approach 
used 
in 
our 
study 
were 
in 
perfect 
agreement 
and 
showed
 their 
value 
as 
stand-alone 
diagnostic 
tools 
for 
the 
genus 
Clavibacter.
 Whereas 
the 
sequence-based 
approach 
is 
more 
time 
consuming, 
it
 is 
surely 
a 
valuable 
tool 
when 
MALDI-TOF 
MS 
is 
not 
available. 
But,
 increasing 
applicability 
of 
MALDI-TOF 
MS 
will 
make 
it 
more 
acces
sible 
for 
the 
end 
users. 
Besides, 
low 
costs 
of 
sample 
preparation 
and
 rapid 
measurement 
procedure 
are 
of 
great 
value 
for 
unbiased 
and
 fast 
diagnostics