Sobria Strain Research Articles

Polyinfection in Fish Aeromoniasis: A Study of Co-Isolated Aeromonas Species in Aeromonas veronii Outbreaks.

We studied the phenotypic and genomic characteristics related to the virulence and antibiotic resistance of two Aeromonas strains, which were co-isolated before an outbreak of Aeromonas veronii among diseased seabass on Agathonisi Island, Greece, in April 2015. The first strain, AG2.13.2, is a potentially pathogenic mesophilic variant of Aeromonas salmonicida, and the second, AG2.13.5, corresponds to an Aeromonas rivipollensis related to A. rivipollensis KN-Mc-11N1 with an ANI value of 97.32%. AG2.13.2 lacks the type III secretion system just like other mesophilic strains of A. salmonicida. This characteristic has been associated with lower virulence. However, the genome of AG2.13.2 contains other important virulence factors such as type II and type VI secretion systems, and toxins such as rtxA, aerolysin aer/act, and different types of hemolysins. The strain also carries several genes associated with antibiotic resistance such as the tetE efflux pump, and exhibits resistance to tetracycline, ampicillin, and oxolinic acid. In an in vivo challenge test with gilthead seabream larvae, the A. veronii bv sobria strain AG5.28.6 exhibited the highest virulence among all tested strains. Conversely, both A. salmonicida and A. rivipollensis showed minimal virulence when administered alone. Interestingly, when A. veronii bv sobria AG5.28.6 was co-administered with A. rivipollensis, the larvae survival probability increased compared to those exposed to A. veronii bv sobria AG5.28.6 alone. This finding indicates an antagonistic interaction between A. veronii bv sobria AG5.28.6 and A. rivipollensis AG2.13.5. The co-administration of A. veronii bv sobria AG5.28.6 with Aeromonas salmonicida did not yield distinct survival probabilities. Our results validate that the primary pathogen responsible for European seabass aeromoniasis is Aeromonas veronii bv sobria.

Structure and gene cluster annotation of the O-antigen of Aeromonas sobria strain K928 isolated from common carp and classified into the new Aeromonas PGO1 serogroup

Aeromonas sobria strain K928 was isolated from a common carp during a Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreak on a Polish fish farm and classified into the new provisional PGO1 serogroup. The lipopolysaccharide of A. sobria K928 was subjected to mild acid hydrolysis, and the O-specific polysaccharide, which was isolated by gel-permeation chromatography, was studied using sugar and methylation analyses and 1H and 13C NMR spectroscopy. The following structure of the branched O-specific polysaccharide repeating unit of A. sobria K928 was established.→2)[α-D-Fucp3NRHb-(1→3)]-α-L-Rhap-(1→3)-β-L-Rhap-(1→4)-α-L-Rhap-(1→3)-β-D-FucpNAc-(1→The O-antigen gene cluster was identified and characterized in the genome of the A. sobria K928 strain after comparison with sequences in the available databases. The composition of the O-antigen genetic region was found to be consistent with the O-polysaccharide structure, and its organization was proposed.

ANTIBACTERIAL EFFICACY OF LEAF EXTRACTS DERIVED FROM FICUS ELASTICA ROXB. EX HORNEM. (MORACEAE) AND ITS CULTIVARS AGAINST AEROMONAS SOBRIA STRAIN

The range of healing targets for particular Ficus species compiled from local medicines can be competitive with that of broad-spectrum traditional remedies. In the current study, we studied the antimicrobial activity of the ethanolic extracts derived from the leaves of Ficus elastica Roxb. ex Hornem. and its cultivars (F. elastica 'Rubra', 'Robusta', 'Burgundy', 'Variegata') against Aeromonas sobria to evaluate the possible use of this plant in preventing infections caused by this fish pathogen in aquaculture. The current study was conducted as a part of an ongoing project between five universities undertaken in the frame of a cooperation program aimed at the assessment of medicinal properties of tropical and subtropical plants, cultivated in vitro. The leaves of F. elastica and its cultivars, cultivated under glasshouse conditions, were sampled at M. M. Gryshko National Botanic Garden (NBG), National Academy of Science of Ukraine. Specifically, the leaves of F. elastica and its cultivars, i.e. F. elastica 'Rubra', 'Robusta', 'Burgundy', 'Variegata' were sampled for our study. Aeromonas sobria (K825) strain, originated from freshwater fish species such as common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum), respectively, was isolated in the Department of Fish Diseases, The National Veterinary Research Institute in Pulawy (Poland). Antimicrobial susceptibility of the tested Aeromonas sobria was performed by the Kirby-Bauer disc diffusion method (1966) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (2014), with our some modifications. Our results of the antimicrobial screening revealed, that F. elastica and its cultivars possessed mild antibacterial properties against the A. sobria strain. The ethanolic extract obtained from leaves of F. elastica 'Variegata' exhibited the maximum antimicrobial activity against A. sobria. Thus, F. elastica and its cultivars (F. elastica 'Rubra', 'Robusta', 'Burgundy', 'Variegata') disclosed mild bioactivity, and this plant can be regarded as a potential source of antibacterial agents. The results of the current study provide a new perspective for the use of various species belonging to the Ficus genus as medicinal plants to improve the antibacterial responses in salmonid aquaculture. Keywords: Ficus elastica Roxb. ex Hornem., extract, antimicrobial efficacy, Kirby-Bauer disk diffusion technique, fish pathogens, susceptibility, resistance.

Structure and Gene Cluster Annotation of the O-Antigen of Aeromonas Sobria Strain K928 Isolated from Common Carp and Classified into the New Aeromonas Pgo1 Serogroup

Structure and Gene Cluster Annotation of the O-Antigen of Aeromonas Sobria Strain K928 Isolated from Common Carp and Classified into the New Aeromonas Pgo1 Serogroup

Aeromonas sobria Serine Protease Degrades Several Protein Components of Tight Junctions and Assists Bacterial Translocation Across the T84 Monolayer.

Aeromonas sobria is a Gram-negative pathogen that causes food-borne illness. In immunocompromised patients and the elderly, A. sobria opportunistically leads to severe extraintestinal diseases including sepsis, peritonitis, and meningitis. If A. sobria that infects the intestinal tract causes such an extraintestinal infection, the pathogen must pass through the intestinal epithelial barrier. In our earlier study using intestinal cultured cells (T84 cells), we observed that an A. sobria strain with higher A. sobria serine protease (ASP) production caused a marked level of bacterial translocation across the T84 intestinal epithelial monolayer. Herein, we investigated the effect of ASP on tight junctions (TJs) in T84 cells. We observed that ASP acts on TJs and causes the destruction of ZO-1, ZO-2, ZO-3, and claudin-7 (i.e., some of the protein components constituting TJs), especially in the strains with high ASP productivity. Based on the present results together with those of our earlier study, we propose that ASP may cause a disruption of the barrier function of the intestinal epithelium as a whole due to the destruction of TJs (in addition to the destruction of adherens junctions) and that ASP may assist invasion of the pathogens from the intestinal epithelium into deep sites in the human body.

Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 Which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.

Comparative Assessment of the Antibacterial Efficacy of Leaf Extract Obtained from Ficus benjamina L. (Moraceae) and its Cultivars against Aeromonas sobria Strain

The present study is in vitro study to evaluate the antimicrobial activity of the ethanolic extracts derived from leaves of Ficus benjamina L. and its cultivars (Safari, Baroque, Amstel Gold, Reginald) against Aeromonas sobria to assess the possible use of this plant in preventing infections caused by this fish pathogen in aquaculture. Antimicrobial susceptibility of the tested Aeromonas sobria was performed by the Kirby-Bauer disc diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). Our results revealed, that F. benjamina and its cultivars possessed antibacterial properties against Aeromonas sobria strain. The ethanolic extract obtained from leaves of F. benjamina ’Safari‘ exhibited the maximum antimicrobial activity against Aeromonas sobria (the mean of inhibition zone diameter was 26.19 ±1.32 mm). Aeromonas sobria strain was susceptible to the F. benjamina ’Amstel Gold‘ (15.25 ±1.25 mm) and ’Reginald‘ (16.25 ±1.10 mm). Aeromonas sobria strain was the most resistant to F. benjamina (12.5 ±0.80 mm) and F. benjamina ’Baroque‘ (13.63 ±0.75 mm) leaf extracts. The results of this study provide a new perspective for the use of various Ficus species as medicinal plants to improve the antibacterial responses in aquaculture. Scanning electron microscopy has been employed to observe epicuticular wax structures which can be used to assure the correct identification of plant raw materials. Further studies including the use of other medicinal plants as food additives in aquaculture, the assessment of their antioxidant effects on various tissues of salmonids are in progress.

Evaluation of quorum-sensing inhibitory effects of extracts of three traditional medicine plants with known antibacterial properties.

Today an alternative approach to control bacterial infections is the use of natural and traditional plant compounds to interfere with their quorum-sensing (QS) systems. In this study, antibacterial and anti-QS sensing activity of Syzygium aromaticum, Dionysia revoluta Boiss. and Eucalyptus camaldulensis Dehnh. were evaluated. These plants are local to the Middle East region and have since ancient times been used for their antibacterial activity. Plant compounds were extracted with n-hexane, methanol and 96% ethanol mixed solvent. Antibacterial activity of this herbal extracts against five Gram-negative and Gram-positive bacteria were assessed. The effective sub–minimum inhibitory concentration (MIC) of this extract on bacterial QS systems were investigated by a violacein quantification assay in the Chromobacterium violaceum CV026 biosensor strain, inhibition of exogenously QS signal molecules in Aeromonas veronii bv. Sobria strain BC88 and Pseudomonas aeruginosa isolated from a patient with cystic fibrosis in vitro. Results found that Syzygium aromaticum 0.39 to 0.048 mg/mL, Dionysia revoluta Boiss. 3.1 to 0.39 mg/mL and E. camaldulensis 0.78 to 0.097 mg/mL showed anti-QS activities by reducing the violacein formation depletion of QS signals produced in A. veronii and P. aeruginosa at sub-MICs. Regarding the anti-QS effects of these herbal extracts, their effective components may be candidates for use in combating bacterial infections at sub-MICs.

Draft Genome Sequence of Aeromonas sobria Strain CHT-30, Isolated from a Diseased Rainbow Trout (Oncorhynchus mykiss) in Peru.

The Gram-negative bacterium Aeromonas sobria is an opportunistic pathogen that affects humans and animals, including fish. Here, we report the draft genome of strain CHT-30, which was isolated from a diseased rainbow trout in Peru. The genome size is 4.91 Mb, with a G+C content of 57.7%, and the genome includes 4,820 coding sequences.

High-throughput sequencing of the 16S rRNA gene to analyze the gut microbiome in juvenile and adult tropical gar (Atractosteus tropicus)

Tropical gar (Atractosteus tropicus) is freshwater and estuarine fish, inhabiting the Earth since the Mesozoic era and undergoing limited physiological variation ever since. Besides its recognized cultural andscientific relevance, the species has seen remarkable growth in its economic impact due to pisciculture. In this study, we present the first report of the whole taxonomic composition of microbial communities in gut contents in juveniles and adults of A. tropicus, by sex and origin (wild and cultivated). For this study, 508 genera were identified, with the most and least abundant being Cetobacterium and Paludibacter, respectively. Fusobacteria, Proteobacteria, Firmicutes, and Bacteroidetes phyla are the core gut microbiome of A. tropicus juvenile and adult by sex and origin. Deinococcus-Thermus phylum sequence was only identified in wild-type males. In the phylogenetic trees reconstruction Lactococcus lactis strains CAU929 and CAU6600, Cp6 and CAU9951,Cetobacterium strain H69, Aeromonas hydrophila strain P5 and WR-5-3-2, Aeromonas sobria strain CP DC28 and Aeromonas hydrophila were identified, some of them with probiotic potential within the three dominant phyla in core gut microbiome in A. tropicus adults, especially in wild-type organisms. Myroides genus was recognized in microbiota gut of the cultivated juvenile A. tropicus. Nevertheless, Alpha diversity indicated that the highest gut microbiota abundance and richness is found in cultivated juvenile and wild-type adult A. tropicus female, rather than adult wild-type males and the least gut microbiota abundance and richness is found in a cultivated adult of A. tropicus for both sexes.

Structure of the O-specific polysaccharide of Aeromonas veronii bv. sobria strain Pt393 isolated from rainbow trout (Oncorhynchus mykiss), which contains a rarely occurring sugar 4-acetamido-4,6-dideoxy-d-galactose, tomosamine

The O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide of Aeromonas veronii bv. sobria strain Pt393, which is pathogenic to the rainbow trout (Oncorhynchus mykiss), after mild acid hydrolysis followed by GPC. The high-molecular-weight OPS fraction was studied with chemical methods, mass spectrometry, and 1H and 13C NMR spectroscopy techniques, including 2D 1H,1H COSY, TOCSY, NOESY, 1H-detected heteronuclear 1H,13C HSQC, and HMBC experiments. It was found that the O-specific polysaccharide was built of a tetrasaccharide repeating unit composed of α-GalpNAc, α-FucpNAc, β-QuipNAc, and α-Fucp4NAc (4-acetamido-4,6-dideoxy-d-galactose, tomosamine) residues. The following structure of the OPS of A. sobria strain Pt393 was established:→4)-α-d-GalpNAc-(1 → 3)-α-l-FucpNAc-(1 → 3)-β-d-QuipNAc-(1 → 3)-α-d-Fucp4NAc-(1→

Virulence characteristics and antimicrobial resistance of Aeromonas veronii biovar sobria 312M, a clinical isolate.

Aeromonas are bacteria widely distributed in the environment, and some species are able to cause infections in humans, of which diarrhea is the most common. The objective of this study was to evaluate the presence of virulence and antimicrobial resistance associated characteristics in A. veronii biovar sobria strain 312M isolated from diarrheal stools. For this, the genome sequencing and phenotypical tests were performed. The draft genome annotation revealed several complete pathways associated with carbon metabolism and a mucin-desulfating sulfatase which may contribute to intestine colonization, and a large number of virulence-associated genes encoding structures associated with adhesion, toxins, and secretion systems. The strain exhibited swimming and swarming motility, biofilm formation, and hemolytic activity. It was resistant to ampicillin, ampicillin/sulbactam, and amoxicillin-clavulanic acid. Although a cphA gene encoding a narrow-spectrum carbapenase was identified in the strain genome, no carbapenemase activity was detected in the antimicrobial susceptibility test. When compared with other A. veronii with complete genomes, the main differences in virulence characteristics are related to lateral flagella and type III and VI secretion systems; the antimicrobial resistance spectrum also varied among strains. The results indicated that A. veronii biovar sobria 312M presents high virulence potential and resistance to limited classes of antimicrobials.

Structural and Serological Studies of the O6-Related Antigen of Aeromonas veronii bv. sobria Strain K557 Isolated from Cyprinus carpio on a Polish Fish Farm, which Contains l-perosamine (4-amino-4,6-dideoxy-l-mannose), a Unique Sugar Characteristic for Aeromonas Serogroup O6

Amongst Aeromonas spp. strains that are pathogenic to fish in Polish aquacultures, serogroup O6 was one of the five most commonly identified immunotypes especially among carp isolates. Here, we report immunochemical studies of the lipopolysaccharide (LPS) including the O-specific polysaccharide (O-antigen) of A. veronii bv. sobria strain K557, serogroup O6, isolated from a common carp during an outbreak of motile aeromonad septicemia (MAS) on a Polish fish farm. The O-polysaccharide was obtained by mild acid degradation of the LPS and studied by chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-antigen was composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc). The following structures of the O-polysaccharides (O-PS 1 and O-PS 2) were established: O-PS 1: →2)-α-l-Rhap4NAc-(1→; O-PS 2: →2)-α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc-(1→. Western blotting and an enzyme-linked immunosorbent assay (ELISA) showed that the cross-reactivity between the LPS of A. veronii bv. sobria K557 and the A. hydrophila JCM 3968 O6 antiserum, and vice versa, is caused by the occurrence of common α-l-Rhap4NAc-(1→2)-α-l-Rhap4NAc and α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc disaccharides, whereas an additional →4)-α-d-GalpNAc-associated epitope defines the specificity of the O6 reference antiserum. Investigations of the serological and structural similarities and differences in the O-antigens provide knowledge of the immunospecificity of Aeromonas bacteria and are relevant in epidemiological studies and for the elucidation of the routes of transmission and relationships with pathogenicity.

Strong Genomic and Phenotypic Heterogeneity in the Aeromonas sobria Species Complex.

Aeromonas sobria is a mesophilic motile aeromonad currently depicted as an opportunistic pathogen, despite increasing evidence of mutualistic interactions in salmonid fish. However, the determinants of its host-microbe associations, either mutualistic or pathogenic, remain less understood than for other aeromonad species. On one side, there is an over-representation of pathogenic interactions in the A. sobria literature, of which only three articles to date report mutualistic interactions; on the other side, genomic characterization of this species is still fairly incomplete as only two draft genomes were published prior to the present work. Consequently, no study specifically investigated the biodiversity of A. sobria. In fact, the investigation of A. sobria as a species complex may have been clouded by: (i) confusion with A. veronii biovar sobria because of their similar biochemical profiles, and (ii) the intrinsic low resolution of previous studies based on 16S rRNA gene sequences and multilocus sequence typing. So far, the only high-resolution, phylogenomic studies of the genus Aeromonas included one A. sobria strain (CECT 4245 / Popoff 208), making it impossible to robustly conclude on the phylogenetic intra-species diversity and the positioning among other Aeromonas species. To further understand the biodiversity and the spectrum of host-microbe interactions in A. sobria as well as its potential genomic diversity, we assessed the genomic and phenotypic heterogeneity among five A. sobria strains: two clinical isolates recovered from infected fish (JF2635 and CECT 4245), one from an infected amphibian (08005) and two recently isolated brook charr probionts (TM12 and TM18) which inhibit in vitro growth of A. salmonicida subsp. salmonicida (a salmonid fish pathogen). A phylogenomic assessment including 2,154 softcore genes corresponding to 946,687 variable sites from 33 Aeromonas genomes confirms the status of A. sobria as a distinct species divided in two subclades, with 100% bootstrap support. The phylogenomic split of A. sobria in two subclades is corroborated by a deep dichotomy between all five A. sobria strains in terms of inhibitory effect against A. salmonicida subsp. salmonicida, gene contents and codon usage. Finally, the antagonistic effect of A. sobria strains TM12 and TM18 suggests novel control methods against A. salmonicida subsp. salmonicida.

N-Acyl Homoserine Lactone-Mediated Quorum Sensing in Aeromonas veronii biovar sobria Strain 159: Identification of LuxRI Homologs.

16 Quorum sensing (QS) is a mechanism that plays important roles in gene expression in response to an 17 expanding bacterial population. In many Gram-negative bacteria, N-acyl homoserine lactone is mainly 18 secreted as the diffusible signalling molecules. This QS system has been shown to control diverse array 19 of virulence and secondary metabolism. Recently, whole genome sequencing of Aeromonas veronii 20 biovar sobria strain 159 was performed. Genome comparison with closely-related Aeromonas species 21 showed that A. veronii strain 159 shares a high degree of genome synteny with A. hydrophila ATCC 22 7966. A detailed genome analysis and gene annotation led us to the findings that A. veronii strain 159 23 harbors QS system which relies on its signal generator, AveI and the transcriptional regulator, AveR. 24 This Aeromonas strain was found to secrete N-butanoylacyl homoserine lactone (C4-HSL). Its LuxIR 25 homologs are similar to proteins of LuxIR famililies among Aeromonas species. This study aims to 26 gain further insights into AveIR system and to compare with AhyIR from A. hydrophila ATCC 7966 27 and AsaIR from A. salmonicida. 28 29 30 31

Characterization of Aeromonas sobria Isolated from Fish Rohu (Labeo rohita) Collected from Polluted Pond

One of the major threats to fish aquaculture sector is the infection by Aeromonas Spp. The current study assesses the phenotypic characteristics and biochemical characterization of the A. sobria strains from the fish cases with septicaemia in order to understand the frequency and occurrence of this infection in the state of Jammu and Kashmir. Clinically the infected fish Rohu (Labeo rohita), one of the Indian Major carps (IMC), was observed for symptoms like loss of escape reflex and skin darkness associated with skin haemorrhages. We isolated 30 colonies of A. sobria strain from 10 cultured Labeo rohita collected from a controlled fish pond in District Poonch of the state. The pond was affected by mismanagement practices, elevated pollution levels and anthropogenic activities. Microscopic examination revealed that the strain was rod-shaped and gram negative. The revealed percent probability identification of A. sobria from the biochemical characterization in Vitek system was 93% with GN card. This study could give us clues for understanding A. sobria harbouring in fish species and shall help in better understanding of the threat prevalent to the fish species of the region by this infection.

Detection and characterization of potentially pathogenic Aeromonas sobria isolated from fish Hypophthalmichthys molitrix (Cypriniformes: Cyprinidae)

The current study focuses on the detection and characterization of potentially pathogenic Aeromonas sobria from fish silver carp (Hypophthalmichthys molitrix). Assessment of clinical, microbiological, pathological and biochemical characteristics of A. sobria were taken into account in order to understand the epidemiology, frequency and occurrence of this infection. Clinically the infected fish (H. molitrix) was observed for various types of symptoms. A total of 33 colonies of A. sobria strain were isolated from 20 cultured H. molitrix, collected from controlled fish pond. Microscopic examination revealed that the strains were rod-shaped, Gram negative bacteria. The revealed percent probability identification of A. sobria from the biochemical characterization in VITEK system was 93% with gram negative (GN) card. The histopathology of Gills caused by this bacterium, A. sobria indicate haemorrhagic gill epithelia and epithelial hyperplasia. Lamelar epithelial hypertrophy and hyperplasia with degenerative changes of the epithelium and hypertrophic epitheliocystis infected cells on gills of H. molitrix were observed during the present study.

Structure of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas sobria strain Pt312

The O-specific polysaccharide (OPS) obtained by mild-acid degradation of the lipopolysaccharide from Aeromonas sobria strain Pt312 was studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY, 1H-detected 1H,13C HSQC, and HMBC experiments. The sequence of the sugar residues was determined using 1H,1H NOESY and 1H,13C HMBC experiments. It was found that the OPS was built up of disaccharide repeating units composed of GlcpNAc and non-stoichiometrically O-acetylated Rhap residues, and had the structure. [Display omitted]

Tissue distribution, expression, and antimicrobial activity of Anas platyrhynchos avian β-defensin 6

In this study, a novel avian β-defensin (AvBD) was isolated from the Chaohu duck. The complete nucleotide sequence of the gene contained a 204-bp open reading frame that encoded 67 amino acids (aa), including a signal peptide of 20 aa, a propiece of 5 aa, and a mature peptide of 42 aa. The homology, characterization, and comparison of this gene with AvBD from other avian species confirmed that it was duck AvBD6. Also, the preproprotein of AvBD6 from chicken, goose, and duck was highly conserved with 100% aa homology. The AvBD6 mRNA was widely expressed in the investigated tissues of healthy 5-mo-old ducks, with the exception of the skin, kidney, and bursa. The AvBD6 mRNA was highly expressed in the spleen, lung, stomach, tongue, and egg yolk, successively; moderately expressed in the bone marrow and liver; and expressed to lower degrees in the trachea, heart, intestine, muscle, esophagus, and testis. We produced recombinant AvBD6 by expressing the gene in Escherichia coli. The yield of soluble glutathione S-transferees (GST)-AvBD6 in the inclusion bodies increased significantly as the incubation temperature was decreased from 37 to 30°C. As expected, the GST-AvBD6 exhibited strong bactericidal properties [minimum inhibitory concentration (MIC) = 0.5 to 2 μM] against the Aeromonas veronii biovar sobria strain BJCP-5, E. coli, and Enterococcus faecalis [ATCC 29212); it possessed medium bactericidal properties (MIC = 4 to 8 μM) against Staphylococcus aureus, Salmonella enteritidis, Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (CMCC 63501;; and it possessed low bactericidal properties (MIC = 16 μM) against Candida albicans (ATCC 10231). Additionally, the antimicrobial activity of GST-AvBD6 proved to be the same as that of synthetic AvBD6. The A. veronii biovar sobria strain BJCP-5 cells that were treated with GST-AvBD6 showed lysis and shrinkage under scanning electron microscopy. The mechanisms of AvBD6-mediated killing of A. veronii biovar sobria involved both cell lysis and nonlysis. Our results indicate that AvBD6 plays an important role in the innate immunity of the Chaohu duck.

Structural analysis of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas veronii bv. sobria strain K49

The O-specific polysaccharide obtained by mild-acid degradation of the lipopolysaccharide from Aeromonas veronii bv. sobria strain K49 was studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy. The sequence of the sugar residues was determined using 1H,1H NOESY and 1H,13C HMBC experiments. The O-specific polysaccharide was found to be a high molecular mass polysaccharide composed of repeating units of the structure:→2)-β-d-Quip3NAc-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-d-FucpNAc-(1→ESI MS confirmed the pentasaccharide structure of the repeating unit, as the molecular mass peaks seen in the spectrum differed by 812.34u, a value corresponding to the calculated molecular mass of the O-unit.