Immune enhancement effects of inactivated vaccine against extracellular products of Aeromonas caviae AC-CY on crucian carp.

Aeromonas caviae is a zoonotic pathogen that can cause disease in aquatic organisms and mammals, including humans, and it is widespread in nature, especially in freshwater environments. Previous research has reported that extracellular products (ECPs) secreted by pathogens during growth are effective protective antigens that can induce the host immune response and protect the host from pathogens. However, little is known about how ECPs enhance immunity. Here, we prepared extracellular products by the cellophane plate method, determined the total protein concentration, and analysed the protein composition of the extracellular products by SDS-PAGE. Subsequently, their enzyme activity and pathogenicity were evaluated separately. Crucian carp were randomly divided into four groups to receive formalin-inactivated A. caviae vaccine (FKC), ECPs mixed with the same amount of Freund's complete adjuvant, the same amount of ECPs mixed with an equal volume of A. caviae inactivated vaccine (FKC + ECPs), sterile PBS alone via intraperitoneal injection. On Days 7, 14, 21, and 28 after immunization, the expression levels of IgM, SOD, and CAT and the lysozyme (LYS) activity in the serum were detected by ELISA, and the relative expression levels of the TNF-α, IFN-γ, IL-1ß, and IL-10 genes in the liver, kidney, spleen, intestine, and gills were measured by qPCR. The extracellular products generated five clearly visible protein bands and exhibited lipase, protease, amylase, DNase and lysozyme but no urease or lecithinase activities. In addition, the median lethal doses of A. caviae and ECPs to crucian carp were 411.64 µg/fish and 1.6 × 105 CFU/mL, respectively. Compared with those of the control group, the IgM, SOD, and CAT contents and serum LYS activity were significantly increased in the experimental groups, and the qRT-PCR results showed that the relative expression levels of TNF-α, IFN-γ, IL-1ß, and IL-10 genes in the liver, kidney, spleen, and intestine were significantly increased after injection immunization. In addition, the relative immune protection rates of the three experimental groups were 60%, 65%, and 45%, all of which were significantly higher than those of the control group. Collectively, our findings show that the extracellular products of A. caviae can be used as a vaccine to significantly improve the immune level of crucian carp and have obvious anti-infection ability. This may represent a promising approach to prevent and control infection by A. caviae and provides strong theoretical support for the development of new inactivated vaccines.

A potential alternative to traditional antibiotics in aquaculture: Yeast glycoprotein exhibits antimicrobial effect in vivo and in vitro on Aeromonas caviae isolated from Carassius auratus gibelio.

In aquaculture, antibiotics are commonly used to provide protection against pathogens; however, this practice has become controversial due to increased occurrences of microbial resistance, and alternatives are needed. This study aimed to investigate the antimicrobial activity of yeast glycoprotein (YG) against Aeromonas caviae. Pathogens were isolated from liver of diseased Carassius auratus gibelio. Based on morphological and biochemical analysis, together with 16S rRNA gene sequencing, the isolated strains were identified as A. caviae and concluded as clones of a single strain and named L2. Further pathogenicity analysis revealed that A. caviae possessed ß-haemolysis, and its median lethal dose for C. gibelio was 1.33 × 106  CFU/ml. Hepatic adenylate kinase and pyruvate kinase activities of C. gibelio were inhibited post-A. caviae infection. Antimicrobial drug test suggested that A. caviae was a multidrug-resistant organism but could be inhibited by YG in vitro. Minimum inhibitory and bactericidal concentration of YG was 83.3 mg/ml and 166.7 mg/ml, respectively. Microbiota sequencing results showed that YG supplement could obviously decrease the relative abundance of Aeromonas and increase the microbial diversity. Our study revealed that A. caviae from C. gibelio was a multidrug-resistant bacteria strain, and could be significantly inhibited by YG in vivo and in vitro, thus providing important insights into ecological control and pathogenesis of A. caviae in aquaculture.

Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan.

Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial-resistant (AMR) Aeromonas strains (A. hydrophila GSH8-2 and A. caviae GSH8M-1) carrying the carabapenemase blaKPC-2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8-2 and GSH8M-1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired blaKPC-2 -positive IncP-6-relative plasmids (pGSH8-2 and pGSH8M-1-2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to blaKPC-2 , pGSH8M-1-2 carries an IS26-mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities.

Regulation of 3-hydroxyhexanoate composition in PHBH synthesized by recombinant Cupriavidus necator H16 from plant oil by using butyrate as a co-substrate.

A (R)-3-hydroxyhexanoate (3HH) composition-regulating technology for poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) production was developed using recombinant Cupriavidus necator H16 with butyrate as a co-substrate. A new (R)-3-hydroxyhexanoyl-CoA ((R)-3HH-CoA) synthesis pathway was designed and enhanced by replacing the PHA synthase gene (phaC1) of C. necator by the phaCAcNSDG (encoding the N149S and D171G mutant of PHA synthase from Aeromonas caviae) and deactivation of the phaA gene (encoding (ß-ketothiolase) from C. necator H16 chromosome). The effect of butyrate as co-substrate was assessed in high-cell-density fed-batch cultures of several C. necator mutants, and the 3HH fraction was successfully increased by adding butyrate to the culture. Moreover, overexpression of BktB (encoding the second ß-ketothiolase with broad substrate specificity) enhanced the (R)-3HH-CoA synthesis pathway in the phaA deactivated mutant of C. necator by promoting the condensation of acetyl-CoA and butyryl-CoA into 3-ketohexanoyl-CoA. Consequently, PHBH containing 4.2-13.0 mol% 3HH was produced from butyrate and palm kernel oil by the genetically modified C. necator H16 strains.

Isolation and identification of Aeromonas caviae strain KS-1 as TBTC- and lead-resistant estuarine bacteria.

Tributyltin chloride (TBTC)- and lead-resistant estuarine bacterium from Mandovi estuary, Goa, India was isolated and identified as Aeromonas caviae strain KS-1 based on biochemical characteristics and FAME analysis. It tolerates TBTC and lead up to 1.0 and 1.4 mM, respectively, in the minimal salt medium (MSM) supplemented with 0.4 % glucose. Scanning electron microscopy clearly revealed a unique morphological pattern in the form of long inter-connected chains of bacterial cells on exposure to 1 mM TBTC, whereas cells remained unaltered in presence of 1.4 mM Pb(NO3)2 but significant biosorption of lead (8 %) on the cell surface of this isolate was clearly revealed by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. SDS-PAGE analysis of whole-cell proteins of this lead-resistant isolate interestingly demonstrated three lead-induced proteins with molecular mass of 15.7, 16.9 and 32.4 kDa, respectively, when bacterial cells were grown under the stress of 1.4 mM Pb (NO3)2. This clearly demonstrated their possible involvement exclusively in lead resistance. A. caviae strain KS-1 also showed tolerance to several other heavy metals, viz. zinc, cadmium, copper and mercury. Therefore, we can employ this TBTC and lead-resistant bacterial isolate for lead bioremediation and also for biomonitoring TBTC from lead and TBTC contaminated environment.

Expression of Aeromonas caviae polyhydroxyalkanoate synthase gene in Burkholderia sp. USM (JCM15050) enables the biosynthesis of SCL-MCL PHA from palm oil products.

AIMS: Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaC(Ac)) was transformed into this strain. METHODS AND RESULTS: The resulting transformant incorporated approximately 1 ± 0·3 mol% of 3HHx in the polymer when crude palm kernel oil (CPKO) or palm kernel acid oil was used as the sole carbon source. In addition, when the transformed strain was cultivated in the mixtures of CPKO and sodium valerate, PHA containing 69 mol% 3HB, 30 mol% 3-hydroxyvalerate and 1 mol% 3HHx monomers was produced. Batch feeding of carbon sources with 0·5% (v/v) CPKO at 0 h and 0·25% (w/v) sodium valerate at 36 h yielded 6 mol% of 3HHx monomer by controlled-feeding strategies. CONCLUSIONS: Burkholderia sp. USM (JCM15050) has the metabolic pathways to supply both the short-chain length (SCL) and medium-chain length (MCL) PHA monomers. By transforming the strain with the Aer. caviae PHA synthase with broader substrate specificity, SCL-MCL PHA was produced. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study demonstrating the ability of transformant Burkholderia to produce P(3HB-co-3HHx) from a single carbon source.

Characterization and functional analyses of R-specific enoyl coenzyme A hydratases in polyhydroxyalkanoate-producing Ralstonia eutropha.

A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R-specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded by phaJ4 from Pseudomonas aeruginosa (phaJ4(Pa)). The recombinant forms of the three proteins, termed PhaJ4a(Re) to PhaJ4c(Re), actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C(4) to C(8). PhaJ4a(Re) and PhaJ4b(Re) showed >10-fold-higher catalytic efficiency than PhaJ4c(Re). The functions of the new PhaJ4 proteins were investigated using previously engineered R. eutropha strains as host strains; these strains are capable of synthesizing poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Deletion of phaJ4a(Re) from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4b(Re) or phaJ4c(Re), indicating that only PhaJ4a(Re) was one of the major enzymes supplying the (R)-3HHx-CoA monomer through ß-oxidation. Introduction of phaJ4a(Re) or phaJ4b(Re) into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4c(Re) did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co-3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially when phaJ4a(Re) or phaJ4b(Re) was tandemly introduced with phaJ(Ac) from Aeromonas caviae.