Genome sequence of Lactococcus garvieae 21881, isolated in a case of human septicemia

Lactococcus garvieae is a Gram-positive bacterium considered an important opportunistic emerging human pathogen and also a well-recognized fish pathogen. Here, we present the draft genome sequence of Lactococcus garvieae strain 21881 (2,164,557 bp, with a G+C content of 37.9%), which represents the first report of a genome sequence on Lactococcus garvieae.     

Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures

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.     

Effectiveness of bivalent vaccines against Aeromonas hydrophila and Lactococcus garvieae infections in rainbow trout Oncorhynchus mykiss (Walbaum)

Lactococcus garvieae and Aeromonas hydrophila are bacterial pathogens affecting salmonids and other fish species and cause of heavy losses in aquaculture. Diseases caused by these bacteria can be controlled satisfactory by immunization using monovalent vaccines. In this study, the protective efficacy of two bivalent vaccines against L. garvieae and A. hydrophila was evaluated in rainbow trout (Oncorhynchus mykiss). Bivalent formulations, containing formalin-inactivated bacteria, were prepared as an aqueous bacterin and as an adjuvanted vaccine using montanide ISA-763. Protection against L. garvieae and A. hydrophila was tested at day 30 and 90 post-vaccination. High levels of protection were achieved for the aqueous and adjuvanted bivalent vaccines against L. garvieae (RPS of 100% and 95.3%) and A. hydrophila (RPS of 100% and 95.3%) at day 30 post-vaccination. Significant differences (p < 0.05) were found between the RPS at days 30 and 90 post-immunization with a decrease in the protection levels for the aqueous bivalent vaccine against L. garvieae (RPS 76.2%) and A. hydrophila (RPS 85%), but not for the adjuvanted vaccine (RPS of 90% against L. garvieae and 95% against A. hydrophila). In addition, high antibody levels were observed in the vaccinated fish at day 15 post-immunization using both vaccines. Our results demonstrate that these bivalent vaccines can effectively protect rainbow trout against L. garvieae and A. hydrophila and could offer an appropriate strategy to prevent these infections in rainbow trout farms.     

Dopamine depresses the immune ability and increases susceptibility to Lactococcus garvieae in the freshwater giant prawn, Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (16.2 +/- 2.1 g) were individually injected with saline, or dopamine at 0.5, 5.0, or 50.0 pmol prawn(-1). The results show that a transient period of immunosuppression occurred between 2 and 8 h after injection of dopamine for all immune parameters except circulating haemocytes and all immune parameters returned to control values within 8-16 h after receiving dopamine. Injection of dopamine also significantly increased the mortality of M. rosenbergii challenged with the pathogen L. garvieae. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility to L. garvieae in M. rosenbergii.     

Genome sequences of Lactococcus garvieae TB25, isolated from Italian cheese, and Lactococcus garvieae LG9, isolated from Italian rainbow trout

Lactococcus garvieae is a fish pathogen and an emerging zoonotic opportunistic pathogen as well as a component of natural microbiota in dairy products. Here, we present the first report of a genome sequence of L. garvieae TB25, isolated from a dairy source, and that of L. garvieae LG9, isolated from rainbow trout.     

Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail.

The present study describes the in vitro and in vivo survival of Lactococcus garvieae bacteriophages and the potential of the phage for controlling experimental L. garvieae infection in yellowtail. Anti-L. garvieae phages persisted well in various physicochemical (water temperature, salinity, pH) and biological (feed, serum and alimentary tract extracts of yellowtail) conditions, except for low acidity. In the in vivo, the phage PLgY-16 was detected in the spleens of yellowtail until 24 h after intraperitoneal (i.p.) injection, or the phage was recovered from the intestine of yellowtail 3 h after the oral administration of phage-impregnated feed but undetectable 10 h later. Simultaneous administration of live L. garvieae and phage enhanced recovery of the phage from the spleen or intestine. The survival rate was much higher in yellowtail that received i.p. injection of the phage after i.p. challenge with L. garvieae, compared with that of control fish without phage injection. When fish were i.p. injected with phage at different hours after L. garvieae challenge, higher protective effects were demonstrated in fish that received phage treatment at the earlier time. Protection was also obtained in yellowtail receiving phage-impregnated feed, in which fish were challenged by an anal intubation with L. garvieae. Anal-intubated L. garvieae were detected constantly in the spleens of the control fish, while they were detected sporadically and disappeared from the phage-treated fish 48 h later. On the other hand, orally administered phage was detected at high plaque-forming units from the intestines and spleens of the phage-treated fish until 48 h later. These results indicate that intraperitoneally or orally administered anti-L. garvieae phage prevented fish from experimental L. garvieae infection, suggesting potential use of the phage for controlling the disease.     

Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan

AIMS: To investigate the existing antimicrobial susceptibility and genetic characteristics of Lactococcus garvieae isolates from cultured Seriola in Japan. METHODS AND RESULTS: Minimum inhibitory concentrations (MICs) of 14 antimicrobial agents for 170 isolates were determined using the agar dilution method. Seventy-five isolates (44.1%) were simultaneously resistant to erythromycin (EM) (MIC>or=2 microg ml-1), lincomycin (LCM) (MIC>or=128 microg ml-1) and oxytetracycline (OTC) (MIC>or=4 microg ml-1). Resistance to EM was grouped as intermediate- and high-level resistant by MIC values. All resistant isolates possessed ermB and tet(S) genes. The number of different bands between pulsed-field gel electrophoresis patterns of 25 isolates and two ATCC strains (isolated in 1974), determined using two enzymes (ApaI and SmaI), did not exceed 3. CONCLUSIONS: The present resistance pattern observed with ermB and tet(S) is similar to that observed in previous reports. Moreover, the genetic characteristics of L. garvieae isolates from a wide area in Japan in 2002 and ATCC strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that EM-, LCM- and OTC-resistant isolates have been present for 15 years and that L. garvieae strains with same origin have spread among Seriola spp. in Japan since 1974.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases.

Clinical and macroscopic findings (anorexia, lethargy, loss of orientation and exophthalmia) indicate that Streptococcus iniae and Lactococcus garvieae infections of trout share some common features, but histopathology reveals notable differences between the 2 diseases. Meningitis and panophthalmitis are the main lesions among S. iniae infected trout, whereas L. garvieae infection results in a hyperacute systemic disease. Differences in the LD50s of the 2 pathogens and the sudden onset of signs and death correlate with the histopathological findings, indicating the severity of L. garvieae infection of trout.     

Cell-surface properties of Lactococcus garvieae strains and their immunogenicity in the yellowtail Seriola quinqueradiata

The cell-surface properties of strains of Lactococcus garvieae were examined. Two capsular types were found, one with a highly developed capsule (KG9408) and one with a micro-capsule (MS93003) carrying fimbriae-like components projecting from the cell surface. One strain (NSS9310) had neither cell capsular nor fimbriae-like structures on its cell surface. The strains with the highly developed capsule were more virulent to fish than either the micro-capsular or non-capsular strains. The KG9408, MS93003 and NSS9310 strains could be clearly differentiated by their susceptibility to bacteriophages. Protection against L. garvieae infection was induced in the yellowtail Seriola quinqueradiata by immunization with formalin-killed L. garvieae KG9408 and MS93003 cells. Although protection was also induced by immunization with NSS9310, the level of protection was significantly lower than that with KG9408 and MS93003 vaccines. Passive immunization with yellowtail immune sera raised against KG9408 and MS93003 conferred strong protection on yellowtail with rapid bacterial clearance after challenge with L. garvieae. Immunoblotting analysis of protein antigens extracted from L. garvieae strains using rabbit anti-KG9408 and anti-MS93003 sera and yellowtail anti-KG9408 and anti-MS93003 sera indicated that some bands in KG9408 and MS93003 strains were not detectable in NSS9310.     

Effect of potassium permanganate stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

This work is part of a continuing series of investigations on the effect of commonly used aquaculture chemicals on the immune resistance and susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae. The methodology has been described in earlier publications of the series. Potassium permanganate at 1.0 mg l(-1) in tryptic soy broth (TSB) had no effect on the growth rate of L. garvieae. The mortality of M. rosenbergii challenged with 4 x 10(6) colony-forming units (cfu) prawn(-1) of TSB-grown L. garvieae was significantly greater than that of challenged controls. Addition of potassium permanganate at 1.0 mg l(-1) in TSB significantly increased the virulence of L. garvieae to M. rosenbergii. Exposure of M. rosenbergii to potassium permanganate prior to challenge with TSB-grown L. garvieae at 4 x 10(6) and 3 x 10(6) cfu prawn(-1) revealed that 96 h mortality was significantly lower for prawns held in water containing 0.3 mg l(-1) of the chemical than for prawns in water containing 1.0 mg l(-1) or no chemical. Potassium permanganate caused no significant changes in total hemocyte counts and differential hemocyte counts, compared to the control treatments. However, a concentration of 1.0 mg l(-1) or more for 96 h resulted in decreased phenoloxidase activity, phagocytic activity and clearance efficiency. Respiratory burst increased with exposure to 0.3 mg l(-1). In conclusion, treatment with potassium permanganate at 0.3 mg 1(-1) was effective in reducing M. rosenbergii mortality from L. garvieae infection, but higher concentrations had a negative effect, probably due to reduced prawn defenses.     

Genome sequence of Lactococcus garvieae 8831, isolated from rainbow trout lactococcosis outbreaks in Spain

Lactococcus garvieae is the etiological agent of lactococcosis, one of the most important disease threats to the sustainability of the rainbow trout farming industry. Here, we present the draft genome sequence of Lactococcus garvieae strain 8831, isolated from diseased rainbow trout, which is composed of 2,087,276 bp with a G+C content of 38%.     

Genome sequence of Lactococcus garvieae UNIUD074, isolated in Italy from a lactococcosis outbreak

Lactococcus garvieae is the etiological agent of lactococcosis disease, affecting many cultured fish species worldwide. In addition, this bacterium is currently considered a potential zoonotic microorganism since it is known to cause several opportunistic human infections. Here we present the draft genome sequence of the L. garvieae strain UNIUD074.     

SdrI of Staphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site

The transferability of a large plasmid that harbors a tetracycline resistance gene tet (S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients ( Lactococcus garvieae and Listeria monocytogenes ), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L . garvieae . A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes , even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.     

Efficacy of protein A-HRP in an immunological study of black rockfish (Sebastes schlegeli Higendorf) humoral immune responses

The efficacy of protein A-horse radish peroxidase (HRP), as compared to that of mouse polyclonal antibody raised against purified Ig, in detection of black rockfish (Sebastes schlegeli Higendorf) immunoglobulin (Ig) was examined. Protein A affinity chromatography successfully purified Ig from black rockfish serum; the purified-Ig could be visualised as two protein bands (MW 70 and 25kDa) following resolution with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In SDS-PAGE immunoblot profiles of the purified-Ig, the mouse polyclonal antibody recognised both the light chain and heavy chains of rockfish Ig, whereas protein A-HRP immunostained only the heavy chain of rockfish Ig. These results suggest that protein A-HRP may be used to detect rockfish antibody-antigen complexes in immunoassays. In a 2-DE immunoblot assay for exploring antigenic profiles of Lactococcus garvieae KG9408, protein A-HRP successfully detected specific antibodies to antigenic proteins of L. garvieae in the rockfish Ig. In addition, enzyme linked immunosorbent assay (ELISA) showed a high correlation between the results obtained for positivity of L. garvieae when protein A-HRP and the mouse polyclonal antibody-was used to analyse samples from 25 diseased rockfish. These results collectively indicate that protein A-HRP has a high affinity for Ig, and may be useful for new investigations into the humoral immune responses of rockfish.     

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

AIMS: To clarify the epidemiological relationship between Lactococcus garvieae isolates from the Seriola in Japan and isolates from other animals. METHODS AND RESULTS: A total of 32 isolates obtained from aquatic (the genus Seriola and trout) and terrestrial animals (cow, pig, cat, dog and horse) was used to evaluate its pathogenicity to yellowtail and mouse, phenotype (KG+ and KG-), its susceptibility to three bacteriophages and the pattern of pulsed field gel electrophoresis (PFGE). Lactococcus garvieae isolated from Seriola showed strong pathogenicity to yellowtail, while isolates from trout showed weak pathogenicity and those obtained from terrestrial animals showed no distinct pathogenicity. Only, the isolates from the genus Seriola in Japan showed susceptibility to the bacteriophages. The results of PFGE pattern indicate that the isolates obtained from the Seriola predict homogeneity, while there is no similarity among the isolates obtained from different animals. CONCLUSION: This experiment indicates that L. garvieae isolated from Seriola in Japan appears to be very different from the isolates obtained from other animals, and the isolates prevalent among the genus Seriola in Japan might be homogeneous. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that a particular genetic group that has specially adapted and acquired virulence toward yellowtail were prevalent among the genus Seriola in Japan.     

Trichlorfon, an organophosphorus insecticide, depresses the immune responses and resistance to Lactococcus garvieae of the giant freshwater prawn Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory bursts (release of superoxide anions), superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (18.2+/-2.1 g) were individually reared in water containing concentrations of trichlorfon of 0, 0.2, or 0.4 mg L(-1) for a 144-h period. No significant differences in the THC were observed among prawns at the beginning and following 144 h of exposure to 0, 0.2, and 0.4 mg L(-1) trichlorfon. However, phenoloxidase activity significantly decreased when the prawns were exposed to trichlorfon at 0.2 and 0.4 mg L(-1), and no significant differences were observed between the two concentrations at any sampling time. The total production of superoxide anion by M. rosenbergii significantly increased with exposure to 0.2 and 0.4 mg L(-1) trichlorfon, and no significant differences were observed between the two concentrations during the 144-h exposure period. However, M. rosenbergii exposed to 0.2 and 0.4 mg L(-1) trichlorfon showed decreased SOD activity from 48 to 144 h. Phagocytic activity and clearance efficiency to L. garvieae significantly decreased when prawns were exposed to 0.2 and 0.4 mg L(-1) trichlorfon for 48 h. In another experiment, prawns were challenged with 5 x 10(5) colony-forming units (cfu)prawn(-1) of L. garvieae, then reared in water containing different concentrations of trichlorfon. The onset of mortality was earlier in prawns exposed to trichlorfon compared to those exposed to the zero control. The cumulative mortality of prawns directly increased with ambient trichlorfon concentrations in the range of 0-0.4 mg L(-1) after 168 h. It is concluded that exposure of M. rosenbergii to trichlorfon at 0.2 mg L(-1) or more caused cytotoxicity resulting in depression of the immune response, and increased its susceptibility to L. garvieae infection.     

First report of Streptococcus agalactiae and Lactococcus garvieae from a wild bottlenose dolphin (Tursiops truncatus)

The isolation and characterization of two bacterial species, Streptococcus agalactiae and Lactococcus garvieae, previously unreported in wild marine mammals are described from a freshly dead bottlenose dolphin, Tursiops truncatus, from Kuwait Bay, Kuwait, in September 2001. Conventional and rapid identification systems were used to determine that isolates from muscle and kidney were S. agalactiae and L. garvieae, respectively. The isolates were gram-positive, catalase-negative, oxidase-negative, nonhemolytic cocci. The S. agalactiae was serotyped to group antigen B, whereas the L. garvieae could not be assigned to any serogroup. These Kuwait isolates displayed considerable homogeneity with corresponding American Type Culture Collection (ATCC) type isolates. Although the dolphin S. agalactiae isolate was nonhemolytic, it was biochemically similar to S. agalactiae isolated from mullet sampled in the concurrent Kuwait Bay fish kill. Some biochemical heterogeneity was observed between the dolphin isolates and corresponding mammalian ATCC type isolates, especially with Voges Proskauer, alanine-phenylanaline-proline arylamidase, and alpha-galactosidase tests. Nile tilapia, Oreochromis niloticus, experimentally infected with the dolphin S. agalactiae and L. garvieae isolates experienced 90% and 0% mortalities, respectively. This is the first isolation of S. agalactiae and L. garvieae from a wild marine mammal, and the microbial characteristics established here provide pertinent information for the future isolation of these bacteria.