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Keywords:

  • Lactococcus garvieae;
  • Lactococcus lactis;
  • genome;
  • SSH

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors’ contribution
  8. References

Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene–targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our findings may be useful for developing more stable and accurate tools for the discrimination of L. garvieae from other closely related species.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors’ contribution
  8. References

Members of the genus Lactococcus have been primarily isolated from food-related sources and are therefore generally regarded as safe. However, Lactococcus garvieae and Lactococcus lactis species have clinical significance in humans and animals. Lactococcus garvieae is considered to be the etiological agent of lactoccocosis in various fish species worldwide (Eldar et al., 1996; Perez-Sanchez et al., 2011). In addition, it has been isolated from animals, such as cattle, water buffalo, cats, and dogs, and from several cases of endocarditis, osteomyelitis, liver abscess, and gastrointestinal diseases in humans (Collins et al., 1983; Reimundo et al., 2011). For this reason, L. garvieae is considered an emerging pathogen in both veterinary and human medicine. L. lactis has been occasionally isolated from the human urinary tract, wound infections, and patients with endocarditis (Mannion & Rothburn, 1990; Aguirre & Collins, 1993; Zechini et al., 2006).

Traditionally, L. garvieae has been identified using a protocol based on conventional culture and biochemical characteristics (Casalta & Montel, 2008). However, the discrimination of this microorganism from other lactic acid bacteria, such as L. lactis, Streptococcus thermophilus, or Enterococcus-like strains, is still quite difficult (Ogier & Serror, 2008). Several PCR-based methods that target the 16S rRNA gene have been developed for the molecular identification of L. garvieae (Zlotkin et al., 1998; Aoki et al., 2000; Odamaki et al., 2011). However, these assays lack specificity and have shown false-positive results with other bacterial species, such as Tetragenococcus solitarius (Jung et al., 2010). Although the entire genome of L. lactis has been fully sequenced (Bolotin et al., 2001; Siezen et al., 2010; Gao et al., 2011), the genetic content of L. garvieae remains unknown despite its emerging clinical significance. Suppressive subtractive hybridization (SSH), a PCR-based DNA subtraction method, enables the identification of genomic sequence differences between two closely related bacterial species (Huang et al., 2007). This technique has been successfully used to discover species-specific genes that differentiate Bacillus anthracis, Streptococcus pneumoniae, and Streptococcus oralis from closely related species (Kim et al., 2008; Park et al., 2010abc). In this study, SSH was used to identify genomic differences between L. garvieae and L. lactis and was applied to the development of molecular identification methods to distinguish L. garvieae from other members of the genus Lactococcus and closely related species.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors’ contribution
  8. References

Bacterial strains and culture

The bacterial strains used in this study are listed in Table 1. Lactococcus strains used for the construction of an SSH library were L. garvieae KCTC 3772T and Lactococcus lactis ssp. lactis KCTC 3769T, obtained from the Korea Collection for Type Culture (KCTC, Daejeon, Korea). Eleven L. garvieae strains used for PCR amplification were obtained from Belgian Coordinated Collections of Micro-Organisms (BCCM/LMG, Gent, Belgium) or were isolated from fish samples in our laboratory. Other Lactococcus, Streptococcus, and Enterococcus strains were purchased from KCTC, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany), American Type Culture Collection (ATCC, Manassas, VA), and Korean Collection for Oral Microbiology (KCOM, Gwangju, Korea). All bacterial strains were grown on brain heart infusion agar (Difco Laboratories, Detroit, MI) at 37 °C for 20 h except for the oral streptococci strains, which were grown on blood agar plates (Asan Pharm Co., Seoul, Korea).

Table 1. Lactococcus and other bacterial species (n = 32) used in this study
SpeciesStraingarF58Fa and garF58RgarF64Fa and garF64RSA1B10-1-Fb and SA1B10-1-RLG-1c and pLG-2cG-Fd and Lc-R
  1. T, type strain.

  2. a

    This study.

  3. b

    Aoki et al. (2000).

  4. c

    Zlotkin et al. (1998).

  5. d

    Odamaki et al. (2011).

Lactococcus garvieaeKCTC 3772T+++++
LMG 8501+++++
LMG 9472+++++
LMG 8162+++++
CAU 1001+++++
CAU 1002+++++
CAU 1003+++++
CAU 1004+++++
CAU 1005+++++
CAU 1006+++++
CAU 1007+++++
CAU 1008+++++
Lactococcus lactis subsp. lactisKCTC 3769T+
Lactococcus lactis subsp. hordniaeKCTC 3768T
Lactococcus lactis subsp. cremorisDSMZ 20069T
Lactococcus chungangensisKCTC 13185T
Lactococcus plantarumDSMZ 20686T
Lactococcus raffinolactisKCTC 3982T
Streptococcus pneumoniaKCTC 5080T
Streptococcus mitisKCTC 13047T
Streptococcus oralisKCTC 13048T
Streptococcus sanguinisKCTC 3284T
Streptococcus parasanguinisKCTC 13046T
Streptococcus pyogenesKCTC 3984T+
Streptococcus anginosusATCC 33397T+
Streptococcus intermediusKCTC 3268T
Streptococcus gordoniiKCTC 3286T
Streptococcus infantisKCOM 1375
Streptococcus australisKCOM 1439
Streptococcus sinensisKCOM 1017
Enterococcus casseliflavusKCTC 3638T+
Enterococcus solitariusKCTC 3923T++

Genomic DNA preparation

Bacterial genomic DNA used for PCR was extracted from cultivated bacteria using the cetyltrimethylammonium bromide method, as described previously (Kim et al., 2008). Extracted DNA was purified using an UltraClean Microbial DNA isolation kit (Mo Bio Laboratories, Solana Beach, CA) and was quantified using an Infinite200 NanoQuant instrument (Tecan, Männedorf, Switzerland) at a wavelength of 260 nm.

Construction of a subtractive genomic DNA library

SSH was performed to identify L. garvieae-specific genomic DNA using a PCR-Select Bacterial Genome subtraction kit (Clontech, Palo Alto, CA). Lactococcus garvieae KCTC 3772T was used as tester DNA and L. lactis ssp. lactis KCTC3769T as driver DNA. The procedures of SSH were performed according to the manufacturer's instructions, with some modifications (Park et al., 2010c). PCR-selected, tester-specific DNA fragments were subsequently inserted into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA), which was transformed into competent Escherichia coli OneShotTm TOP10 cells (Invitrogen). The transformed E. coli cells were plated onto selective Luria–Bertani medium containing ampicillin/IPTG/X-Gal (Sigma-Aldrich Co., St. Louis, MO), and white colonies were screened for the insert fragments after incubation at 37 °C for 18 h.

Southern blot analysis

To verify the presence of cloned inserts, white colonies were cultured in Luria-Bertani broth (LB), and recombinant plasmid DNA was isolated using a QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany). The inserts were amplified via PCR using primers complimentary to the adaptor sequences at both ends of an insert. PCR products were purified using a QIAquick PCR Purification kit (Qiagen) and were resuspended in 50 μL distilled water. After denaturation at 100 °C for 5 min, the DNA samples were dispensed into separate wells of a BioDot apparatus (BioRad, Hercules, CA), transferred onto a positively charged Hybond-N+ membrane (Amersham Biosciences, Little Chalfont, Bucks, UK), and fixed via irradiation under 1000 mJ UV for 3 min in a Stratalinker® (Stratagene, La Jolla, CA). Probe labeling and hybridization were performed using the ECL Direct Nucleic Acid Labeling and Detection system (Amersham Bioscience) according to the manufacturer's instructions. The membrane was exposed to Hyper Film™ ECL for visualization.

DNA sequencing analyses

The tester-specific clones were sequenced using M13F and/or M13R primers. Cycle sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing kit, and the sequencing reactions were analyzed on an automated DNA sequencer (model 3730; Applied Biosystems, Foster City, CA). The sequences generated from the automatic sequencer were edited by removing the vector and adaptor sequences. Sequence assembly and further editing were performed with the clustal_x 1.81 program (Thompson et al., 1994), and blastn, blastx, and tblastx analyses against the database of the National Center for Biotechnology Information (NCBI) were performed for each sequence to determine homology with other microorganisms and to annotate their functions. The nucleotide sequences obtained in this study were deposited in the dbGSS (database of Genome Survey Sequences) of NCBI GenBank under accession numbers JM426692JM426710 for SSH libraries of L. garvieae (Table 2).

Table 2. Significant results of Lactococcus garvieaeSSH library–specific sequences
Clone nameGenBank accession no.Nucleotide blastCoverage (%)Maximum identity (%)E-valueblastxCoverage (%)Maximum identity (%)E-value
CAUA03No significant similarity foundHypothetical protein L3780053821.00E−05
CAUA06JM426692No significant similarity foundTransposase88801.00E−45
CAUA05Bacillus thuringiensis serovar tenebrionis plasmid pBMB165 hypothetical protein, Rep165 (rep165), and replication-associated proteins genes24918.00E−07Transposase88819.00E−13
CAUB02No significant similarity foundAlcohol dehydrogenase83699.00E−17
CAUB21JM426693No significant similarity foundTransposase74612.00E−07
CAUC08JM426694No significant similarity foundAspartate carbamoyltransferase45612.00E−12
CAUD04JM426695No significant similarity foundTransposase87801.00E−45
CAUD16No significant similarity foundTransposase74612.00E−07
CAUD17JM426696No significant similarity foundTransposase91817.00E−31
CAUD18JM426697No significant similarity foundGlucose-6-phosphate 1-dehydrogenase89629.00E−22
CAUE01Bacillus thuringiensis serovar tenebrionis plasmid pBMB165 hypothetical22901.00E−05Transposase68742.00E−06
CAUE19JM426698No significant similarity foundPolyprenyl-phosphate glycosyltransferase72886.00E−23
CAUE22JM426699No significant similarity foundPolyprenyl-phosphate glycosyltransferase80861.00E−24
CAUE30JM426701No significant similarity foundSingle-strand DNA-binding protein76551.00E−20
CAUE24JM426700No significant similarity foundTransposase46270.49
CAUF02No significant similarity foundPutative fibronectin-binding protein-like protein A75804.00E−16
CAUF07JM426702No significant similarity foundTransposase80845.00E−40
CAUF08JM426703No significant similarity foundCell wall surface anchor family protein98624.00E−08
CAUF09JM426704No significant similarity foundGlutamate/gamma-aminobutyrate antiporter96542.00E−29
CAUF10JM426705No significant similarity foundAminopeptidase N99872.00E−119
CAUF27No significant similarity foundDNA gyrase subunit B77973.00E−19
CAUF43No significant similarity foundHypothetical protein L15897282762.00E−13
CAUF58JM426706No significant similarity foundABC transporter ATP-binding protein68704.00E−12
CAUF60JM426707No significant similarity foundTransposase91801.00E−45
CAUF64JM426708Streptococcus pneumoniae NV104, complete genome98761.00E−102Ribosome recycling factor, UMP kinase50/4578/943E−55/3.00E−54
CAUF82JM426709No significant similarity found  Transposase85617.00E−13
CAUF84JM426710Lactococcus lactis subsp. cremoris MG1363, complete genome44936.00E−78Transposase35961.00E−23

Primer design and PCR amplification

PCR primers were designed for the clones CAUF58 (garF58F, 5′-CGGAGTAGCCGATAATTCCA-3′ and garF58R, 5′-GCAGGTACCCTGAAAAAGGA-3′) and CAUF64 (garF64F, 5′-GTGCTGAACGTCACCTTGAA-3′ and garF64R, 5′-CGTTTGCCATGATTTTTCCT-3′) using primer3 software (Rozen & Skaletsky, 2000). PCRs were performed with 100 ng genomic DNA template in 20-μL reaction mixtures containing 1 μM each primer, 2 μL 10× reaction buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, and 2.5 U Taq polymerase. Amplification was carried out in a GeneAmp PCR system 9700 (Applied Biosystems) under the following conditions: initial denaturation at 94 °C for 5 min, followed by 30 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s, with a final extension at 72 °C for 10 min. The primer specificities were evaluated using 12 L. garvieae, six other Lactococcus, 12 Streptococcus, and two Enterococcus strains and were compared with the specificities of previously reported primers targeting the 16S rRNA gene [pLG-1 and pLG-2 (Zlotkin et al., 1998), SA1B10-1-F and SA1B10-1-R (Aoki et al., 2000), and LcG-F and Lc-R (Odamaki et al., 2011)] using the published PCR conditions. After PCR amplification, 5 μL of each PCR product was resolved on a 1.2% Seakem LE agarose gel (FMC Bioproducts, Rockland, ME) and was visualized on the GelDoc xR image-analysis system (BioRad) after ethidium bromide staining.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors’ contribution
  8. References

DNA signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species (Phillippy et al., 2007). In this study, the DNA signatures specific for L. garvieae were investigated through the identification of sequences present in L. garvieae but absent in a closely related species. Lactococcus lactis was chosen as a reference organism because it is most closely related to L. garvieae in the phylogenetic tree, and its full genome has been determined (Cho et al., 2008).

Using SSH, 192 clonal libraries were generated and tested via reverse Southern blotting analysis using L. garvieae KCTC 3772T as the tester probe and L. lactis ssp. lactis KCTC 3769T as the driver probe to eliminate false-positive clones. Twenty-seven of 192 (14%) clones carried inserts that hybridized to the probe for the Lgarvieae genome but not to that of the L. lactis genome; this percentage is much higher than those of B. anthracis (4.3%) (Kim et al., 2008) and S. oralis (5.8%) (Park et al., 2010a), but almost identical to that of S. pneumoniae (14.1%) (Park et al., 2010c).

The 27 DNA signatures specific to L. garvieae are presented in Table 2. Edited sequences were analyzed using Nucleotide blast analysis. Four (CAUA05, CAUE01, CAUF64, and CAUF84) of the 27 sequences were identified as significantly homologous to sequences from other bacterial species (75%–93% identities). In part, CAUA05 and CAUE01 showed maximum identity with Bacillus thuringiensis serovar tenebrionis plasmid pBMB165 hypothetical protein Rep165 (rep165) and replication-associated proteins genes (91% identity; 1E−06 and 90% identity; 2E−05, respectively); however, the query coverage was very low, ranging from 22% to 24%. blastx analysis of those sequences suggested that this hypothetical protein might be a transposase of the IS116//IS110/IS902 insertion sequence (IS) protein family of S. pneumoniae (81% identity; 9E−13 and 74% identity; 2E−06, respectively).

An IS is a short DNA sequence that acts as a simple transposable element. Different prokaryotic genomes contain different types of IS families; L. lactis does not seem to have this type of IS family (Bolotin et al., 2001), suggesting that this might be a novel transposase introduced from S. pneumoniae via horizontal gene transfer. CAUF64 (GenBank accession number JM426708) showed significant identity with two neighboring genes, pyrH and rrf, of S. pneumoniae NV104 (76% identity; 2E−105). blastx analysis of those sequences showed that this hypothetical protein corresponded to part of the ribosome recycling factor (50% identity; 3E−55) and the uridine 5′-monophosphate (UMP) kinase (94% identity; 3E−54). CAUF84 (GenBank accession number JM426710) was notably matched to transposase gene sequences of Lactococcus lactis ssp. cremoris at both the nucleotide (93% identity; 5E−78) and protein levels (35% identity; 1E−23). The remaining 23 sequences had no identities with any nucleotide sequences in the current NCBI GenBank database. The whole-genome sequences of L. lactis strain subsp. lactis KF147 and CV56 have been reported (Siezen et al., 2010; Gao et al., 2011), but those of L. garvieae have not yet been completed. Thus, there is insufficient nucleotide and protein information in GenBank.

Using the full genome information of L. lactis subsp. lactis IL1403 and S. pneumoniae TIGR4 (Bolotin et al., 2001; Feng et al., 2009), Aguado-Urda et al. (2010) investigated the genomic differences among L. garvieae, L. lactis, and S. pneumoniae using open reading frame (ORF) microarrays. Among 256 genes identified via microarray, seven common genes, namely uracil permease, single-strand DNA-binding protein, aminopeptidase N, DNA gyrase subunit B, ABC transporter ATP-binding protein, ribosome recycling factor, and UMP kinase, were common to our results. The consistency of these data indicates that SSH could be used effectively to exploit DNA signatures instead of expensive microarray-based methods or whole-genome sequencing.

In recent years, molecular genetic analyses based on the 16S rRNA gene have provided a powerful means for characterizing species (Stackebrandt et al., 1991; Fox et al., 1992; Stackebrandt & Goebel, 1994). However, the 16S rRNA gene sequences from members of closely related bacterial species may be so highly conserved that they cannot be used to distinguish between strains at the species level (Stackebrandt et al., 2002). Indeed, the nucleotide sequences of the 16S rRNA genes from the genus Lactococcus exhibit a high degree of similarity, making use of the 16S rRNA gene alone insufficient for discrimination among these species. In the 16S rRNA gene phylogenetic tree, L. garvieae is the most closely related to L. lactis. However, the ability to distinguish between these species is important in the dairy industry and because L. garvieae is a well-known fish pathogen (Cho et al., 2008).

In this study, new PCR assays were developed based on two of 27 DNA signatures identified by SSH and compared with three PCR assays that are currently being used for the detection of L. garvieae. Based on the nucleotide sequences of the genetic loci carrying the novel nucleotide sequence (clone CAUF58; GenBank accession number JM426706) and pyrH gene (clone CAUF64), two specific primer sets were designed and their specificities were evaluated with 32 reference strains. Clone CAUF58, suspected to encode ABC transporter ATP-binding protein, was selected from 23 novel DNA sequences unique to L. garvieae. Clone CAUF64 was chosen from four clones that corresponded to genes in other bacterial species. The pyrH gene of clone CAUF64 matched only S. pneumoniae and S. oralis strains with a maximum identity of 76%, and the query coverage reached 98%. The pyrH encodes uridylate kinase, which is known to be a homohexamer with allosteric effectors of guanosine 5′-triphosphate (GTP) and uridine 5′-triphosphate (UTP) (Serina et al., 1995).

The PCR results are summarized in Table 1. Both primer sets amplified the expected PCR amplicon with a size of 201 bp (clone CAUF58; garF58F and garF58R) or 397 bp (clone CAUF64; garF64F and garF64R) in all Lgarvieae strains but not in any of the other strains of Lactococcus or in Streptococcus and Enterococcus strains (Fig. 1). Primers targeting the 16S rRNA gene have been previously used for L. garvieae screening (Zlotkin et al., 1998; Aoki et al., 2000). However, in the current study, false-positive amplifications with the primer sets SA1B10-1-F and SA1B10-1-R3, LG-1 and pLG-2, and cG-F and Lc-R5 were observed for genomic DNA of L. lactis subsp. lactis, Enterococcus casseliflavus, Enterococcus solitarius, Streptococcus pyogenes, and Streptococcus anginosus. Therefore, the new DNA signatures CAUF58 and CAUF64 show higher specificity for PCR-based detection of L. garvieae compared with those of the current primers.

image

Figure 1. Electrophoretic examination of Lactococcus garvieae-specific PCR products generated with primers garF58F and garF58R (a) and garF64F and garF64R (b). M: GenedireX 100-bp DNA ladder, Lanes 1: L. garvieaeKCTC 3772T, 2: L. garvieaeLMG 8162, 3: L. garvieaeLMG 8501, 4: L. garvieaeLMG 9472, 5: L. garvieaeCAU 1001, 6: L. garvieaeCAU 1002, 7: L. garvieaeCAU 1003, 8: L. garvieaeCAU 1004, 9: L. garvieaeCAU 1005, 10: L. garvieaeCAU 1006, 11: L. garvieaeCAU 1007, 12: L. garvieaeCAU 1008, 13: Lactococcus lactis ssp. lactisKCTC 3769T, 14: Lactococcus lactis ssp. hordniaeKCTC 3768T, 15: Lactococcus lactis subsp. cremorisDSMZ 20069T, 16: Lactococcus chungangensisKCTC 13185T, 17: Lactococcus plantarumDSMZ 20686T, 18: Lactococcus raffinolactisKCTC 3982T, 19: Streptococcus pneumoniaeKCTC 5080T, 20: Streptococcus mitisKCTC 13047T, 21: Streptococcus oralisKCTC 13048T, 22: Streptococcus sanguinisKCTC 3284T, 23: Streptococcus parasanguinisKCTC 13046T. To simplify the figure, other bacterial species are not shown because the results for these strains were the same as those shown in lanes 13–23.

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Lactococcus garvieae, the leading agent of lactococcosis, affects many fish species worldwide. In addition, this bacterium is considered a potential zoonotic microorganism because it is known to cause human infections, and L. garvieae outbreaks in humans have recently been reported in Italy (Reimundo et al., 2011). Our data indicate that SSH can be exploited for the development of more stable and robust chromosome-specific DNA signatures that will supplement the previously reported diagnostic markers including 16S rRNA for accurate identification of L. garvieae.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors’ contribution
  8. References