Curr Microbiol (2014) 68:551–557 DOI 10.1007/s00284-013-0512-5

Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli Iva Jebava • Victoria Chuat • Sylvie Lortal Florence Valence



Received: 10 October 2013 / Accepted: 29 October 2013 / Published online: 21 December 2013 Ó Springer Science+Business Media New York 2013

Abstract We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species ofl actobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.

Electronic supplementary material The online version of this article (doi:10.1007/s00284-013-0512-5) contains supplementary material, which is available to authorized users. I. Jebava (&) Department of Dairy, Fat and Cosmetic Science, Institute of Chemical Technology, Prague, Czech Republic e-mail: [email protected] V. Chuat  S. Lortal  F. Valence CIRM-BIA, UMR 1253—Science et Technologie du Lait et de l’OEuf, Institut National de la Recherche Agronomique, Rennes, France

Introduction Lactobacillus helveticus, a part of the L. acidophilus taxonomic subgroup, is hardly distinguishable by simple physiological and biochemical testing from other closely related thermophilic homofermentative lactobacilli such as L. acidophilus, L. amylovorus, L. crispatus, L. johnsonii, L. gasseri, and especially from the phylogenetically closest species L. gallinarum [10, 12, 17]. Accurate identification of this industrially relevant species is essential in basic and applied research [1]. Lactobacillus helveticus, known also as a probiotic and bioactive peptides producing culture, is mainly used as a starter or adjunct culture in cheese and some fermented drinks [15, 24, 34]. Contrary to L. helveticus which originates principally from dairy sources, L. gallinarum is mainly found in poultry and also in some dairy and non-dairy products like sourdough along with L. helveticus [11, 20, 21, 29, 32]. Nowadays, some simple molecular tools are available for identifying L. helveticus. The first reported DNA-based identification method was based on hybridization of a cloned 2 kb fragment from a 34 kb plasmid in L. helveticus CNRZ1094 [6]. Regarding PCR identification of L. helveticus, specific primers to amplify gene coding for the surface layer (S-layer) protein were also proposed [30]. Later, another PCR based on primers targeting genes coding for aminopeptidase C (pepC), aminopeptidase N (pepN), and trypsin-like serine protease (htrA) was described [9]. For simultaneous detection of L. helveticus and other thermophilic starter cultures in Grana Padano cheese, a multiplex PCR and a PCR assay targeting the genes coding for a cellenvelope associated proteinase (prtH) or the phenylalanyltRNA synthase alpha subunit (pheS) were developed [2, 5]. Remarkably, none of these methods used L. gallinarum as a negative control, except for the work of Ventura et al. [30].

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Table 1 Bacterial strains used in this study Species

Source

Strain

Equivalent

Biotope

Origin

L. helveticus

CCDM

BAII

nd

nd

Czech Republic

CCDM

112

nd

Highland isolate

Czech Republic

CCDM

121

nd

L. helveticus var. pragenesis, starter

Czech Republic

CCDM

125

nd

Starter

Czech Republic

CCDM

136

nd

Highland isolate

Czech Republic

CCDM

139

nd

Highland isolate

Czech Republic

CCDM

140

nd

Highland isolate

Czech Republic

CCDM

380

nd

nd

Czech Republic

CCDM

466

nd

Highland isolate

Czech Republic

CCDM

467

nd

Highland isolate

Czech Republic

CCDM CCDM

468 499

nd nd

Czech Republic Czech Republic France

L. acidophilus

CIRM-BIA

99

ITG LH77

Highland isolate Highland isolate Starter, Comte´ production

CIRM-BIA

100

CNRZ 303

Starter, Comte´ production

France

CIRM-BIA

101

CIP 103146T

CIRM-BIA

102

CNRZ 241

Starter, Emmental production Starter, Comte´ production

France France

France

CIRM-BIA

103

CNRZ 32

Starter, Comte´ production

CIRM-BIA

104

ISLC 5

Starter, Parmesan production

Italy

CIRM-BIA

105

CIP 615

nd

Japan

CIRM-BIA

106

CNRZ 414

Isolate from Koumis (cow’s milk)

Russia

CIRM-BIA

107

ITG LH1

Serum

France

DPC

4571

nd

Swiss cheese whey

Ireland

ICT

BROI

nd

Isolate from raw cow’s milk

Czech Republic

ICT

CH1

nd

Commercial starter

Czech Republic

ICT

KUM

nd

Isolate from Koumis (mare‘s milk)

Kazakhstan

CCDM

151

nd

nd

nd

CIRM-BIA CIRM-BIA

438 439

CNRZ 55 CNRZ 1880

Isolate from mouse excrement Human isolate

United Kingdom nd

CIRM-BIA

444

CNRZ 216

Isolate from mouse digestive tract

France

ICT

3006

nd

nd

nd

ICT

3007

nd

nd

nd

CIRM-BIA

773

CNRZ 449

Starter, Yogurt production

France

CIRM-BIA

776

nd

Starter, Yogurt production

France

CIRM-BIA

658T

CNRZ 208

Starter, Yogurt production

nd

ICT

30117

nd

nd

nd

CIRM-BIA

208

CNRZ 232

nd

nd

CIRM-BIA

210

CNRZ 226

nd

nd

CIRM-BIA

220T

CNRZ 207

Starter, Emmental production

nd

ICT

30139

nd

nd

nd

CCDM

1070T

nd

nd

nd

CIRM-BIA

523

CNRZ 1925

Isolate from chicken

USA

CIRM-BIA CIRM-BIA

664 668T

CNRZ 1924 CNRZ 1931

Conjunctivitis Isolate from chicken

France USA

CIRM-BIA

1322

nd

nd

nd

CIRM-BIA

1323

nd

nd

nd

L. amylolyticus

CIRM-BIA

669T

CNRZ 1928

Isolate from fermented corn

nd

L. johnsonii

CIRM-BIA

650

CNRZ 251

Starter, Gruyere production

France

CIRM-BIA

651

CNRZ218

Isolate from mouse digestive tract

France

L. delbruecki subsp. bulgaricus

L. delbruecki subsp. lactis

L. crispatus

L. gallinarum

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I. Jebava et al.: Peptidoglycan Hydrolases as Species-Specific Markers

553

Table 1 continued Species

L. rhamnosus

Source

Strain

Equivalent

CIRM-BIA

674T

CIRM-BIA

572

CIRM-BIA

604 T

Biotope

Origin

CNRZ 1937

Isolate from human blood

USA

nd

Raw cow0 s milk isolate

Brasil

nd

Raw cow0 s milk isolate

Brasil nd

CIRM-BIA

607

CNRZ 212

nd

ICT

30105

nd

nd

nd

CIRM-BIA

666T

CNRZ 209

Fermented sugar-beet

nd

CIRM-BIA

697

CNRZ 1627

Starter

Italy

CIRM-BIA

872

CNRZ 17

Starter, Gruyere production

France

L. plantarum

ICT

3011

nd

nd

nd

L. paracasei

ICT

30125

nd

nd

nd

L. reuteri

ICT

30111

nd

nd

nd

L. ultunensis

CCDM

838

nd

nd

nd

L. fermentum

CCDM Czech collection of dairy microorganisms, Prague, Czech Republic, CIRM-BIA Centre international de resources Microbiennes–Bacte´ries d0 Inte´reˆt Alimentaire, INRA Rennes, France, CNRZ Centre National de Recherche Zootechnique collection, INRA Jouy-en-Josas, France CIP Collection of Institut Pasteur, Paris, France ICT Institute of Chemical Technology, Prague, Czech Republic, ISLC Instituto Sperimentale Lattiero Caseario di Lodi, Italy, ITG Institut Technique du Gruye`re, La Roche-sur-Foron, France, nd not determined

To our knowledge, a simple PCR method to differentiate L. helveticus from its genomically closest species, L. gallinarum, has not been reported yet. Although the 16S rRNA gene sequencing is increasingly used to identify lactic acid bacteria, high sequence similarity between closely related species makes their discrimination at the genomic level difficult. Particularly, genotypic differentiation between L. helveticus and L. gallinarum is still not evident [7, 11, 14, 18, 23]. The first complete genome of L. helveticus DPC 4571 enabled to perform bioinformatic analysis of orthologous genes between related species [4]. Among the potentially genomic markers the peptidoglycan hydrolases, essential enzymes for cell growth and division, have shown important homology within L. helveticus strains [16, 28]. Peptidoglycan hydrolases are ubiquitous and contrary to cell wall structure [33]; they do not participate on different autolytic capacity among L. helveticus strains. Two conserved genes coding for peptidoglycan hydrolases in L. helveticus, Lhv_0190 and Lhv_0191, are known to be organized in an operon, but display different levels of sequence homology between themselves. The objective of this study was to develop a PCR method based on Lhv_0190 and Lhv_0191 genes to differentiate L. helveticus from other closely related homofermentative lactobacilli, especially from L. gallinarum.

Materials and Methods Bacterial Strains and Growth Conditions A total of 25 L. helveticus strains, characterized previously by Jebava et al. [16] from CIRM-BIA (Centre International

de Ressources Microbienne—Bacte´rie d0 Inte´reˆt Alimentaire, Rennes, France) and CCDM (Czech Collection of Dairy Microorganisms, Prague, Czech Republic), were used in this study. In addition, 35 strains of other Lactobacillus spp. were included as negative controls (Table 1). Cultures were grown in MRS broth (Difco Laboratories, MI, USA) at 42 °C for the following species: L. helveticus, L. delbrueckii, L. fermentum, L. acidophilus, and L. ultunensis; at 37 °C for L. johnsonii, L. amylolyticus, L. crispatus, and L. gallinarum, and at 30 °C for the other species. Bacterial growth was monitored at 650 nm using a spectrophotometer Beckman DU 7400 (Beckman Coulter, Nyon, Switzerland). Genomic DNA Extraction Genomic DNA was extracted from strains grown for 12 h at 42 °C in MRS broth by DNeasy Blood&Tissue Kit (QIAGEN, Hilden, Germany) as described previously [16]. In Silico Analysis of 16S rDNA Sequences All sequences were acquired from GenBank (NCBI, http:// www.ncbi.nlm.nih.gov/sites/gquery) and aligned using MEGA 5 software (http://www.megasoftware.net/) [22, 26] using the neighbor-joining method. Accession Nos of compared partial 16S rRNA gene sequences of type strains were: L. acidophilus DSM 20079T (equiv. NBRC 13951, GenBank ID: AB680529.1), L. amylovorus DSM 20531T (GenBank ID: AY944408.1), L. crispatus DSM 20584T (GenBank ID: AF257097.1), L. gallinarum DSM 10532T (equiv. JCM 2011T, GenBank ID: AB596947.1), L. gasseri DSM 20243T (equiv. CIP 102991T GenBank ID:

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I. Jebava et al.: Peptidoglycan Hydrolases as Species-Specific Markers

Table 2 Specific primers designed for Lhv_0190 and Lhv_0191 using Primer3 software and applied in this work Sequence (50 –30 )

Position

PCR product length (bp)

Lhv_0190Fwd:

CAGTTGTGTTGACTTCCACAAT

35–57

542

Lhv_0190Re:

CAAATTGTGGCTGGTGATTCT

555–576

Lhv_0191Fwd:

GGGCTGATTACAGTGGCTAAT

43–64

Lhv_0191Re:

CTTGCCCTTTTCGGTGTAAA

769–789

Locus

Primer

Lhv_0190 Lhv_0191

HE573914.1), L. delbrueckii subsp. bulgaricus DSM 20081T (BCRC 10696T, GenBank ID: AY773948.1), L. helveticus DSM 20075T (GenBank ID: AY369116.1), L. johnsonii DSM 10533T (equiv. CIP 103602T, GenBank ID: AJ002515.1), L. salivarius DSM 20555T (equiv. JCM 1231, GenBank ID: AB370881.1), and L. ultunensis DSM 16047T (GenBank ID: AB370881.1). Primer Design Lhv_0190 and Lhv_0191 genes, coding for two peptidoglycan hydrolases of L. helveticus DPC 4571 (GenBank ID: NC_010080.1), were aligned with their ortholog genes in sequenced strains: L. helveticus DSM 20075 (GenBank ID: NZ_ACLM00000000.1), L. acidophilus NCFM (GenBank ID: NC_006814.3), L. delbrueckii subsp. bulgaricus ATCC 11842 (GenBank ID: NC_008054.1), L. crispatus 125-2-CHN (GenBank ID: NZ_ACPV00000000.1), L. crispatus ST1 (GenBank ID: NC_014106.1), and L. ultunensis DSM 16047 (GenBank ID: NZ_ACGU00000000.1). The comparison of two PGHs genes between L. helveticus and L. gallinarum cannot be performed due to the lack of L. gallinarum complete genome. The identical regions between ortholog genes of L. helveticus DPC 4571 and L. helveticus DSM 20075 as well as the different regions between L. helveticus DPC 4571 and other ortholog genes of other species (L. acidophilus, L. delbrueckii, L. crispatus, and L. ultunensis) were determined. Using Primer3Plus software [27], two pairs of specific primers (Table 2) were designed in the conserved regions of L. helveticus strains and, at same time, in the most variable regions of other species. The first set of primers (Lhv_0190Fwd and Lhv_0190Re) was used to amplify the sequence of the Lhv_0190 gene coding for the N-acetylglucosaminidase and the second set (Lhv_0191Fwd and Lhv_0191Re) to detect the Lhv_0191 gene coding for the N-acetylmuramyl-L-alanine amidase. PCR Amplification The composition of the PCR reaction mix was as follows: 20 ng genomic DNA, 2.5 lL 10 9 buffer containing MgCl2 (QIAGEN), 1 lL 10 mM dNTP Mix (QIAGEN), 1 lL of each primer (50 lM), and 2.5 U Taq DNA

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polymerase in a final volume of 25 lL. PCR was performed using a VeritiTM 96-well thermal cycler (Applied Biosystems, Foster City, CA, USA). Similar conditions for single gene amplification and multiplex PCR were used. The PCR conditions were: denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 45 s. A final extension step was performed for 10 min at 72 °C before the storage of the samples at 4 °C. Negative (no DNA in sample) and positive (DNA from L. helveticus DPC 4571) controls were included in all PCR sets. All PCR products were analyzed by electrophoresis in a 1 % (w/v) agarose gel (Interchim, Montlucon, France) in a Mini-Sub cell GT system (BioRad, CA, USA). After electrophoresis, the gels were stained with ethidium bromide and visualized by UV light.

Results and Discussion In silico analysis of partial 16S rDNA gene was used to study genomic relationships among closely related obligatory homofermentative lactobacilli [25]. It showed that L. helveticus is mostly related to L. gallinarum (Fig. 1). Their genomic proximity explains the difficulty to perform accurate identification based only on 16S rRNA gene sequencing [8, 18, 31]. In order to perform the differentiation between these two closely related species, a PCR method based on the detection of genes coding for peptidoglycan hydrolases was designed. Since the complete genome of L. gallinarum is not available, the second closely related species, L. acidophilus (Fig. 1), was used to compare genes coding for peptidoglycan hydrolases. Lhv_0190 and Lhv_0191 sequences in L. helveticus DPC 4571 were compared to the orthologous genes, Lba_0176 and Lba_0177, in L. acidophilus NCFM. Lhv_0190 and Lhv_0191 and their orthologous genes in L. acidophilus NCFM showed 21.9 and 24.82 % variability in nucleotide sequences, respectively (for detail sequence analysis see Supplementary materials). This comparison revealed sufficient differences to construct species-specific primers to identify L. helveticus strains by PCR. Lhv_0190 and Lhv_0191 sequences in L. helveticus DPC 4571 were also compared to their orthologous genes, HMPREF0518_0918

I. Jebava et al.: Peptidoglycan Hydrolases as Species-Specific Markers

555

Fig. 1 Evolutionary relationship of selected homofermentative lactobacilli based on comparison of 16S rRNA. The analysis involved ten nucleotide sequences and, in total, 1,366 nucleotidic positions in the final dataset. All positions containing gaps and missing data were eliminated

Fig. 2 PCR products obtained by multiplex PCR for selected closely related lactobacilli. Lanes. 1-L. acidophilus CIRM-BIA 438, 2-L. delbrueckii subsp. bulgaricus CIRM-BIA 658T, 3-L. delbrueckii subsp. lactis CIRM-BIA 220T, 4-L. crispatus CIRM-BIA 1070T, 5-L. gallinarum CIRM-BIA 668T, 6-L. helveticus DPC 4571, 7-L.

amylolyticus CIRM-BIA 669T, 8-L. johnsonii CIRM-BIA 674T, 9-L. rhamnosus CIRM-BIA 607T, 10-L. fermentum CIRM-BIA 666T, 11-L. ultunensis CCDM 838, 12 negative control, M molecular weight marker

and HMPREF0518_0919, in L. helveticus DSM 20075. The intra-species homology achieved 92.16 and 99.54 % identity for L. helveticus DPC 4571 and L. helveticus DSM 20075 genes. The search for orthologous genes in other closely related species of L. helveticus to perform in silico analyses was limited by the few data available in genomic databases. Nevertheless, orthologous genes in L. delbrueckii, L. crispatus, and L. ultunensis could be compared to L. helveticus DPC 4571 genes. For Lhv_0190, the lowest homology was found at the positions 30–60 and 550–580, and so the primers were designed at positions 35–57 and 555–576 (Lhv_0190Fwd and Lhv_0190Re, Table 2). A set of primers for Lhv_0191 was constructed within two regions showing minimal homology at the positions 43–64 and 769–789 (Lhv_0191Fwd and Lhv_0191Re, Table 2). Attention was paid to compatibility of these sets of primers regarding the size of the PCR products generated, preserving the possibility to carry out a multiplex PCR reaction.

When using a single PCR for each set of primers, only one PCR product of the predicted size, 542 bp for Lhv_0190 and 747 bp for Lhv_0191, was obtained (data not shown). Following a multiplex PCR, using the two sets of primers together, two PCR products were obtained: the first one of 542 bp corresponding to Lhv_0190 gene and the second one of 747 bp corresponding to Lhv_0191 gene. The PCR method was applied to 25 strains of L. helveticus previously identified by sequencing the whole 16S rRNA gene [16]. Specific amplifications at the expected size of 542 and 747 bp were observed for all tested strains of L. helveticus, with two pairs of primers used as either single or in multiplex PCR (Fig. 2). To confirm the specificity of the proposed PCR method, the primers were applied to 33 strains belonging to 13 closely related species of lactobacilli (Table 1). There was no amplification observed except for L. gallinarum (Fig. 2). For this species, one amplicon of 542 bp corresponding to Lhv_190 gene and one additional amplicon

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greater than 1,500 bp were observed. These two amplicons were consistently observed for three other tested strains of L. gallinarum. To our knowledge, this proposed multiplex PCR is the first using peptidoglycan hydrolase genes for species identification of Lactobacillus species, even though the taxonomic interest of peptidoglycan hydrolase zymograms was highlighted previously [19]. For other species, e.g., Vibrio anguillarum, a PCR method based on the detection of amiB encoding the Nacetylmuramyl-L-alanine amidase was successfully applied for species discrimination [13]. Bacterial identification based on conserved peptidoglycan hydrolase gene sequence could be certainly used more generally to distinguish closely related bacterial species. Considering the decreasing cost and increasing availability of sequencing data, determination of nucleotide sequences of peptidoglycan hydrolases and their use to define species-specific primers could become widespread complementing 16S rRNA sequencing. The targeted genes, i.e., peptidoglycan hydrolases, could be used analogically for identification of other homofermentative lactobacilli. The accurate identification of bacterial species has a great relevance in food analysis, microbial ecology, and also in clinical diagnosis. The proposed multiplex PCR offered sensitive and rapid identification of L. helveticus and could be applied to detect L. helveticus in dairy products, such as cheese and fermented milks. Lactobacillus gallinarum is not important from a technological point of view, but until now, its potential presence in dairy products has not been taken into account [3, 29]. However, L. gallinarum identification itself could become very important in future research because of its potential antimicrobial properties [3].

Conclusion The detection of microorganisms is highly dependent on the specificity of the probes used for PCR. Most commonly used 16S rRNA, nucleotide sequences of small-subunit rRNA, may not always be high enough to distinguish closely related species even by subsequent sequencing. Therefore, the proposed multiplex PCR, highly specific for the identification of L. helveticus, should prove to be more powerful to discriminate between L. helveticus and L. gallinarum than the 16S rRNA sequencing used alone. A routine PCR technique with more accessible materials should increase the feasibility and accessibility of correct identification of these closely related species. Acknowledgments This work was supported by research grants from INRA and the Ministry of Education, Youth and Sport of the Czech Republic (Grant No. MSMT6046137305). We are grateful to Tom Beresford for providing the strain DPC 4571.

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Peptidoglycan hydrolases as species-specific markers to differentiate Lactobacillus helveticus from Lactobacillus gallinarum and other closely related homofermentative lactobacilli.

We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, espe...
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