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Food Microbiology 46 (2015) 288e298

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Food Microbiology

journal homepage: www.elsevier .com/locate/ fm

Genetic discrimination of foodborne pathogenic and spoilage Bacillusspp. based on three housekeeping genes

S. Caama~no-Antelo, I.C. Fern�andez-No, K. B€ohme, M. Ezzat-Alnakip, M. Quintela-Baluja,J. Barros-Vel�azquez, P. Calo-Mata*

Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences/College of Biotechnology, University of Santiago deCompostela, Rúa Carballo Calero s/n, Campus Lugo, E-27002 Lugo, Spain

a r t i c l e i n f o

Article history:Received 5 June 2013Accepted 17 August 2014Available online 27 August 2014

Keywords:BacillusPhylogeneticstuf geneBacterial identificationFood safety

* Corresponding author. Tel.: þ34 647344274; fax:E-mail addresses: p.calo.mata@gmail.com, p.calo.m

http://dx.doi.org/10.1016/j.fm.2014.08.0130740-0020/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Bacillus genus includes foodborne pathogenic and spoilage-associated species, such as Bacillus cereus,Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus. Bacillus is also a heterogeneous genus thatincludes closely related species that are difficult to discriminate among, especially when well-conservedgenes such as 16S rRNA and 23S rRNA are considered. The main goal of the present work was to study theusefulness of three housekeeping genes, the TU elongation factor (tuf), the DNA gyrase b subunit (gyrB)and the RNA polymerase b subunit (rpoB) genes, for use in differentiating among the most importantfoodborne Bacillus spp. sequences from 20 foodborne isolated Bacillus strains, and sequences belongingto different Bacillus spp. retrieved from the GenBank were analysed. In general terms, gyrB, rpoB and tufgene regions for the strains considered in this study exhibited interspecific similarities of 57.8%, 67.23%and 77.66% respectively. Novel tufGPF and tufGPR universal primers targeted to the tuf gene weredesigned and proved to be useful for the amplification of all Bacillus spp considered. In conclusion, the tufgene can be considered to be a good target for the differential characterisation of foodborne Bacillusspecies, especially for differentiating B. subtilis and B. cereus from other closely related species.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The Bacillus genus is an extensive and heterogeneous genusconsisting of more than 259 recognized species (http://www.bacterio.cict.fr) widely distributed across many terrestrial and aquatichabitats (Oguntoyinbo, 2007). Besides the Listeria genus, Bacillus isone of the major Gram positive bacteria often involved in food is-sues related to food poisoning and food spoilage. For this reason,their early detection is of crucial importance to ensure food safetyand to avoid economic losses.

Because of endospore formation and survival when food isimproperly cooked, many species in this taxon such as Bacillus ce-reus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus maybe pathogenic and/or cause the spoilage of certain foods (Logan,2011) under unsuitable storage conditions after cooking. In thecase of B. cereus, spores germinate and the vegetative cells multiply,resulting in the production of enterotoxins that have been linked totwo types of poisoning: the diarrhoeal type and the emetic type

þ34 982252195.ata@usc.es (P. Calo-Mata).

(Stenfors Arnesen et al., 2008). Thus, B. cereus has been recognizedas a causative agent of food poisoning. On the other hand, B. subtilishas been implicated in foodborne illness with vomiting as thecommonest symptom, but accompanying diarrhoea was frequentlyreported too. Although rarely causes food poisoning, someB. subtilis strains may occasionally cause food poisoning, such as theoutbreak in a kindergarten caused by milk powder (Pavic et al.,2005). B. licheniformis can cause foodborne illness, with nausea,vomiting, diarrhoea and stomach cramps occurring 5e12 h afterconsumption of contaminated foods. Foodborne outbreaks usuallyinvolve foods such as cooked meats and vegetables, ice cream,desserts, raw milk and industrially-produced baby food contami-nated with these bacteria. Microbial counts, where made, rangedfrom 3 � 105 to 1 �108 CFU g�1 of the implicated food but ingesteddoses are unknown (Salkinoja-Salonen et al., 1999). B. pumilusrarely causes human infection, however in 2006 the presence ofB. pumilus in rice was found to be responsible for three cases of foodpoisoning. The symptoms that resulted from infection includeddizziness, headache, chills, back pain, stomach cramps and diar-rhoea (From et al., 2007).

To date, identification and discrimination of species has beenbased on analysis of morphological, biochemical, and

Table 1Partial gene sequences from GenBank used for phylogenetic tree.

Bacillus strain GenBank sequence for the genes:

16 rRNA 23 rRNA gyrB rpoB tuf

B. subtilis subsp. spizizenii CP002905CP002183

CP002905CP002183

CP002905CP002183

CP002905CP002183

CP002905CP002183

B. subtilis subsp. subtilis NC00964CP002906

NC00964CP002906

NC00964CP002906

NC00964CP002906

NC00964CP002906

B. subtilis NC002468 e NC002468 NC002468 e

B. licheniformis NC006322AE017333CP000002

NC006322AE017333CP000002

NC006322AE017333CP000002

NC006322AE017333CP000002

NC006322AE017333CP000002

B. amyloliquefaciens subsp. Plantarum NC017061HE774679

NC017061HE774679

NC017061HE774679

NC017061HE774679

NC017061HE774679

B. amyloliquefaciens NC014551NC016784NC009725CP002927

NC014551NC016784NC009725CP002927

NC014551NC016784NC009725CP002927

NC014551NC016784NC009725e

NC016784NC009725CP002927e

B. pumilus NC009848 NC009848 NC009848 NC009848 NC009848B. cereus NC011658

NC004722CP001283CP001407CP001186CP001176CP017194CP000227

NC011658NC004722CP001283CP001407CP001186CP001176CP017194CP000227

NC011658NC004722CP001283CP001407CP001186CP001176CP017194CP000227

NC011658NC004722CP001283CP001407CP001186CP001176CP000227e

NC011658NC004722CP001283CP001407CP001186CP001176CP017194CP000227

B. thuringiensis NC008600CP002508CP000485AE017355CP001907CP001903

NC008600CP000485AE017355CP001907CP001903

NC008600CP002508CP000485AE017355CP001907CP001903

NC008600CP002508CP000485AE017355CP001907CP001903

CP000485AE017355CP001907CP001903e

e

B. anthracis CP001215 CP001215 CP001215 CP001215 CP001215

“e” means that there isn't information about this region for this strain.

Table 2Reference strains considered in this study.

Code Bacterial strain Origin NCBI code

BaA81 Bacillus amyloliquefaciensATCC 23842

CECT 493 KC510253

BaA83 Bacillus amyloliquefaciens DSM 23117 KC510254BaC23 Bacillus cereus ATCC 14893 CECT 4014 KC510255BaC27 Bacillus cereus ATCC 9634 CECT 193 KF028778BaC28 Bacillus cereus ATCC 14579 CECT 5050 KC510256BaC29 Bacillus cereus ATCC 14579 CECT 148 KC510257BaC55 Bacillus spp. (Bacillus cereus group) Food extracteda KC510266BaL51 Bacillus licheniformis ATCC 14580 CECT 20 KC510259BaL3 Bacillus licheniformis Food extracteda KC510267BaL33 Bacillus licheniformis Food extracteda KC510268BaL42 Bacillus licheniformis Food extracteda KC510258BaP33 Bacillus pumilus ATCC 14884 CECT 152 KC510260BaP14 Bacillus pumilus Food extracteda KC510269BaS05 Bacillus subtilis subsp. Spizizenii

ATCC 6633CECT 356 KC510261

BaS06 Bacillus subtilis ATCC 6051 CECT 39 KC510262BaS07 Bacillus subtilis ATCC 11774 CECT 35 KC510263BaS73 Bacillus subtilis Food extracteda KC510271BaS69 Bacillus spp. (Bacilus subtilis group) Food extracteda KC510270BaT71 Bacillus thuringiensis ATCC 10792 CECT 197 KC510264BaT74 Bacillus thuringiensis DSM 6101 DSM 6101 KC510265

“e” means that this sequence doesn't appear in the NCBI.a Spoiled pasteurized and refrigerated seafood product.

S. Caama~no-Antelo et al. / Food Microbiology 46 (2015) 288e298 289

immunological characteristics. In recent years, methods based onDNA analysis have been developed for rapid detection and identi-fication of bacteria (Kolbert and Persing, 1999; Mohania et al.,2008). Analysis of 16S rRNA gene sequence (1483e1489 bp) is themost commonly used method for the identification of bacteria andfor elucidating bacterial phylogenetic relationships (Kolbert andPersing, 1999; Joung and Cote, 2002). However, due to high ge-netic homology inside certain bacterial genera, the 16S rRNA maynot be especially useful for the discrimination of closely-relatedspecies (Fox et al., 1992) this being the case of some Bacillus spp.such as the B. cereus and B. subtilis groups.

Thus, the B. cereus group is closely related to species such asBacillus anthracis, Bacillus thuringiensis (Daffonchio et al., 2006),Bacillus mycoides (Drobniewski, 1993; Turnbull, 1999), Bacilluspseudomycoides (Nakamura, 1998), Bacillus weihenstephanensis(Lechner et al., 1998) and Bacillus medusa (Delaporte, 1969). TheB. subtilis group contains the closely related taxa B. subtilis subsp.subtilis (Smith et al., 1964; Nakamura et al., 1999), B. licheniformis(Skerman et al., 1980), Bacillus amyloliquefaciens (Priest et al.,1987), Bacillus atrophaeus (Nakamura, 1989), Bacillus mojavensis,Bacillus vallismortis (Roberts et al., 1996), B. subtilis subsp. spizi-zenii (Nakamura et al., 1999) and Bacillus sonorensis (Palmisanoet al., 2001). However, and despite their genetic relatedness,their pathogenicity and disease spectra differ substantially, thisunderlining the need of achieving a reliable discrimination in thisgenus.

The study of genes other than 16S rRNA, has been considered forspecies discrimination. Thus, the 23S rRNA, the DNA gyrase bsubunit (gyrB) (Chen and Tsen, 2002; La Duc et al., 2004), the RNApolymerase b subunit (rpoB) (Drancourt and Raoult, 2002; Qi et al.,2001) and the TU elongation factor (tuf) (Ghebremedhin et al.,2008) genes have been considered for the identification of certain

bacterial taxons. Such genes may provide well conserved regionswith potential usefulness for the design of primers and molecularprobes for identification purposes in Bacillus spp.

Accordingly, the main objective of this work was to evaluate theusefulness of different DNA regions, mainly the tuf, rpoB and gyrBgenes, to achieve a good discrimination of foodborne Bacillus spp.with relevance for food safety.

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2. Materials and methods

2.1. Species considered and sequence analysis

The sequences of the 16S rRNA, 23S rRNA, gyrB, rpoB and tufgenes of different Bacillus species and strains were downloadedfrom the GenBank sequence database of the National Center forBiotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/)and are shown in Table 1.

All sequences of each individual region of each Bacillus speciesand strain considered were aligned using the CLUSTAL X 1.81 soft-ware (Saitou and Nei, 1987; Thompson et al., 1997). Phylogeneticanalysis was conducted by means of the MEGA 5.0 software (Kumaret al., 2008). Phylogenetic relationships were estimated using theNeighbour-Joining method (Felsenstein, 1985) and maximum like-lihood correction with 1000 bootstrap replicates to constructdistance-based trees. In addition, gene conservation meaning

Fig. 1. Phylogenetic analysis of the 16S rRNA gene of Bacillus spp. from NCBI GenBank usingvalues from Neighbour-Joining analysis. Branch A (B. subtilis/B. amyloliquefaciens), branch B (B

interspecies sequence similarity was evaluated using MEGA 5.0software. In all phylogenetic trees, Lactobacillus brevis NC008497from the GenBank sequence database was included as an outgroup.

2.2. Bacterial strains and growth conditions

A collection of 20 Bacillus species and strains were also studied.Thirteen strains were obtained from the Spanish Type CultureCollection (CECT) and the Germany Culture Collection (DSMZ) andthe other seven strains were obtained from our laboratory collec-tion and were originally isolated from fresh and processed seafoodproducts in previous studies (Table 2). All strains were reactivatedin Brain Heart Infusion (BHI) broth (Becton, Dichinson and Com-pany, Le Pont de Claix, France), and incubated at 30 �C for 24 h.Afterwards, bacterial cultures were grown on Plate Count Agar(PCA, Oxoid, Hampshire, England) at 30 �C for 24 h., and singlecolonies were isolated.

the Neighbour-Joining method. Numbers above and below branches indicate bootstrap. licheniformis), branch C (B. pumilus) and branch D (B. cereus/B. thuringiensis/B. anthracis).

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2.3. DNA extraction

Genomic DNA was prepared from 1 mL of bacterial strains thatwere grown in BHI and incubated at 30 �C for 24 h. Pellets of thisstrains were obtained by spinning at 7500 rpm for 10 min. Bacterialcell lysis was performed by addition of 180 mL of a lysis solution(20 mg/ml lysozyme in 20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100) (SigmaeAldrich, Saint 22 Louis, MO) and after incubation for30 min at 37 �C. Then 25 mL of proteinase K and 200 mL of Buffer ALwere added and the mixture was incubated at 70 �C for 30 min.Genomic DNA was purified using the DNeasy Tissue Mini Kit(Qiagen, Valencia, CA) (Paradis et al., 2005) and quantified using theQuant-iTTM (Invitrogen, Paisley, UK).

2.4. DNA amplification and sequencing of the 16S rRNA and tufgenes

Some primers described in the literature were tested in-silicobut either the alignment or the PCR amplification was poor. The

Fig. 2. Phylogenetic analysis of the 23S rRNA gene of Bacillus spp. from NCBI GenBank usingvalues from Neighbour-Joining analysis. Branch A (B. amyloliquefaciens) branch B (B. subtilis),B. anthracis).

primers tested included: forward primer T1 (50-AAYATGATIA-CIGGIGCIGCICARATGGA-30) and reverse primer T2 (50-CCIA-CIGTICKICCRCCYTCRCG-30) (Campos et al., 2006), forward primerTUF-1 (50-GATGCTGCTCCAGAAGA-30) and reverse primer TUF-2(50-ACCTTCTGGCAATTCAATC-30) (Ventura et al., 2003) forwardprimer Keu1 (50-AAYATGATIACIGGIGCIGCICARATGGA-30) andreverse primer Keu2 (50-AYRTTITCICCIGGCATIACCAT-30)(Chavagnat et al., 2002) and forward primer tuf371 (50-CWGGTCGTGGKACIGTTG-30) and reverse primer tuf384r (50-NGTMCCACGACCWGTIAT-30) (Chavagnat et al., 2002).

Hence, a new pair of primers was designed for the amplificationof Bacillus spp. considered in this study. For it, and considering theresults after of the alignments of the different genes studied, the tufgenewas chosen and the following set of primers were designed ontwo well-conserved regions of tuf gene using the Primer Express2.0 software (Applied Biosystems, Foster City, CA, USA): forwardprimer tufGPF (50-ACGTTGACTGCCCAGGACAC-30) and reverseprimer tufGPR (50-GATACCAGTTACGTCAGTTGTACGGA-30). Bacterialamplifications of the tuf gene by polymerase chain reaction (PCR)

the Neighbour-Joining method. Numbers above and below branches indicate bootstrapbranch C (B. licheniformis), branch D (B. pumilus) and branch E (B. cereus/B. thuringiensis/

S. Caama~no-Antelo et al. / Food Microbiology 46 (2015) 288e298292

were performed with the designed primers. All amplifications as-says comprised 100 ng of template DNA, 5 mL of each oligonucle-otide primer, 25 mL of a master mix (Biomix, Bioline, London, UK),and double distilled water to achieve a final volume of 50 mL. PCRassays were carried out on a MyCycler Thermal Cycler (BioRadLaboratories, Hercules, CA, USA) as described elsewhere (B€ohmeet al., 2010). Amplification conditions were as follows: after pre-incubation at 95 �C for 8 min, 35 cycles, each with 30 s of dena-turation at 95 �C, 1 min annealing at 55 �C and 30 s of extension at72 �C were completed, these being followed with a final elongationstep at 72 �C for 10 min (Chavagnat et al., 2002). PCR products werevisualized by ethidium bromide staining of 2.5% horizontal agaroseelectrophoresis gels run at 100 V.

The 16S rRNA gene was amplified by PCR using the universalprimer pair P8FPL/P806R (Relman et al., 1992) as described byFern�andez-No et al. (2013)

Prior to sequencing, PCR products were purified with the Exo-SAP3 IT Kit (GE Healthcare, Uppsala, Sweden). Direct sequencing

Fig. 3. Phylogenetic analysis of the gyrB of Bacillus spp. from NCBI GenBank using the Neigfrom Neighbour-Joining analysis. Branch A (B. amyloliquefaciens) branch B (B. subtilis), branB. anthracis).

was performed with the BigDye Terminator v3.1 Cycle SequencingKit (Applied Biosystems). The same primers used for PCR were alsoused for sequencing of both strands of the PCR products. Thesequencing reactions were analysed in an automatic sequencingsystem (ABI 3730XL DNA Analyzer, Applied Biosystems, Foster City,CA, USA) provided with the POP-7 system.

Forward and reverse strands were carefully reviewed by eyeusing the Chromaslite 2.00 software (Griffitch University, Queens-land, Australia). Then, sequence alignment using the Clustal X 1.81software and phylogenetic analysis using the MEGA 5.0 softwarewere conducted as mentioned above.

3. Results

3.1. Sequence analysis of the different genes included in this study

In this work, a battery of five genes, some of themwidely used inphylogenetic studies, were compared. The sequences of 16S rRNA

hbour-Joining method. Numbers above and below branches indicate bootstrap valuesch C (B. licheniformis), branch D (B. pumilus) and branch E (B. cereus/B. thuringiensis/

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(1538e1555 bp), 23S rRNA (2923e2931 bp), gyrB (1917e1935 bp),rpoB (3552e3591 bp) and tuf (1185e1191 bp) genes of differentBacillus species and strains were downloaded from the GenBanksequence database (Table 1). After sequence alignment and phylo-genetic analysis with the MEGA 5.0 software, interspecies sequencesimilarities of 92.06% for the 16S rRNA gene (124 variable sites out of1565), 88.43% for the 23S rRNA gene (239 variable sites out of 2039),57.80% for the gyrB gene (819 variable sites out of 1941), 67.23% forthe rpoB gene (1153 variable sites out of 3629), and 77.66% for the tufgene (266 variable sites out of 1191), were found.

When analyzing all genes studied, the tuf gene appeared to bethe best candidate for the discrimination among Bacillus spp. Thisgene exhibited enough well conserved sites for the design of uni-versal primers for the simultaneous amplification of differentspecies, and, at the same time, enough variability to allow speciesdifferentiation.

3.2. Comparative phylogenetic analysis of Bacillus species asdetermined by the five genetic markers

After phylogenetic analysis of the five genes, a phylogenetic treefor each gene was constructed (Figs. 1e5). The phylogenetic tree for

Fig. 4. Phylogenetic analysis of the rpoB gene of Bacillus spp. from NCBI GenBank using thvalues from Neighbour-Joining analysis. Branch A (B. amyloliquefaciens) branch B (B. subtilis),B. anthracis).

the 16S rRNA gene showed four clearly separated branches (Fig. 1):branch A, which included the species B. amyloliquefaciens andB. subtilis; branch B, which included B. licheniformis species; branchC, which included B. pumilus species and branch D, which includedthe B. cereus group (B. cereus, B. thuringiensis and B. anthracis).

In contrast, the phylogenetic trees for the 23S rRNA, gyrB, rpoBand tuf genes exhibited five main branches clearly differentiated (A,B, C, D and E in Figs. 2e5, respectively): branch A, which includedthe species B. amyloliquefaciens; branch B, which included thespecies B. subtilis; branch C, which including B. licheniformis spe-cies; branch D, which included B. pumilus species and branch E,which included the B. cereus group (B. cereus, B. thuringiensis and B.anthracis). This analysis clearly revealed the difficulties in differ-entiating between strains belonging to the B. cereus group due tosimilarities in their nucleotide sequences in all the five genesstudied.

When comparing the phylogenetic trees for the five genemarkers, we found that the differentiation of all species included inthe study was similar in all of them. However, when taking intoaccount the sequence alignment for each gene and the phylogenies,the tuf genewas found to provide new information thatmay help toclarify some weak points detected when using the 16S rRNA gene.

e Neighbour-Joining method. Numbers above and below branches indicate bootstrapbranch C (B. licheniformis), branch D (B. pumilus) and branch E (B. cereus/B. thuringiensis/

Fig. 5. Phylogenetic analysis of the tuf gene of Bacillus spp. from NCBI GenBank using the Neighbour-Joining method. Numbers above and below branches indicate bootstrap valuesfrom Neighbour-Joining analysis. Branch A (B. amyloliquefaciens) branch B (B. subtilis), branch C (B. licheniformis), branch D (B. pumilus) and branch E (B. cereus/B. thuringiensis/B.anthracis).

S. Caama~no-Antelo et al. / Food Microbiology 46 (2015) 288e298294

Furthermore, the potential use of the tuf gene as a genetic markerfor the study of Bacillus spp. relies on the presence of enough wellconserved sites for primers and probes design, and enough vari-ability, as to allow the differentiation of species. Accordingly, thismarker was considered the best choice for primer and probe designin foodborne Bacillus spp.

3.3. Amplification, sequencing and phylogenetic analysis of Bacillusspp. based on the tuf and 16s rRNA genes

Due to the lack of adequate primers on the tuf gene of Bacillusspp. currently available, two novel universal primers were designedfor the amplification of the tuf gene. The design of the universalprimers was targeted to well conserved sites in the alignment, alsotrying that the amplification product was as long as possible as tobe sufficiently different to allow species differentiation. Thus, twoprimers were designed on two well conserved regions of the tufgene: tufGPF (50-ACGTTGACTGCCCAGGACAC-30) and tufGPR (50-GATACCAGTTACGTCAGTTGTACGGA-30). Such primers weredesigned based on the alignment of Bacillus spp. sequencesdeposited in the GenBank sequence database for the tuf gene(Table 1) using the Primer Express 2.0 software. Sequence similaritysearches were carried out with the Blast tool of the NCBI andresulted in identities of 95e100% with the Bacillus/Lactobacillus/

Streptococcus group and between 85 and 100% with a wide range ofbacterial species.

Then, once the primers for the tuf gene were designed, thesewere used for the amplification of all Bacillus strains studied(Table 2), this resulting in the amplification of a single product of791 bp. Afterwards, sequencing was performed in both directions,and the primers used for PCR were also employed for thesequencing of both strands of the PCR products. Subsequently, thesequences were analysed for each species and deposited in theGenBank sequence database (Table 2).

All tuf gene sequences were aligned using Clustal X software.The alignment included wild Bacillus strains, Bacillus strains fromculture collections and Bacillus sequences downloaded from theGenBank. Then, cluster analysis was performed using MEGA soft-ware in order to determine phylogenetic relationships among allBacillus strains (Fig. 6). The tuf gene cluster was compared with 16SrRNA gene cluster for the same strains. In the case of the 16S rRNAgene, all sequences were obtained from a previous work(Fern�andez-No et al., 2013). All these sequences were obtained afterPCR amplification of the 16S rRNA gene with the universal primerpair P8FPL/P806R (Relman et al., 1992). All 16s rRNA sequences ofall Bacillus species were aligned and cluster analysis was performedto determinate phylogenetic relationships among all species(Fig. 7).

Fig. 6. Phylogenetic analysis of the amplified tuf gene of Bacillus spp. from isolation, collection Bacillus spp strains and strains downloaded from NCBI GenBank using the Neighbour-Joining method. Numbers above and below branches indicate bootstrap values from Neighbour-Joining analysis. Branch A (B. amyloliquefaciens) branch B (B. subtilis), branch C(B. licheniformis), branch D (B. pumilus) and branch E (B. cereus/B. thuringiensis/B. anthracis).

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When comparing the tuf gene and 16S rRNA phylogenetic trees,some differences could be observed. In the case of the tuf genephylogenetic tree, five branches could be observed: branch A,which included the species B. amyloliquefaciens; branch B, whichincluded the species B. subtilis; branch C, which includedB. licheniformis species; branch D, which included B. pumilus speciesand branch E, which included the B. cereus group (B. cereus, B.thuringiensis and B. anthracis). However, in the 16S rRNA phyloge-netic tree, only four branches could be differentiated: branch A,which included the species B. amyloliquefaciens and B. subtilis;branch B, which included B. licheniformis species; branch C, whichincluded B. pumilus species and branch D, which included theB. cereus group (B. cereus, B. thuringiensis and B. anthracis).

4. Discussion

The increase in microbial foodborne illnesses in recent years hasincreased the concern about food safety. Among them, certain Ba-cillus spp. such as B. cereus, B. licheniformis, B. subtilis and B. pumilusare relevant due to their spoilage and/or pathogenic nature, alsobeing able to resist certain preservation methods such as pasteuri-zation. In our study, several molecular targets have been explored intheir usefulness for the molecular identification of Bacillus spp. inorder to determine which of these markers is the most suitable forthe discrimination of closely related Bacillus species of food interest.

In general, the 16S rRNA gene sequence analysis is the mostcommonly used method for bacterial identification and for

Fig. 7. Phylogenetic analysis of the 16S rRNA gene of Bacillus spp. from isolation, collection Bacillus spp strains and strains downloaded from NCBI GenBank using the Neighbour-Joining method. Numbers above and below branches indicate bootstrap values from Neighbour-Joining analysis. Branch A (B. subtilis/B. amyloliquefaciens), branch B (B. licheniformis),branch C (B. pumilus) and branch D (B. cereus/B. thuringiensis/B. anthracis).

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constructing bacterial phylogenetic relationships (Kolbert andPersing, 1999; Mohania et al., 2008). Although the 16S rRNA hasbeen useful in phylogenetic studies at the genus level, its use hasbeen questioned in studies at the species level (Martínez-Murcia elal., 1992; Bavykin et al., 2004). Moreover, previous reports haveconfirmed the difficulties in distinguishing certain Bacillus spp.(Chen and Tsen, 2002;Wang et al., 2007; B€ohme et al., 2011). In this

regard, the 16S rRNA sequence similarities of the Bacillus speciesincluded in this study resulted to be very high: 92%. Whencomparing with the other genes studied, the 23S rRNA showedsequence similarities of ca. 88%. In contrast, the tuf, rpoB and gyrBgenes provided more sequence variability, with sequence similar-ities of ca. 78%, 67%, and 58%, respectively. In this sense, althoughthe gyrB or rpoB genes appeared to be good candidates for Bacillus

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species differentiation, little conserved regions were found, thisdifficulting the design of primers for their universal amplification.In contrast, the tuf gene exhibited enough conserved regions forprimer design and enough variable regions for species-specificidentification, either by coupling sequencing or restriction frag-ment length polymorphism analysis. These results agree withprevious studies that pointed out the tuf gene as a good candidatefor inferring relationships among closely related species other thanBacillus spp. (B€ohme et al., 2010; Ke et al., 2000; Ludwig et al., 1993).In some of these studies, primers were designed for the amplifi-cation of this gene (Martineau et al., 2001; B€ohme et al., 2010;Picard et al., 2004), however they were not adequate for theamplification of the species of Bacillus used in this study. Accord-ingly, in this study a new pair of primers for the amplification of thetuf genewas designed and used for the amplification of Bacillus spp.relevant for food safety. The phylogenetic trees constructed withthe tuf gene and 16S rRNA gene were compared. Based on the re-sults obtained, the tuf gene resulted to be more discriminant for thedifferentiation of closely related species such as B. subtilis andB. amyloliquefaciens than other genes or that MALDI-TOF (Laschet al., 2009). In contrast, all gene markers showed that thediscrimination between B. cereus and B. thuringiensis is quite diffi-cult despite the variability within the tuf, rpoB and gyrB genes. Inthis regard, similar results were found when using MALDI-TOF forthe discrimination the B. cereus group (Lasch et al., 2009).

5. Conclusions

Bacillus species differentiation based on five molecular genemarkers, 16S rRNA, 23S rRNA, tuf, rpoB and gyrB was carried out forthe interspecific differentiation of Bacillus species. The 16S rRNAand 23S rRNA genes proved to be more conserved than the othergenes with sequence similarities of ca. 92%. and 88%, respectively.In contrast, the gyrB and rpoB genes, with sequence similarities ofca. 58% and 67%, respectively, were the least conserved. Finally, thetuf gene, with sequence similarities of ca. 78% showed to be moreadequate for species differentiation allowing the differentiation ofB. subtilis and B. amyloliquefaciens. As a conclusion, the tuf genemaybe an efficient alternative target for the identification and taxo-nomic analysis of members of the B. subtilis group, being alsoadequate for probe and primer design for the differentiation offoodborne Bacillus species.

Acknowledgements

This work was funded by project 10PXIB261045PR from Xuntade Galicia, projectAGL2010-19646 from the Spanish Ministry ofScience and Technology and project IPT-322 2011-1290-010000cofinanced by the Spanish Ministry of Economy and Competitivityand the European Regional Development Fund 2007-2013 (FEDER).The work of Sonia Caama~no Antelo is supported by the “OtiliaMillares” research doctoral scholarship from Otilia MillaresFoundation.

References

Bavykin, S.G., Lysov, Y.P., Zakhariev, V., Kelly, J., Jackman, J., Stahl, D.A., Cherni1, A.,2004. Use of 16S rRNA, 23S rRNA, gyrB gene sequence analysis to determinephylogenetic relationships of Bacillus cereus group microorganisms. J. Clin.Microbiol. 42, 3711e3730.

B€ohme, K., Fernandez-No, I., Barros-Velazquez, J., Gallardo, J., Calo-Mata, P.,Canas, B., 2010. Species differentiation of seafood spoilage and pathogenicgram-negative bacteria by MALDI.TOF mass fingerprinting. J. Proteome Res. 9,3169e3183.

B€ohme, K., Fernandez-No, I., Barros-Velazquez, J., Gallardo, J., Canas, B., Calo-Mata, P., 2011. Rapid species identification of seafood spoilage and pathogenic

gram-positive bacteria by MALDI-TOF mass fingerprinting. Electrophoresis 32,2951e2965.

Campos, C., Rodriguez, O., Calo Mata, P., Prado, M., Barros-Velazquez, J., 2006.Preliminary characterization of bacteriocins from Lactococcus lactis, Entero-coccus faecium and Enterococcus mundtii strains isolated from turbot (Psettamaxima). Food Res. Int. 39, 356e364.

Chavagnat, F., Haueter, M., Jimeno, J., Casey, M.G., 2002. Comparison of partial tufgene sequences for the identification of lactobacilli. FEMS Microbiol. Lett. 217,177e183.

Chen, M.L., Tsen, H.Y., 2002. Discrimination of Bacillus cereus and Bacillus thur-ingiensis with 16S rRNA and gyrB gene based PCR primers and sequencing oftheir annealing sites. J. Appl. Microbiol. 92, 912e919.

Daffonchio, D., Raddadi, N., Merabishvili, M., Cherif, A., Carmagnola, L., Brusetti, L.,Rizzi, A., Chanishvili, N., Visca, P., Sharp, R., Borin, S., 2006. Strategy for iden-tification of Bacillus cereus, Bacillus thuringiensis strains closely related to Ba-cillus anthracis. Appl. Environ. Microbiol. 72 (2), 1295e1301.

Delaporte, M., 1969. Description de Bacillus medusa nov. Sp. C. R. Acad. Sci. Ser. D.269, 1129e1131.

Drancourt, M., Raoult, D., 2002. rpoB gene sequence-based identification of Staph-ylococcus species. J. Clin. Microbiol. 40, 1333e1338.

Drobniewski, F.A., 1993. Bacillus cereus related species. Clin. Microbiol. Rev. 6,324e338.

Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using bootstrap.Evolution 39, 783e791.

Fern�andez-No, I.C., B€ohme, K., Díaz-Bao, M., Cepeda, A., Barros-Vel�azquez, J., Calo-Mata, P., 2013. Characterisation and profiling of Bacillus subtilis, Bacillus cereusand Bacillus licheniformis by MALDI-TOF mass fingerprinting. Food Microbiol. 33(2), 235e242.

Fox, G.E., Wisotzkey, J.D., Jurtshunk, P.J., 1992. How close is close: 16S rRNAsequence identity may not be sufficient to guarantee species identity. Int. J. Syst.Bacteriol. 42, 166e170.

From, C., Hormazabal, V., Granum, P.E., 2007. Food poisoning associated withpumilacidin-producing Bacillus pumilus in rice. Int. J. Food Microbiol. 115,319e324.

Ghebremedhin, B., Layer, F., K€onig, W., K€oning, B., 2008. Genetic classification dis-tinguishing of Staphylococcus species based on different partial gap, 16 rRNA,hsp60, sodA and tuf gene sequences. J. Clin. Microbiol. 46, 1019e1025.

Joung, K.B., Cote, J.C., 2002. Evaluation of ribosomal RNA gene restriction patternsfor the classification of Bacillus species related genera. J. Appl. Microbiol. 92,97e108.

Ke, D., Boissinot, M., Huletsky, A., Picard, F.J., Frenette, J., Ouellette, M., Roy, P.H.,Bergeron, M.G., 2000. Evidence for horizontal gene transfer in evolution ofelongation factor Tu in Enterococci. J. Bacteriol. 182, 6913e6920.

Kolbert, C.P., Persing, D.H., 1999. Ribosomal DNA sequencing as a tool for identifi-cation of bacterial pathogens. Curr. Opin. Microbiol. 2, 299e305.

Kumar, S., Nei, M., Dudley, J., Tamura, K., 2008. MEGA: a biologist-centric softwarefor evolutionary analysis of DNA and protein sequences. Briefings Bioinforma. 9,299e306.

La Duc, M.T., Satomi, M., Agata, N., Venkateswaran, K., 2004. gyrB as a phylogeneticdiscrimination for members of the Bacillus anthracis-cereus-thuringiensis group.J. Microbiol. Methods 56, 383e394.

Lasch, P., Beyer, W., Nattermann, H., St€ammler1, M., Siegbrecht, E., Grunow, R.,Naumann, D., 2009. Identification of Bacillus anthracis using MALDI-ToF massspectrometry and artificial neural networks. Appl. Environ. Microbiol. 75 (22),7229e7242.

Lechner, S., Mayr, R., Fransis, K.P., Prub, B.M., Kaplan, T., Wiebner-Gunkel, E.,Stewart, G.S., Scherer, S., 1998. Bacillus weihenstephanensis sp. nov. is a newpsychrotolerant species of the Bacillus cereus group. Int. J. Syst. Bacteriol. 48,1373e1382.

Logan, N.A., 2011. Bacillus and relatives in foodborne illness. J. Appl. Microbiol. 112,417e429.

Ludwig, W., Neumaier, J., Klugbauer, N., Brockmann, E., Roller, C., Jilg, S., Reetz, K.,Schachtner, I., Ludvigsen, A., Bachleitner, M., Fischer, U., Schleifer, K.H., 1993.Phylogenetic relationships of bacteria based on comparative sequence analysisof elongation factor Tu ATP-synthase L-subunit genes. Antonie Leeuwenhoek64, 285e304.

Martineau, F., Picard, F.J., Ke, D., Paradis, S., Roy, P.H., Ouellette, M., Bergeron, M.G.,2001. Development of PCR assay for identification of Staphylococci at genus andspecies levels. J. Clin. Microbiol. 39, 2541e2547.

Martínez-Murcia, A.J., Benlloch, S., Collins, M.D., 1992. Phylogenetic interrelation-ships of members of the genera Aeromonas and Plesiomonas as determined by16S ribosomal DNA sequencing: lack of congruence with results of DNAeDNAhybridization. Int. J. Syst. Bacteriol. 42, 412e421.

Mohania, D., Nagpal, R., Kumar, M., Bhardwaj, A., Yadav, M., Jain, S., Marotta, F.,Singh, V., Parkash, O., Yadav, H., 2008. Molecular approaches for identificationcharacterization of lactic acid bacteria. J Dig. Dis 9, 190e198.

Nakamura, L.K., 1989. Taxonomic relationship of black-pigmented Bacillus subtilisstrains a proposal for Bacillus atrophaeus sp. Nov. Int. J. Syst. Bacteriol. 39,295e300.

Nakamura, L.K., 1998. Bacillus pseudomycoides sp. Nov. Int. J. Syst. Bacteriol. 48,1031e1035.

Nakamura, L.K., Roberts, M.S., Cohan, F.M., 1999. Relationship of Bacillus subtilisclades associated with strains 168 W23: a proposal for Bacillus subtilis subsp.subtilis subsp. nov. Bacillus subtilis subsp. spizizenii subsp. Nov. Int. J. Syst.Bacteriol. 49, 1211e1215.

S. Caama~no-Antelo et al. / Food Microbiology 46 (2015) 288e298298

Oguntoyinbo, F.A., 2007. Monitoring of marine Bacillus diversity among the bacteriacommunity of sea water. Afr. J. Biotechnol. 6, 163e166.

Palmisano, M.M., Nakamura, L.K., Duncan, K.E., Istock, C.A., Cohan, F.M., 2001. Ba-cillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated fromsoil in the Sonoran Desert, Arizona. Int. J. Syst. Evol. Microbiol. 51, 1671e1679.

Paradis, S., Boissinot, M., Paquette, N., B�elanger, S.D., Martel, E.A., Boudreau, D.K.,Picard, F.J., Ouelette, M., Roy, P.H., Bergeron, M.G., 2005. Phylogeny of theEnterobacteriaceae based on genes encoding elongation factor TU and F-ATPaseb-subunit. Int. J. Syst. Evol. Microbiol. 55, 2013e2025.

Pavic, S., Brett, M., Petric, N., Lastre, D., Smoljanovic, M., Atkinson, M., Kovacic, A.,Cetinic, E., Ropac, D., 2005. An outbreak of food poisoning in a kindergartencaused by milk powder containing toxigenic Bacillus subtilis Bacillus lichen-iformis. Arch. Leb. 56, 20e22.

Picard, J.P., Ke, D., Boudreau, D.K., Boissinot, M., Huletsky, A., Richard, D.,Ouellette, M., Roy, R.H., Bergeron, M., 2004. Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 Streptococcal species.J. Clin. Microbiol. 42, 3686e3695.

Priest, F.G., Goodfellow, M., Shute, L.A., Berkeley, R.C.W., 1987. Bacillus amylolique-faciens sp. nov., nom. Rev. Int. J. Syst. Bacteriol. 37, 69e71.

Qi, Y., Patra, G., Liang, X., Williams, L.E., Rose, S., Redkar, R.J., DelVecchio, V.G., 2001.Utilization of the rpoB gene as a specific chromosomal marker for real-time PCRdetection of Bacillus anthracis. Appl. Environ. Microbiol. 67, 3720e3727.

Relman, D., Schmidt, T., MacDermott, R., Falkow, S., 1992. Identification of the un-cultured Bacillus of Whipple's disease. J. Med. 327, 293e301.

Roberts, M.S., Nakamura, L.K., Cohan, F.M., 1996. Bacillus vallismortis sp. nov., a closerelative of Bacillus subtilis, isolated from soil in Death Valley, California. Int. J.Syst. Bacteriol. 46, 470e475.

Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for recon-structing phylogenetic trees. Mol. Biol. Evol. 4, 406e425.

Salkinoja-Salonen, M.S., Vuorio, R., Andersson, M.A., K€ampfer, P., Andersson, M.C.,Honkanen-Buzalski, T., Scoging, A.C., 1999. Toxigenic strains of Bacillus lichen-iformis related to food poisoning. Appl. Environ. Microbiol. 65, 4637e4645.

Skerman, V.B.D., McGowan, V., Sneath, P.H.A., 1980. Approved lists of bacterialnames. Int. J. Syst. Bacteriol. 30, 225e420.

Smith, N.R., Gibson, T., Gordon, R.E., Sneath, P.H.A., 1964. Type cultures proposedneotype cultures of some species in the genus Bacillus. J. General. Microbiol. 34,269e272.

Stenfors Arnesen, L.P., Fagerlund, A., Granum, P.E., 2008. From soil to gut: Bacilluscereus its food poisoning toxins. FEMS Microbiol. Rev. 32, 557e721.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. TheClustalX windows interface: flexible strategies for multiple sequence alignmentaided by quality analysis tools. Nucleic Acids Res. 24, 4876e4882.

Turnbull, P.C.B., 1999. Definitive identification of Bacillus anthracis e a review.J. Appl. Microbiol. 87, 237e240.

Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T.R., Zink, R., 2003. Analysis,characterization, Loci of the tuf genes in lactobacillus Bifidobacterium speciesand their direct application for species identification. Appl. Environ. Microbiol.69, 6908e6922.

Wang, L., Lee, F., Tai, C., Kasai, H., 2007. Comparison of gyrB gene sequences, 16SrRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group.Int. J. Syst. Evol. Microbiol. 57, 1846e1850.