Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse
Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD50 value in rainbow trout obtained for strain 074 was 2.1×102±84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.
Received:
Accepted:
Revised:
Published Online:
Funding
This study was supported by the:
Ministry of Science and Innovation of Spain
(Award CSD2007-00002 and AGL2009-07003)
AlegríaA.,
Alvarez-MartínP.,
SacristánN.,
FernándezE.,
DelgadoS.,
MayoB.(2009). Diversity and evolution of the microbial populations during manufacture and ripening of casín, a traditional Spanish, starter-free cheese made from cow's milk. Int J Food Microbiol 136:44–51 [View Article][PubMed]
AubinG. G.,
BémerP.,
GuillouzouicA.,
CrémetL.,
TouchaisS.,
FraquetN.,
BoutoilleD.,
ReynaudA.,
LepelletierD.,
CorvecS.(2011). First report of a hip prosthetic and joint infection caused by Lactococcus garvieae in a woman fishmonger. J Clin Microbiol 49:2074–2076 [View Article][PubMed]
BarakatR. K.,
GriffithsM. W.,
HarrisL. J.(2000). Isolation and characterization of Carnobacterium, Lactococcus, and Enterococcus spp. from cooked, modified atmosphere packaged, refrigerated, poultry meat. Int J Food Microbiol 62:83–94 [View Article][PubMed]
BoekhorstJ.,
HelmerQ.,
KleerebezemM.,
SiezenR. J.(2006). Comparative analysis of proteins with a mucus-binding domain found exclusively in lactic acid bacteria. Microbiology 152:273–280 [View Article][PubMed]
BohnsackJ. F.,
TakahashiS.,
DetrickS. R.,
PelinkaL. R.,
HammittL. L.,
AlyA. A.,
WhitingA. A.,
AddersonE. E.(2001). Phylogenetic classification of serotype III group B streptococci on the basis of hylB gene analysis and DNA sequences specific to restriction digest pattern type III-3. J Infect Dis 183:1694–1697 [View Article][PubMed]
BoydE. F.,
BrüssowH.(2002). Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends Microbiol 10:521–529 [View Article][PubMed]
CarvalhoM. G.,
VianniM. C.,
ElliottJ. A.,
ReevesM.,
FacklamR. R.,
TeixeiraL. M.(1997). Molecular analysis of Lactococcus garvieae and Enterococcus gallinarum isolated from water buffalos with subclinical mastitis. Adv Exp Med Biol 418:401–404[PubMed]
ChanJ. F.,
WooP. C.,
TengJ. L.,
LauS. K.,
LeungS. S.,
TamF. C.,
YuenK. Y.(2011). Primary infective spondylodiscitis caused by Lactococcus garvieae and a review of human L. garvieae infections
. Infection [View Article][PubMed]
ChenZ.,
LuongT. T.,
LeeC. Y.(2007). The sbcDC locus mediates repression of type 5 capsule production as part of the SOS response in Staphylococcus aureus
. J Bacteriol 189:7343–7350 [View Article][PubMed]
DàvilaE.,
ZamoraL. M.,
PlaM.,
CarreteroC.,
ParésD.(2006). Identification and antagonistic activity of lactic acid bacteria occurring in porcine blood from industrial slaughterhouses – a preliminary study. Int J Food Microbiol 107:207–211 [View Article][PubMed]
DeivanayagamC. C.,
RichR. L.,
CarsonM.,
OwensR. T.,
DanthuluriS.,
BiceT.,
HöökM.,
NarayanaS. V.(2000). Novel fold and assembly of the repetitive B region of the Staphylococcus aureus collagen-binding surface protein. Structure 8:67–78 [View Article][PubMed]
DevrieseL. A.,
HommezJ.,
LaevensH.,
PotB.,
VandammeP.,
HaesebrouckF.(1999). Identification of aesculin-hydrolyzing streptococci, lactococci, aerococci and enterococci from subclinical intramammary infections in dairy cows. Vet Microbiol 70:87–94 [View Article][PubMed]
el-AdhamiW. A.,
StewartP. R.,
MatthaeiK. I.(1997). The isolation and cloning of chromosomal DNA specific for a clonal population of Staphylococcus aureus by subtractive hybridisation. J Med Microbiol 46:987–997 [View Article][PubMed]
El-BaradeiG.,
Delacroix-BuchetA.,
OgierJ. C.(2007). Biodiversity of bacterial ecosystems in traditional Egyptian Domiati cheese. Appl Environ Microbiol 73:1248–1255 [View Article][PubMed]
EldarA.,
GhittinoC.,
AsantaL.,
BozzettaE.,
GoriaM.,
PrearoM.,
BercovierH.(1996).Enterococcus seriolicida is a junior synonym of Lactococcus garvieae, a causative agent of septicemia and meningoencephalitis in fish. Curr Microbiol 32:85–88 [View Article][PubMed]
EldarA.,
GoriaM.,
GhittinoC.,
ZlotkinA.,
BercovierH.(1999). Biodiversity of Lactococcus garvieae strains isolated from fish in Europe, Asia, and Australia. Appl Environ Microbiol 65:1005–1008[PubMed]
ElliottJ. A.,
CollinsM. D.,
PigottN. E.,
FacklamR. R.(1991). Differentiation of Lactococcus lactis and Lactococcus garvieae from humans by comparison of whole-cell protein patterns. J Clin Microbiol 29:2731–2734[PubMed]
EyngorM.,
ZlotkinA.,
GhittinoC.,
PrearoM.,
DouetD. G.,
ChilmonczykS.,
EldarA.(2004). Clonality and diversity of the fish pathogen Lactococcus garvieae in Mediterranean countries. Appl Environ Microbiol 70:5132–5137 [View Article][PubMed]
FeferJ. J.,
RatzanK. R.,
SharpS. E.,
SaizE.(1998).Lactococcus garvieae endocarditis: report of a case and review of the literature. Diagn Microbiol Infect Dis 32:127–130 [View Article][PubMed]
FlórezA. B.,
MayoB.(2006). Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE. Int J Food Microbiol 110:165–171 [View Article][PubMed]
FurutanN. P.,
BreimanM. A.,
FischerM. A.,
FacklamR. R.(1991).Lactococcus garvieae infection in humans: a cause of prosthetic valve endocarditis [C297]. Proceedings of the 91st America Society of Microbiology Conference109 Dallas: American Society for Microbiology;
HallT. A.(1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
JamesP. R.,
HardmanS. M.,
PattersonD. L.(2000). Osteomyelitis and possible endocarditis secondary to Lactococcus garvieae: a first case report. Postgrad Med J 76:301–303 [View Article][PubMed]
Juíz-RíoS.,
OsorioC. R.,
de LorenzoV.,
LemosM. L.(2005). Subtractive hybridization reveals a high genetic diversity in the fish pathogen Photobacterium damselae subsp. piscicida: evidence of a SXT-like element. Microbiology 151:2659–2669 [View Article][PubMed]
KawanishiM.,
KojimaA.,
IshiharaK.,
EsakiH.,
KijimaM.,
TakahashiT.,
SuzukiS.,
TamuraY.(2005). Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan. Lett Appl Microbiol 40:322–328 [View Article][PubMed]
KawanishiM.,
YoshidaT.,
YagashiroS.,
KijimaM.,
YagyuK.,
NakaiT.,
MurakamiM.,
MoritaH.,
SuzukiS.(2006). Differences between Lactococcus garvieae isolated from the genus Seriola in Japan and those isolated from other animals (trout, terrestrial animals from Europe) with regard to pathogenicity, phage susceptibility and genetic characterization. J Appl Microbiol 101:496–504 [View Article][PubMed]
KawanishiM.,
YoshidaT.,
KijimaM.,
YagyuK.,
NakaiT.,
OkadaS.,
EndoA.,
MurakamiM.,
SuzukiS.,
MoritaH.(2007). Characterization of Lactococcus garvieae isolated from radish and broccoli sprouts that exhibited a KG+ phenotype, lack of virulence and absence of a capsule. Lett Appl Microbiol 44:481–487 [View Article][PubMed]
KimS. R.,
NonakaL.,
SuzukiS.(2004). Occurrence of tetracycline resistance genes tetM and tetS in bacteria from marine aquaculture sites. FEMS Microbiol Lett 237:147–156[CrossRef]
KongJ.,
JosephsenJ.(2002). The ability of the plasmid-encoded restriction and modification system LlaBIII to protect Lactococcus lactis against bacteriophages. Lett Appl Microbiol 34:249–253 [View Article][PubMed]
KongJ.,
JytteJ.,
MaG. R.(2001). [Cloning and structure analysis of a restriction and modification system, LlaBIII from Lactococcus lactis subsp. cremoris W56]. Sheng Wu Gong Cheng Xue Bao 17:663–668[PubMed]
KopermsubP.,
YunchalardS.(2010). Identification of lactic acid bacteria associated with the production of plaa-som, a traditional fermented fish product of Thailand. Int J Food Microbiol 138:200–204 [View Article][PubMed]
LlosaM.,
Gomis-RüthF. X.,
CollM.,
de la Cruz FdF.(2002). Bacterial conjugation: a two-step mechanism for DNA transport. Mol Microbiol 45:1–8 [View Article][PubMed]
MichelC.,
PelletierC.,
BoussahaM.,
DouetD. G.,
LautraiteA.,
TailliezP.(2007). Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis. Appl Environ Microbiol 73:2947–2955 [View Article][PubMed]
MofredjA.,
BarakaD.,
KloetiG.,
DumontJ. L.(2000).Lactococcus garvieae septicemia with liver abscess in an immunosuppressed patient. Am J Med 109:513–514 [View Article][PubMed]
MonederoV.,
MazéA.,
BoëlG.,
ZúñigaM.,
BeaufilsS.,
HartkeA.,
DeutscherJ.(2007). The phosphotransferase system of Lactobacillus casei: regulation of carbon metabolism and connection to cold shock response. J Mol Microbiol Biotechnol 12:20–32 [View Article][PubMed]
MushegianA. R.,
FullnerK. J.,
KooninE. V.,
NesterE. W.(1996). A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals. Proc Natl Acad Sci U S A 93:7321–7326 [View Article][PubMed]
NeuhausF. C.,
BaddileyJ.(2003). A continuum of anionic charge: structures and functions of d-alanyl-teichoic acids in gram-positive bacteria. Microbiol Mol Biol Rev 67:686–723 [View Article][PubMed]
NietoJ. M.,
DevesaS.,
QuirogaI.,
ToranzoA. E.(1995). Histopathology of Enterococcus sp. infection in farmed turbot (Scophthalmus maximus L.). J Fish Dis 18:21–30 [View Article]
NonakaL.,
IkenoK.,
SuzukiS.(2007). Distribution of tetracycline resistance gene, tetM, in Gram-positive and Gram-negative bacteria isolated from sediment and seawater at a coastal aquaculture site in Japan. Microbes Environ 22:355–364[CrossRef]
O'SullivanD. J.,
KlaenhammerT. R.(1993). Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl Environ Microbiol 59:2730–2733[PubMed]
Olivares-FusterO.,
AriasC. R.(2008). Use of suppressive subtractive hybridization to identify Flavobacterium columnare DNA sequences not shared with Flavobacterium johnsoniae
. Lett Appl Microbiol 46:605–612 [View Article][PubMed]
Paludan-MüllerC.,
MadsenM.,
SophanodoraP.,
GramL.,
MøllerP. L.(2002). Fermentation and microflora of plaa-som, a thai fermented fish product prepared with different salt concentrations. Int J Food Microbiol 73:61–70 [View Article][PubMed]
ParsonsJ. A.,
BannamT. L.,
DevenishR. J.,
RoodJ. I.(2007). TcpA, an FtsK/SpoIIIE homolog, is essential for transfer of the conjugative plasmid pCW3 in Clostridium perfringens
. J Bacteriol 189:7782–7790 [View Article][PubMed]
PoncetS.,
MilohanicE.,
MazéA.,
Nait AbdallahJ.,
AkéF.,
LarribeM.,
DeghmaneA. E.,
TahaM. K.,
DozotM. et al.(2009). Correlations between carbon metabolism and virulence in bacteria. Contrib Microbiol 16:88–102 [View Article][PubMed]
RantsiouK.,
UrsoR.,
IacuminL.,
CantoniC.,
CattaneoP.,
ComiG.,
CocolinL.(2005). Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages. Appl Environ Microbiol 71:1977–1986 [View Article][PubMed]
ReckseidlerS. L.,
DeShazerD.,
SokolP. A.,
WoodsD. E.(2001). Detection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant. Infect Immun 69:34–44 [View Article][PubMed]
TeixeiraL. M.,
MerquiorV. L.,
VianniM. C.,
CarvalhoM. G.,
FracalanzzaS. E.,
SteigerwaltA. G.,
BrennerD. J.,
FacklamR. R.(1996). Phenotypic and genotypic characterization of atypical Lactococcus garvieae strains isolated from water buffalos with subclinical mastitis and confirmation of L. garvieae as a senior subjective synonym of Enterococcus seriolicida
. Int J Syst Bacteriol 46:664–668 [View Article][PubMed]
VelaA. I.,
VázquezJ.,
GibelloA.,
BlancoM. M.,
MorenoM. A.,
LiébanaP.,
AlbendeaC.,
AlcaláB.,
MendezA. et al.(2000). Phenotypic and genetic characterization of Lactococcus garvieae isolated in Spain from lactococcosis outbreaks and comparison with isolates of other countries and sources. J Clin Microbiol 38:3791–3795[PubMed]
VillaniF.,
AponteM.,
BlaiottaG.,
MaurielloG.,
PepeO.,
MoschettiG.(2001). Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk. J Appl Microbiol 90:430–439 [View Article][PubMed]
WinstanleyC.(2002). Spot the difference: applications of subtractive hybridisation to the study of bacterial pathogens. J Med Microbiol 51:459–467[PubMed]
ZechnerE. L.,
de la CruzF.,
EisenbrandtR.,
GrahnA. M.,
KoraimannG.,
LankaE.& al., e. (2000). Conjugative DNA transfer processes. The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread87–174ThomasC. M.
Amsterdam: H. A. Publishers;
ZhangY. L.,
OngC. T.,
LeungK. Y.(2000). Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish. Microbiology 146:999–1009[PubMed]
Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse