Emergence of Novel Streptococcus iniae Exopolysaccharide-Producing Strains following Vaccination with Nonproducing Strains

Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium. Present findings indicate that the extracellular polysaccharide is a major antigenic factor, suggesting an evolutionary selection of strains capable of extracellular polysaccharide production.     

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

Strain variation and geographic endemism in Streptococcus iniae

Twenty-six Israeli isolates of Streptococcus iniae from both marine and fresh/brackish water sources were compared with each other and with 9 foreign isolates. All the isolates were tentatively identified according to their biochemical profile. Direct sequencing of approximately 600 bp PCR products of the 16S rDNA confirmed their identification as S. iniae at the molecular level and revealed a new (one-nucleotide) variant among Israeli isolates, in addition to 2 variants that had been previously reported. Strain variation was further examined by subjecting the isolates to randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. The RAPD method allowed separation of the isolates into only 2 groups, one including 5 Israeli fresh/brackish water isolates and one including all the other isolates. The AFLP method grouped the Israeli marine isolates into one homogeneous cluster, although they had been obtained in different years (1995 to 2001) from different species of fish, and from wild (Red Sea) as well as cultured (both Mediterranean and Red Sea) sources. The Israeli fresh/brackish water isolates and foreign isolates separated into distinct entities that clustered at generally high degrees of similarity. The distance between the clusters of the Israeli marine and fresh/brackish water isolates indicates that the S. iniae streptococcosis that has been afflicting the aquaculture industries in the 2 environments in recent years was caused by distinct strains. AFLP showed superior discriminative properties over RAPD in detecting intraspecific variation and proved to be an important tool for the characterization of S. iniae. A correlation between strain variation and geographic endemism was established.     

Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi (Lates calcarifer)

In Streptococcus iniae, lactate metabolism is dependent upon two proteins, lactate permease that mediates uptake and lactate oxidase, a flavin mononucleotide-dependent enzyme that catalyzes oxidation of α-hydroxyacids. A novel variant of the lactate oxidase gene, lctO, in Australian isolates of S. iniae from diseased barramundi was found during a diagnostic screen using LOX-1 and LOX-2 primers, yielding amplicons of 920 bp instead of the expected 869 bp. Sequencing of the novel gene variant (type 2) revealed a 51-nucleotide insertion in lctO, resulting in a 17-amino-acid repeat in the gene product, and three-dimensional modeling indicated formation of an extra loop in the monomeric protein structure. The activities of the lactate oxidase enzyme variants expressed in Escherichia coli were examined, indicating that the higher-molecular-weight type 2 enzyme exhibited higher activity. Growth rates of S. iniae expressing the novel type 2 enzyme were not reduced at lactate concentrations of 0.3% and 0.5%, whereas a strain expressing the type 1 enzyme exhibited reduced growth rates at these lactate concentrations. During a retrospective screen of 105 isolates of S. iniae from Australia, the United States, Canada, Israel, Réunion Island, and Thailand, the type 2 variant arose only in isolates from a single marine farm with unusually high tidal flow in the Northern Territory, Australia. Elevated plasma lactate levels in the fish, resulting from the effort of swimming in tidal flows of up to 3 knots, may exert sufficient selective pressure to maintain the novel, high-molecular-weight enzyme variant.Streptococcus iniae is a major pathogen of farmed fish, resulting in severe economic losses globally estimated at U.S. $150 million annually (27). S. iniae is essentially a blood pathogen, with infection resulting in a generalized septicemia and meningitis (1). During infection the pathogen avoids phagocytosis by means of an antiopsonic capsule (4, 18, 22) and by binding host serum components including immunoglobulins (3) and fibrinogen (2). Little is known, however, about the metabolism of S. iniae during infection although lactic acid bacteria may produce l-lactate from fermentation of glucose. In S. iniae a lactate oxidase gene, lctO, has been characterized previously (11). The product of the lctO gene in S. iniae is a flavin enzyme (l-lactate 2-monooxygenase, EC 1.13.12.4), which catalyzes the oxidation of lactate to pyruvate, coupled with reduction of O₂ to H₂O₂ (11). Lactate oxidase has been extensively characterized, both structurally and functionally, in the cold-water marine pathogen Aerococcus viridans (9, 30, 35, 36); thus, the catalytic activities of these enzymes are relatively well understood. In S. iniae, lactate can be utilized as an energy source through an aerobic but nonrespiratory mode of metabolism (11), a mechanism that is coupled to hydrogen peroxide production in Streptococcus pyogenes (26).Since the discovery of the lactate oxidase gene in S. iniae, its presence has been routinely used for PCR-based diagnosis, overcoming the lack of specificity of commercial biochemical diagnostic kits and other molecular methods. Confirmation of isolate identity as S. iniae by commercial bacterial identification kits is problematic because the biochemical profile is absent from databases supplied with the kits or because the databases are unable to identify atypical strains with confidence (25). Identification of isolates by molecular methods such as PCR is more reliable since isolates with atypical biochemical profiles can confidently be identified. PCR has been used to amplify sections of the 16S rRNA gene (37), the chaperonin HSP60 (12), and the 16S-23S rRNA gene intergenic spacer region (5) for identification of S. iniae. The development of the lactate oxidase gene (lctO) PCR assay by Mata et al. (21) reported that the primer pair LOX-1/LOX-2 could be used successfully to aid in the identification of S. iniae via the generation of a specific 870-bp product. Moreover, the LOX-1/LOX-2 primer pair overcame the problem of nonspecific amplification of Streptococcus difficilis that had previously been reported with the 16S rRNA gene primer pair described previously (21, 37).In Australia, S. iniae causes major economic loss in farmed barramundi (Lates calcarifer, Bloch) (1, 6). Barramundi, also known as Asian sea bass, are perciform euryhaline fish native to Australia and tropical southeast Asia. In Australia, barramundi have both cultural and commercial significance in terms of their iconic status among indigenous populations and the recent rapid growth of commercial farming. The value of intensive barramundi culture in Australia increased from Australian $15.5 million in 2004 to Australian $23.5 million in 2006 (34). There is also increasing farmed output of L. calcarifer in Malaysia, Indonesia, Taiwan, and Vietnam (33) and small to medium recirculating aquaculture ventures in the United States and United Kingdom using imported fingerlings.During routine diagnostic screening of S. iniae isolated throughout Australia from diseased barramundi, a novel variant of the lctO gene was found that resulted in amplicons of 920 bp following PCR using the LOX-1/LOX-2 primer pair. Isolates expressing the novel lactate oxidase gene were isolated only from a single site in the Northern Territory, Australia. In the present study, the novel lctO variant is investigated genetically and phenotypically in order to better understand how the larger gene product may have arisen from this single site.     

Interactions of Streptococcus iniae with phagocytic cell line

Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1β, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection.     

Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.     

Agglutinin Response to Pertussis Vaccination in the Child

Children were immunized with plain pertussis vaccine made by three manufacturers in 1967. After a primary course of three injections at monthly intervals, starting at 3-4 months of age, the agglutinin response was poor. Even after a “booster” dose, given five months later, not all of the vaccines had stimulated a response to all three pertussis agglutinogens. A further investigation with current vaccines of different kinds administered according to more than one schedule is recommended.     

Possible Transmission of Streptococcus iniae from Wild Fish to Cultured Marine Fish

Streptococcus iniae was isolated from diseased wild fish collected near a mariculture facility where gilthead sea bream and European sea bass exhibited a similar infection. Species-specific PCR and ribotyping confirmed that wild and cultured fish were infected by a single S. iniae clone. Wild fish are therefore potential amplifiers of pathogenic S. iniae strains.     

Identification of Streptococcus iniae by commercial bacterial identification systems

The fish pathogen Streptococcus iniae cannot be identified by most commercial bacterial identification systems. The results presented here indicate that over 70% of our S. iniae isolates have been identified using the Biolog(R) GP microplate panels and Microlog(R) database. The isolates were confirmed as S. iniae by specific PCR methods and have been found to conform to the result obtained with the type strain S. iniae ATCC 29178.     

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies. The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.     

cadDX Operon of Streptococcus salivarius 57.I

A CadDX system that confers resistance to Cd²⁺ and Zn²⁺ was identified in Streptococcus salivarius 57.I. Unlike with other CadDX systems, the expression of the cad promoter was negatively regulated by CadX, and the repression was inducible by Cd²⁺ and Zn²⁺, similar to what was found for CadCA systems. The lower G+C content of the S. salivarius cadDX genes suggests acquisition by horizontal gene transfer.     

Evidence for Rare Capsular Switching in Streptococcus agalactiae

The polysaccharide capsule is a major antigenic factor in Streptococcus agalactiae (Lancefield group B streptococcus [GBS]). Previous observations suggest that exchange of capsular loci is likely to occur rather frequently in GBS, even though GBS is not known to be naturally transformable. We sought to identify and characterize putative capsular switching events, by means of a combination of phenotypic and genotypic methods, including pulsed-field gel electrophoretic profiling, multilocus sequence typing, and surface protein and pilus gene profiling. We show that capsular switching by horizontal gene transfer is not as frequent as previously suggested. Serotyping errors may be the main reason behind the overestimation of capsule switching, since phenotypic techniques are prone to errors of interpretation. The identified putative capsular transformants involved the acquisition of the entire capsular locus and were not restricted to the serotype-specific central genes, the previously suggested main mechanism underlying capsular switching. Our data, while questioning the frequency of capsular switching, provide clear evidence for in vivo capsular transformation in S. agalactiae, which may be of critical importance in planning future vaccination strategies against this pathogen.Streptococcus agalactiae (group B streptococcus [GBS]) is primarily a colonizing agent of the genitourinary and gastrointestinal tracts, but it is also a leading cause of bacterial sepsis and meningitis in neonates and is increasingly associated with invasive infections in adults (39). The capsular polysaccharide is a major GBS virulence factor and also the main target of antibody-mediated killing (11). In the last decade, conjugated multivalent vaccines have been developed and proved to be highly immunogenic, raising the possibility of the prevention of perinatal GBS disease through maternal immunization (38).Nine capsular types are recognized: Ia, Ib and II to VIII, along with a new provisional serotype IX, recently proposed (19). Comparison of the capsular locus genes suggested that the structural diversity of the capsular polysaccharide is associated with the genetic diversity of the capsular locus, possibly driven by horizontal gene transfer (9, 24). Capsular serotyping has been the classical method used in epidemiological studies to differentiate GBS isolates, although further characterization of GBS diversity includes the use of a broad range of DNA-based typing methods, such as restriction fragment length polymorphisms (RFLP), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Both PFGE and MLST have provided new clues about the population structure of S. agalactiae, particularly the recognition of diverse lineages among serotype III that were shown to differ in virulence potential and tropism (16, 25, 26, 31, 41). Although the distinction of lineages within a particular serotype has proved useful, a complete correlation between capsular type and the lineages defined by MLST was not found (4, 21, 22). Moreover, whole-genome comparative analysis of isolates expressing different serotypes showed that they sometimes share more genes than strains of the same serotype, suggesting a serotype-independent clustering of strains (43). These observations support the hypothesis that closely and divergently related clones may share the genes coding for a particular capsular type, suggesting that exchange of capsular genes in vivo may have occurred (16, 21, 22). We refer to these phenomena here as capsular switching in vivo, recognizable by the expression of different serotypes and the presence of different capsular loci in otherwise indistinguishable isolates when sampling a set of 11 loci distributed in the genome.The changes at the capsular locus were proposed to be driven by the equilibrium between the selective pressure imposed by host immunity and conservation of a particular capsular polysaccharide, as an adaptive advantage of virulent clones (4, 9, 21). Capsular switching by homologous recombination would be facilitated by the organization of the locus encoding the capsular polysaccharide synthesis genes (cps), where the highly variable serotype determining region (cpsG-cpsK) is flanked by conserved genes (9, 24). This led to the suggestion that genetic exchange of the central part of the cps operon could be driving capsular switching (9, 22). According to Luan et al., who specifically addressed this issue, horizontal transfer of capsular genes occurs at a high level within a population without restriction to genetic background. The authors of that study also suggest that since only advantageous combinations of genotype-serotype persist, these altered serotypes, due to capsular switching, are recognized at a lower frequency among stable clones (21).Capsular switching is well established in other streptococcal species such as Streptococcus pneumoniae, where spontaneous in vivo capsular transformation events were observed and characterized (28, 34). In contrast to GBS, S. pneumoniae is naturally transformable, and this is widely believed to be responsible for the ease with which this species exchanges DNA. Capsular switching may have serious impact in pneumococcal vaccination programs since it may provide the selective pressure for virulent genotypes to switch capsules and escape vaccine coverage (6), and a similar response could be seen with a future introduction of GBS vaccination (38).The aim of the present study was to evaluate the concordance between serotype and the clusters defined by PFGE and to further characterize any putative transformants to establish unequivocally that capsular switching occurs in GBS. We combined PFGE with the analysis of multiple genes spread across the GBS genome in order to identify capsular transformants and concluded that capsular switching events occur less frequently than previously thought.     

Recovery of Streptococcus iniae from Diseased Fish Previously Vaccinated with a Streptococcus Vaccine

Streptococcus iniae was recovered from diseased rainbow trout (Oncorhynchus mykiss, Walbaum) previously vaccinated against streptococcosis. PCR and serological methods indicate the presence of a new serotype in the diseased fish.     

Sil: A Streptococcus iniae Bacteriocin with Dual Role as an Antimicrobial and an Immunomodulator That Inhibits Innate Immune Response and Promotes S. iniae Infection

Streptococcus iniae is a Gram-positive bacterium and a severe pathogen to a wide range of economically important fish species. In addition, S. iniae is also a zoonotic pathogen and can cause serious infections in humans. In this study, we identified from a pathogenic S. iniae strain a putative bacteriocin, Sil, and examined its biological activity. Sil is composed of 101 amino acid residues and shares 35.6% overall sequence identity with the lactococcin 972 of Lactococcus lactis. Immunoblot analysis showed that Sil was secreted by S. iniae into the extracellular milieu. Purified recombinant Sil (rSil) exhibited a dose-dependent inhibitory effect on the growth of Bacillus subtilis but had no impact on the growths of other 16 Gram-positive bacteria and 10 Gram-negative bacteria representing 23 different bacterial species. Treatment of rSil by heating at 50°C abolished the activity of rSil. rSil bound to the surface of B. subtilis but induced no killing of the target cells. Cellular study revealed that rSil interacted with turbot (Scophthalmus maximus) head kidney monocytes and inhibited the innate immune response of the cells, which led to enhanced cellular infection of S. iniae. Antibody blocking of the extracellular Sil produced by S. iniae significantly attenuated the infectivity of S. iniae. Consistent with these in vitro observations, in vivo study showed that administration of turbot with rSil prior to S. iniae infection significantly increased bacterial dissemination and colonization in fish tissues. Taken together, these results indicate that Sil is a novel virulence-associated bacteriostatic and an immunoregulator that promotes S. iniae infection by impairing the immune defense of host fish.     

Streptococcus iniae infections in Red Sea cage-cultured and wild fishes

Streptococcus iniae was isolated from 2 moribund wild Red Sea fishes, Pomadasys stridens (Pomadasyidae) and Synodus variegatus (Synodontidae), both collected in shallow waters along the Israeli coast of the Gulf of Eilat. The site is approximately 2 km from a mariculture cage farm in which streptococcal infections were diagnosed in previous years in the red drum Sciaenops ocellatus. This is the first report of S. iniae in Red Sea fishes. Biochemical and molecular similarities between the isolates from cultured fishes and those from the wild specimens suggest that a single strain is involved, and that 'amplification' and dispersal of this pathogen from captive to feral fishes have occurred. At the molecular level, the pathogen is different from the S. iniae isolates that have been afflicting the Israeli freshwater aquaculture in recent years. Although S. iniae prevalence in the wild fish populations of the area remains to be determined, the northernmost region of the Gulf of Eilat, virtually landlocked and with generally calm seas and weak currents, seems to be particularly vulnerable to the impact of diseases that develop in this mariculture system.     

Streptococcus iniae type II infections in rainbow trout Oncorhynchus mykiss

Clinical and pathological findings (anorexia, hemorrhage, lethargy, loss of orientation and exophthalmia) indicated that Streptococcus iniae type II is responsible for a fatal disease in rainbow trout. Histopathological findings revealed that S. iniae type II produces a systemic disease, including a diffuse necrotizing myositis. The distribution of viable bacteria in infected tissues substantiated the pathological findings, confirming that S. iniae type II is responsible for a generalized septic disease of rainbow trout.     

Cloning and analysis of the L-lactate utilization genes from Streptococcus iniae.

The presence of lactate oxidase was examined in eight Streptococcus species and some related species of bacteria. A clone (pGR002) was isolated from a genomic library of Streptococcus iniae generated in Escherichia coli, containing a DNA fragment spanning two genes designated lctO and lctP. We show that these genes are likely to be involved in the L-lactic acid aerobic metabolism of this organism. This DNA fragment has been sequenced and characterized. A comparison of the deduced amino acid sequence of LctP protein demonstrated that the protein had significant homology with the L-lactate permeases of other bacteria. The amino acid sequence of the LctO protein of S. iniae also showed a strong homology to L-lactate oxidase from Aerococcus viridans and some NAD-independent lactate dehydrogenases, all belonging to the family of flavin mononucleotide-dependent alpha-hydroxyacid-oxidizing enzymes. Biochemical assays of the gene products confirm the identity of the genes from the isolated DNA fragment and reveal a possible role for the lactate oxidase from S. iniae. This lactate oxidase is discussed in relation to the growth of the organism in response to carbon source availability.