Justice C.F. Baiano

Towards Control of Streptococcus iniae

Recently identified virulence factors can lead to new approaches, such as vaccination programs at fish farms.

Isolation of a novel strain of Bacillus pumilus from penaeid shrimp that is inhibitory against marine pathogens.

A bacterium was isolated from the mid-gut of healthy black tiger shrimp, Penaeus monodon, based on a large zone of inhibition in mixed culture on solid medium. The isolate was a Gram-positive, motile spore former, with an optimum pH range for growth in tryptone soya broth containing 2% NaCl of between pH 6 and 9. The bacterium was highly salt tolerant with concentrations between 0% and 8% having no detrimental effect on growth. The isolate was identified as Bacillus pumilus based on physiological capabilities using the API50CHB and Biolog systems. Amplification and sequencing of the 16S rRNA gene followed by phylogenetic analysis confirmed its identity. The Bacillus pumilus isolate was strongly inhibitory against the marine bacterial pathogens Vibrio alginolyticus, V. mimicus and V. harveyi, and weakly inhibitory against V. parahaemolyticus in cross-streaking assays on solid medium. The organism was marginally self-inhibitory, and inhibited B. licheniformis and B. subtilis. The suitability of the B. pumilus isolate for use as a probiotic in farmed shrimp was further supported by the absence of any of the known B. cereus enterotoxin genes. Based on these in vitro results, in vivo safety and efficacy trials are underway to determine suitability of the novel strain as a commercial probiotic.

Identification and molecular characterisation of a fibrinogen binding protein from Streptococcus iniae.

BackgroundBinding of serum components by surface M-related proteins, encoded by the emm genes, in streptococci constitutes a major virulence factor in this important group of organisms. The present study demonstrates fibrinogen binding by S. iniae, a Lancefield non-typeable pathogen causing devastating fish losses in the aquaculture industry and an opportunistic pathogen of humans, and identifies the proteins involved and their encoding genes.ResultsFibrinogen binding by S. iniae significantly reduced respiratory burst activity of barramundi peritoneal macrophages in primary cultures compared to BSA-treated or untreated controls, indicating a potentially important role for fibrinogen binding cell-surface proteins in avoiding phagocytic attack in fish. We describe a novel emm-like gene, simA, encoding a 57 kDa fibrinogen binding M-like protein in S. iniae. These SiM proteins and their corresponding tetrameric structures from some sequevar types (~230 kDa) bound fibrinogen in Western blots. simA was most closely related (32% identity) to the demA gene of S. dysgalactiae. Genome walking and sequencing determined the genetic organization of the simA region had similarities to the mgrC regulon in GCS and to S. uberis. Moreover, a putative multigene regulator, mgx was orientated in the opposite direction to the simA gene in common with S. uberis, but contrary to findings in GAS and GCS. In GAS, diversity among emm-genes and consequent diversity of their M-related proteins results in substantial antigenic variation. However, an extensive survey of S. iniae isolates from diverse geographic regions and hosts revealed only three variants of the gene, with one sequevar accounting for all but two of the 50 isolates analysed.ConclusionThese proteins play a role in avoiding oxidative attack by phagocytic cells during infection of fish by S. iniae, but genetic diversity amongst these key surface proteins has not yet arisen. This lack of diversity coupled with a functional role in macrophage resistance suggests that these proteins may constitute important targets for future vaccines against S. iniae in fish.

Two hepcidin-like antimicrobial peptides in Barramundi Lates calcarifer exhibit differing tissue tropism and are induced in response to lipopolysaccharide

Genes encoding two hepcidin-like antimicrobial peptides were discovered in Barramundi, Lates calcarifer (barramundi, Giant sea perch). Analysis of the coding regions indicated that genes for each hepcidin comprised 3 exons and 2 introns. The deduced amino acid sequences for each molecule resulted in a protein comprising a signal sequence of 24 aa in each case, coupled to a prepropeptide of 75 aa for hepcidin 1 and 78 aa for hepcidin 2. A cleavage site was identified in each prepropetide at amino acid 64 with the cleavage motif--QKR/QS--resulting in mature peptides of 25 and 28 amino acids respectively. Each mature peptide contained 8 conserved cysteine residues and 3 dimensional modeling predicted a β-hairpin and β-sheet structure characteristic of human Liver Expressed Antimicrobial Peptide (LEAP). Analysis of the deduced amino acid sequences by BLAST with phylogenetic supported indicated that hepcidin 1 was a HAMP1-type peptide closely related to hepcidins identified in other Perciformes (Micropterus and Pseudosciaena), whilst hepcidin 2 was a HAMP2-type peptide most similar to a hepcidin previously identified in black rock fish (Sebastes schlegeli). Both hepcidin genes were inducible in barramundi following intraperitoneal injection with lipopolysaccharide, with elevated expression detected in liver and head kidney 3 h post IP injection for hepcidin 1 and in liver only for hepcidin 2. The elevated expression was transient with return to normal levels within 24-48 h. No significant expression of either peptide was detected in spleen, skin or gill following IP injection with LPS.

Genetic variability amongst Streptococcus iniae isolates from Australia

Keywords: Australia, barramundi, genetic diversity,genotype, pulsed-field gel electrophoresis, Strepto-coccus iniae.Streptococcus iniae is a beta-haemolytic, Gram-positive coccus that causes generalized septicaemiaand meningoencephalitis in fish (Perera, Johnson,Collins & Lewis 1994). Significant mortality in adiverse range of commercial fish species has causedsevere losses for the aquaculture industries in theUS, Japan, Israel, Bahrain, Europe, Indonesia andAustralia (Agnew & Barnes 2007; Bromage,Thomas & Owens 1999; Colorni, Diamant, Eldar,Kvitt & Zlotkin 2002; Goh, Driedger, Gillett, Low,Hemmingsen, Amos, Chan, Lovgren, Willey, Shaw& Smith 1998; Kusuda & Kawai 1998; Perera et al.1994; Shoemaker, Klesius & Evans 2001; Stoffre-gen, Backman, Perham, Bowser & Babish 1996;Yuasa, Kitancharoen, Kataoka & Al-Murbaty1999). In Australia, S. iniae causes substantial lossesto the barramundi, Lates calcarifer (Bloch), aqua-culture industry, resulting in a number of farmsclosing during recent years. Moreover, recentattempts to control the disease through vaccinationhave met with limited success. In addition to lossesto aquaculture, S. iniae has been reported to causedisease in humans handling fresh infected fish (Lau,Woo, Luk, Fung, Hui, Fong, Chow, Wong & Yuen2006; Lau, Woo, Tse, Leung, Wong & Yuen 2003;Weinstein, Litt, Kertesz, Wyper, Rose, Coulter,Mcgeer, Facklam, Ostach, Willey, Borczyk & Low1997) and is thus of increasing clinical significance.Human infections have been attributed to certaingenotypes based on pulsed-field gel electrophoresis(Fuller, Bast, Nizet, Low & de Azavedo 2001), butthere would appear to be more than one genotypeinvolved in human infections (Lau et al. 2003). Theaim of the present study was therefore to charac-terize Australian isolates of S. iniae genotypicallyand compare them with reference fish and humanisolates from the Centers for Disease Control andPrevention (CDC), Atlanta, USA in order toelucidate the diversity of isolates within the Aus-tralian aquaculture industry.Streptococcus iniae isolates were obtained fromveterinary laboratories across Australia, and fromthe CDC, Atlanta, USA (Table 1). Bacterial isolateswere routinely cultured on Columbia agar basecontaining 2% defibrinated sheep blood at 35 C.Identity of all strains received was determined byGram stain and API 20 Strep (BioMerieux, MarcyLEtiole, France). Identity was confirmed by PCRamplification of the lactate oxidase gene (lox)asdescribed previously (Mata, Blanco, Dominguez,Fernandez-Garayzabal & Gibello 2004) from geno-mic DNA purified as follows: S. iniae fromovernight vegetable peptone cultures (10 mL) werewashed three times in phosphate-buffered saline(pH 7.4), resuspended in 500 lL lysozyme(100 mg mL

Evolution of the Capsular Operon of Streptococcus iniae in Response to Vaccination

ABSTRACT Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.

Host-directed evolution of a novel lactate oxidase in Streptococcus iniae isolates from barramundi (Lates calcarifer).

ABSTRACT 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.