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Keywords:

  • 16S rRNA;
  • detection;
  • fish;
  • pathogen;
  • quantitative polymerase chain reaction

Abstract

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

Aims:  To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples.

Methods and Results:  A real-time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean CT value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R2 = 0·99) over a 7-log-unit dynamic range down to ten L. garvieae cells.

Conclusions:  This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel-based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments.

Significance and Impact of the Study:  This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.


Introduction

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

Lactococcus garvieae, a Gram-positive coccus, is considered to be the aetiological agent of lactococcosis in various fish species worldwide (Elder et al. 1996; Kusuda et al. 1991; Vendrell et al. 2006). Besides fish, L. garvieae has been isolated from cows and buffalo (Collins et al. 1983; Carvalho et al. 1997) and has also been identified as a pathogen in humans (James et al. 2000; Mofredj et al. 2000; Vinh et al. 2006). In addition, L. garvieae has recently been isolated in the dairy environment, in raw cow’s milk and traditional Italian cheeses (El-Baradei et al. 2007). Together, these facts indicate the expanding importance of L. garvieae.

The identification of L. garvieae has been performed in a scheme based on biochemical and antigenic characteristics (Holt et al. 1994). However, the discrimination of this micro-organism from other Gram-positive cocci, such as the human pathogen Lactococcus lactis ssp. lactis (Elliott et al. 1991; Fefer et al. 1998) or the Enterococcus-like strains isolated from diseased fish (Eldar et al. 1999; Toranzo et al. 1994; Wallbanks et al. 1990), is still quite difficult.

A real-time quantitative polymerase chain reaction (qPCR), one of the most promising PCR techniques, has been developed as a rapid, simple and convenient method because of its specificity and sensitivity in which it is used to amplify and simultaneously quantify the targeted DNA molecule. Recently, several qPCR assays have been used for the detection and quantification of microbial pathogens, such as Vibrio vulnificus (Gordon et al. 2008), Brucella abortus (Newby et al. 2003), Salmonella (Daum et al. 2002), Escherichia coli (Ahmed et al. 2008) and Yersinia enterocolitica (Lambertz et al. 2008).

In the current study, a qPCR method based on the 16S rRNA gene was performed and evaluated to develop a simple and robust approach for the identification and quantification of pathogenic bioserotypes of L. garvieae from food, as well as various environments.

Materials and methods

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

Bacterial strains

The bacterial strains used in this study are listed in Table 1. The strains were obtained from the Korean Collection for Type Cultures (KCTC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and the Belgian Co-ordinated Collections of Micro-organisms (BCCM/LMG). Enterococci and vagococci were grown aerobically in trypticase soy yeast extract medium (TSYE, DSMZ) at 37°C, and lactococci were grown at 30°C. Streptococcus gordonii, Streptococcus mitis, Streptococcus pyogenes, Streptococcus parasanguinis, Streptococcus sanguinis and Streptococcus pneumoniae were grown under microaerophilic conditions in TSYE medium at 37°C; Streptococcus intermedius and Streptococcus oralis were grown under the same conditions in chopped meat medium (Difco Laboratories, Detroit, MI, USA) and Corynebacterium blood medium (DSMZ), respectively.

Table 1.   Bacterial strains (n = 43) tested and PCR results
No.SpeciesDesignationIsolation sourcePCR
  1. +, PCR product was amplified with the CAU12F-15R primer set; −, PCR product was not amplified with the CAU12F-15R primer set; DSM, Deutsche Sammlung von Mikroorganismen; KCTC, Korean Collection for Type Cultures.

 1Lactococcus garvieaeKCTC 3772TBovine mastitis+
 2L. garvieaeLMG 8501Bovine mastitis+
 3L. garvieaeLMG 12889Diseased yellowtail+
 4L. garvieaeLMG 9472Raw milk+
 5L. garvieaeLMG 8162+
 6L. garvieaeLMG 14494Turtle eye+
 7Lactococcus pisciumKCTC 3639TDiseased rainbow yearling
 8Lactococcus lactis ssp. lactisKCTC 3769T
 9Lactococcus lactis ssp. hordniaeKCTC 3768TLeaf hopper
10Lactococcus lactis ssp. cremorisDSM 20069T
11Lactococcus raffinolactisKCTC 3982TRaw milk
12Lactococcus plantarumDSM 20686TFrozen peas
13Lactococcus chungangensisKCTC 13185TActivated sludge foam
14Streptococcus oralisKCTC 13048THuman mouth
15Strep. oralisDSM 20066Human throat
16Strep. oralisDSM 20395Human plaque
17Strep. oralisDSM 20379Human plaque
18Strep. oralisATCC 9811THuman mouth
19Streptococcus gordoniiKCTC 3286TSubacute bacterial endocarditis
20Streptococcus mitisKCTC 3556THuman oral cavity
21Streptococcus pyogenesKCTC 3984TScarlet fever
22Streptococcus intermediusKCTC 3268T
23Streptococcus parasanguinisKCTC 13046THuman throat
24Streptococcus sanguinisKCTC 3284TSubacute bacterial endocarditis
25Streptoccus pneumoniaeKCTC 5080T
26Tetragenococcus solitariusKCTC 3553TEar exudate
27Enterococcus hiraeKCTC 3616T
28Enterococcus mundtiiKCTC 3630TSoil
29Enterococcus casseliflavusKCTC 3638TPlant material
30Enterococcus malodoratusKCTC 3641TGouda cheese
31Enterococcus cecorumKCTC 3642TChicken caecum
32Enterococcus saccharolyticusKCTC 3643TStraw bedding
33Enterococcus villorumKCTC 13904TGut of a pig
34Enterococcus haemoperoxidusKCTC 13910TService water
35Enterococcus moraviensisKCTC 13911TService water
36Enterococcus phoeniculicolaKCTC 3818TUropygial (preening) gland
37T. solitariusKCTC 3923TEar exudate
38Enterococcus raffinosusKCTC 5189TBlood culture
39Enterococcus aviumKCTC 5190THuman faeces
40Enterococcus faecalisKCTC 3206T
41Vagococcus salmoninarumLMG 11491TRainbow trout
42Vagococcus lutraeLMG 19537TOtter blood
43Vagococcus fluvialisLMG 9464TChicken faeces

Genomic DNA preparation

Bacterial genomic DNA was prepared using the bead–beat method from bacterial strains and Korean salted fermented seafoods (jeotgal samples), as described previously (Chang et al. 2008). Extracted DNA was purified using an UltraClean Microbial DNA Isolation kit (Mo Bio Laboratories, Solana Beach, CA, USA) and quantified using the NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, DE, USA) at a wavelength of 260 nm.

Conventional PCR for species identification

The 16S rRNA gene sequences of the members in the family Streptococcaceae and Enterococcaceae obtained from GenBank databases were aligned using the ClustalX program (Thompson et al. 1997), and then L. garvieae species-specific oligonucleotide primers were designed using Primer 3 software (Rozen and Skaletsky 2000) with default settings. The specificity and optimization of the primer sets were determined using Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the oligonucleotide properties calculator program (http://www.basic.northwestern.edu/biotools/oligocalc.html). PCR amplifications were carried out in a final volume of 20 μl containing 1 μl (10 ng) of template DNA, 10 mmol l−1 Tris–HCl (pH 9·0), 40 mmol l−1 KCl, 250 μmol l−1 dNTPs, 1 U of Taq polymerase, 1·5 mmol l−1 MgCl2 and 10 pmole of each primer. The detection limits were determined with DNA (0·01–100 ng) from the type strain of L. garvieae. The PCR conditions were an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 1 min and a final extension at 72°C for 7 min. Amplifications were performed in a PTC-220 DNA Engine Dyad PCR machine (MJ Research Inc., Waltham, MA, USA). The amplicons were electrophoresed in 2% SeaKem LE agarose gel (FMC Bioproducts, Philadelphia, PA, USA) and visualized by following ethidium bromide staining.

DNA sequencing analyses

Each amplified PCR product was inserted into a pCR 2.1 cloning vector, transformed to E. coli TOP 10F’ (Invitrogen, Carlsbad, CA, USA) and sequenced using the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3730 automated DNA sequencer. Nucleotide sequence homologies were determined using the Blast suite of programs (Altschul et al. 1997) against the NCBI GenBank nonredundant database (http://www.ncbi.nlm.nih.gov).

Real-time qPCR

Standard curves were generated by plotting the cycle threshold values (CT) of the qPCR performed on dilution series of purified DNA from L. garvieae cells (107 to 1 CFU ml−1) against the log input cells ml−1. Lactococcus garvieae KCTC 3772T concentrations were calculated by the viable cell plate count method. Serial tenfold dilutions of the cultures were plated on TSA (Difco). The plates were subsequently incubated at 30°C for 2 days, and CFU were determined in triplicate. The qPCR amplification was performed in a total volume of 20 μl containing 1 μl of each template DNA, 10 pmole of each primer, 7 μl of nuclease-free water and 10 μl of SYBR Green master mix (Finnzymes Inc., Woburn, MA, USA). All the amplifications were carried out in optical grade 96-well plates on a DNA Engine OPTICON™ 2 system (MJ Research). The PCR conditions used were an initial denaturation step of 15 min at 95°C, followed by 60 cycles of 94°C for 20 s, 58°C for 30 s, 72°C for 45 s and a final extension step of 5 min at 72°C. A melting curve analysis followed PCR amplification from 95 to 65°C at a rate of 0·1°C s−1 with continuous acquisition of fluorescence data. All samples were analysed in triplicate. The qPCR amplicons were confirmed by electrophoresis in 2% SeaKem LE agarose gel and ethidium bromide staining.

Detection and quantification of Lactococcus garvieae

To evaluate qPCR for the rapid detection and quantification of L. garvieae, Korean salted fermented seafoods (jeotgal) made with various marine products were tested. The jeotgal samples used in this study were as follows: ggolddugijeot (octopus), saeujeot (shrimp), myeongranjeot (pollock), changnanjeot (pollock), chungeraljeot (fish eggs), meonggaejeot (ascidian), bajirakjeot (short-necked clam), jogaejeot (shellfish) and galchisokjeot (scabbard intestines). All qPCR were performed as described earlier. The CT of samples obtained by qPCR was used to calculate the total number of CFU ml−1 present in each sample based on the standard curve for the type strain of L. garvieae.

Results

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

Specificity of conventional PCR

Oligonucleotide primer construction for the species-specific discrimination of L. garvieae from other closely related Lactococcus species was based on the information from the full nucleotide sequence of the 16S rRNA gene. Two primers (CAU12F, 5′-ACTCGTGCTATCCTT-3′ and CAU15R, 5′-TGGGTACTCCCAACTTCC-3′) were generated, and their specificities were subsequently tested against 43 strains in the genera Lactococcus, Streptococcus, Enterococcus, Vagococcus and Tetragenococcus, including six clinical and environmental L. garvieae strains isolated from bovine mastitis, diseased yellowtail, raw milk and turtle eye. The expected PCR product of 415 bp was common to all L. garvieae strains. This product was efficiently and distinctly detected, because no amplification of PCR products from any other closely related Gram-positive cocci occurred with the primers, CAU12F and CAU15R (data not shown). The detection limits of the specific PCR assay were determined with genomic DNA extracted from the L. garvieae KCTC 3772T at levels from 0·01 to 100 ng of DNA. The detectable minimum concentration set was determined to be 1 ng of DNA (Fig. 1).

image

Figure 1.  The limit of detection for Lactococcus garvieae genomic DNA using conventional PCR with CAU12F and CAU15R primers. Lane: M, 100 bp DNA ladder (Bioneer Corporation, Daejeon, Korea); 1, 100 ng ml−1; 2, 50 ng ml−1; 3, 10 ng ml−1; 4, 5 ng ml−1; 5, 1 ng ml−1; 6, 0·1 ng ml−1; 7, 0·01 ng ml−1 and 8, 0 ng ml−1.

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Sensitivity of real-time qPCR

DNA obtained from an L. garvieae culture at 107 CFU ml−1 was serially diluted tenfold to determine the sensitivity of qPCR using the L. garvieae-specific primers, CAU12F and CAU15R. Each DNA dilution per PCR mixture was used to construct a standard curve. The minimum level of detection with the new primer pair from purified L. garvieae genomic DNA was 32 fg (Fig. 2), with a mean CT value of 36·75 (Table 2). The melting temperature for the amplicon from the L. garvieae type strain was 84·69°C. Linear regression indicated a high correlation between the log numbers of L. garvieae cells and CT values (R2 = 0·99) in qPCR. The linear range was DNA from 10 to 107 cells per PCR mixture.

image

Figure 2.  The limit of detection for Lactococcus garvieae genomic DNA using quantitative polymerase chain reaction with CAU12F and CAU15R primers. Lane: M, 100 bp DNA ladder marker (Bioneer Corporation); 1, 32 ng ml−1; 2, 3·2 ng ml−1; 3, 0·32 ng ml−1; 4, 32 pg ml−1; 5, 3·2 pg ml−1; 6, 0·32 pg ml−1; 7, 32 fg ml−1; 8, 3·2 fg ml−1 and N, 0 ng ml−1.

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Table 2.   Determination of CT values for a dilution series of Lactococcus garvieae with 107 cells
DNA concentration (ng μl−1)Cell numberCT (mean ± SE)
32 × 10−01 × 10710·28 ± 0·31
32 × 10−11 × 10613·59 ± 0·42
32 × 10−21 × 10517·33 ± 0·86
32 × 10−31 × 10421·45 ± 1·55
32 × 10−41 × 10325·24 ± 2·33
32 × 10−51 × 10231·44 ± 0·86
32 × 10−61 × 10136·75 ± 1·89
32 × 10−71 × 100ND
Negative control0ND

Detection and quantification of Lactococcus garvieae

Nine jeotgal samples were tested for the detection of L. garvieae using the qPCR assay. Two jeotgal samples were positive, and seven samples were negative. The number of bacteria in two jeotgal samples detected by the qPCR assay was quantified using the L. garvieae-specific primer set, CAU12F and CAU15R. The population of L. garvieae in ggolddugijeot (octopus) and galchisokjeot (scabbard intestines) was log10 1·51 ± 0·01 (at CT values 33·25 ± 0·08) and 1·715 ± 0·04 (at CT values 32·35 ± 0·21) cells per gram, respectively (Fig. 3). These results were confirmed by sequence analysis of the amplicons obtained from PCR and qPCR assays, which were 100% identical with the fragment in the pathogenic bioserovar L. garvieae strain, IMAU60022 (GenBank accession no. FJ215671).

image

Figure 3.  Standard curves obtained by quantitative polymerase chain reaction using the CAU12F and CAU15R primers from tenfold serial dilutions of Lactococcus garvieae grown in TSA medium ( ◆ ). Correlation between the standard curves obtained from octopus jeotgal ( □ ) and scabbard jeotgal ( △ ). Error bars represent standard errors.

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Discussion

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

A real-time qPCR technique for the detection and quantification of the fish and human pathogen, L. garvieae, was developed and validated. The applicability was evaluated using seven traditional Korean salted seafoods (jeotgal samples). The microflora in jeotgal has been reported to contain various bacterial communities, such as lactic acid bacteria (Lactobacillus and Leuconostoc), halotolerant and/or halophilic bacteria (Staphylococcus, Halomonas, Micrococcus and Salinicoccus), the halophilic archaeon (Halakalicoccus and Natronococcus), spore-forming bacteria (Paenibacillus and Bacillus) and nonspore-forming bacteria (Planomicrobium and Psychrobacter; Mah et al. 2008; Roh et al. 2007; Yoon et al. 2002). However, L. garvieae has not been previously detected in this food.

Lactococcus garvieae is a Gram-positive coccus, facultatively anaerobic, nonmotile, nonspore forming bacteria that produces α-haemolysis on blood agar. The strains have been identified as the causative agent from disease outbreaks in yellowtail and rainbow trout in many countries (Kusuda et al. 1991). The pathogenicity of L. garvieae species isolated from outbreaks has been reported in several fish species, as well as in mice (Kawanishi et al. 2006).

The isolation of L. garvieae from aquatic animals, humans and terrestrial animals indicates that it has been regarded as an emerging pathogen of increasing clinical significance in the fields of veterinary and human medicine (Eyngor et al. 2006; Fihman et al. 2006;Fortina et al. 2007), although a potential virulence risk between strains is different (Barnes et al. 2002). Recently, L. garvieae strains have been recovered from various fish, dolphins, mice, humans, cows and the dairy environment (El-Baradei et al. 2007; Elder et al. 1996; Elliott et al. 1991; Evans et al. 2006; Eyngor et al. 2006; Kawanishi et al. 2006; Romalde et al. 1996).

Phenotypic and genetic analysis by restriction fragment length polymorphism ribotyping, pulsed-field gel electrophoresis, random amplified polymorphic DNA, API system (BioMérieux), serological correlation and antibiotic susceptibility of bacterial strains obtained from various environments indicated that each epidemiological characterization varies greatly and is closely related to the host (Barnes et al. 2002; Eldar et al. 1999; Eyngor et al. 2004; Fortina et al. 2007; Kawanishi et al. 2006; Ravelo et al. 2003; Vela et al. 2000).

Several molecular diagnostic studies have been developed for L. garvieae-specific detection in aquacultures. The primers, pLG-1 and pLG-2 (c. 1100 bp fragment) from the 16S rRNA gene and SA1B10-1-F and SA1B10-1-R (709 bp fragment) from the dihydropteroate synthase gene, could differentiate it from other closely related genera using a conventional PCR-based method (Zlotkin et al. 1998; Aoki et al. 2000). However, these techniques lack assay specificity for the detection of the L. garvieae genomic DNA and can yield false-positive results, such as Tetragenococcus solitarius (data not shown).

The 16S–23S rDNA intergenic spacer region is considered a good potential target for selecting species-specific molecular assays and has already been successfully used for distinguishing lactococci commonly isolated in a dairy environment. (Blaiotta et al. 2002) concluded that this method could not be applied to qPCR because of the high degree of interspecific polymorphism.

Although conventional PCR-based methods were developed to differentiate L. garvieae strains from closely related species, there is a difficulty in diagnosis from the various environmental samples. Studies on the detection and quantification of pathogenic bacteria by real-time PCR assay from various environmental samples have been reported (Blackstone et al. 2003; Lambertz et al. 2008). However, this technique has not been previously applied to L. garvieae. 16S rRNA-based specific primers have been designed from many bacterial species and applied to flora analysis as the most sensitive and rapid method in a natural environment (Iwaya et al. 2005; Neeley et al. 2005; Walter et al. 2001). Furthermore, the 16S rRNA genes are generally present in high numbers in viable cells, turning out to be more sensitive than the housekeeping gene-based assay (Masco et al. 2007). The primer sequences designed in this study for qPCR analysis were selected from a highly specific region in the L. garvieae 16S rRNA gene sequence. The results from the validation of the new primers indicated that the newly designed primer set is specific and reliable in detecting the pathogen, L. garvieae, in natural environmental conditions.

Among the real-time PCR techniques, SYBR Green I PCR provides the simplest and most economical format for detecting and quantifying PCR products (De Medici et al. 2003). A comparison of the cell counts between the two cell counting methods and the DAPI counting method has established the accuracy of qPCR assays (Sails et al. 2003). This is the first time L. garvieae has been detected by SYBR Green I real-time PCR, although it was detected from only two of the nine samples. These results present its potential use for quantitative studies from L. garvieae-associated environmental samples.

The minimum level of detection by the qPCR was 32 fg of purified genomic DNA, equivalent to approx. ten L. garvieae cells per PCR mixture with CT values of 36·75. This sensitivity for detection was similar to those obtained from other pathogenic bacterial species (Lambertz et al. 2008; Sails et al. 2003; Yang et al. 2003). The PCR-negative samples had CT mean values of >37·15, whereas the PCR-positive samples had CT mean values between 32·35 and 33·25 in nine jeotgal samples. One sample with a CT mean value of 37·15 indicates that it may have contained very low levels of L. garvieae genome equivalents per gram. To confirm the specificity of the primers, the sequences of amplicon obtained from the conventional PCR and qPCR assay were analysed using the NCBI BLAST analysis.

This real-time qPCR method based on the 16S rRNA gene was rapid, sensitive and specific for the detection and quantification of L. garvieae in Korean seafood (jeotgal samples). The ability to detect and quantify with real-time PCR could be used for the screening of L. garvieae strains in food or natural environments.

Acknowledgements

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

This work was supported by the 21C Frontier Microbial Genomics and Applications Centre Program, Ministry of Education, Science and Technology (grant 11-2008-03-002-00), Korea. We are grateful to Dr Nagamani Bora (Aston University, UK) for providing editorial assistance with the English.

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