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Background: Accurate identification of infectious diseases using molecular techniques, such as PCR and NGS, is well-established. This study aims to assess the utility of Bactfast and Fungifast in diagnosing bloodstream infections in ICU settings, comparing them against traditional culture methods. The objectives include evaluating sensitivity and specificity and identifying a wide range of pathogens, including non-culturable species. Methods: We collected 500 non-duplicate blood samples from ICU patients between January 2023 and December 2023. Specimens underwent traditional culture, MALDI-TOF, VITEK®2 compact system, and NGS-based Bactfast and Fungifast analyses. Results: Out of the 500 samples, 26.8% (n=134) showed bacterial growth via traditional culture methods, while 4.8% (n=24) were positive for fungal growth. MALDI-TOF and VITEK®2 compact system yielded comparable results, identifying 26.4% (n=132) of specimens with bacterial growth. NGS-based Bactfast detected bacterial presence in 38.2% (n=191) of samples, including non-culturable bacteria missed by traditional methods. However, NGS-based Fungifast showed concordant fungal detection rates with culture methods. Among identified pathogens by culture method included Klebsiella pneumoniae 20.89% (n=28), Enterococcus faecalis 18.65% (n=25), Escherichia coli 15.67% (n=21), Pseudomonas aeruginosa 12.68% (n=17), Acinetobacter baumannii 10.44% (n=14), various Streptococcus species 7.46% (n=10), Mycobacterium tuberculosis 6.71% (n=9), Mycobacterium abscessus 4.47% (n=6), and Salmonella spp 2.98% (n=4). Non-culture-based NGS identified additional (n=33) pathogens, including Klebsiella pneumoniae 27.27% (n=9), Bacteroides fragilis 21.21% (n=7), Aerococcus viridans 15.15% (n=5), Elizabethkingia anopheles 12.12% (n=4), Aeromonas salmonicida 9% (n=3), Clostridium 9% (n=3), and Bacteroides vulgatus 6% (n=2). Candida albicans was reported in 5% (n=24) of samples by both methods. Conclusion: NGS-based Bactfast and Fungifast demonstrate high sensitivity in identifying a wide array of bacterial and fungal pathogens in ICU patients, outperforming traditional culture methods in detecting non-culturable organisms. These molecular assays offer rapid and comprehensive diagnostic capabilities, potentially improving clinical outcomes through timely and accurate pathogen identification. [ABSTRACT FROM AUTHOR]

Aquaculture is expected to play a vital role in solving the challenge of sustainably providing the growing world population with healthy and nutritious food. Pathogen outbreaks are a major risk for the sector, so early detection and a timely response are crucial. This can be enabled by monitoring the pathogen levels in aquaculture facilities. This paper describes a photonic biosensing platform based on silicon nitride waveguide technology with integrated active components, which could be used for such applications. Compared to the state of the art, the current system presents improvements in terms of miniaturization of the Photonic Integrated Circuit (PIC) and the development of wafer-level processes for hybrid integration of active components and for material-selective chemical and biological surface modification. Furthermore, scalable processes for integrating the PIC in a microfluidic cartridge were developed, as well as a prototype desktop readout instrument. Three bacterial aquaculture pathogens (Aeromonas salmonicida, Vagococcus salmoninarum, and Yersinia ruckeri) were selected for assay development. DNA biomarkers were identified, corresponding primer-probe sets designed, and qPCR assays developed. The biomarker for Aeromonas was also detected using the hybrid PIC platform. This is the first successful demonstration of biosensing on the hybrid PIC platform. [ABSTRACT FROM AUTHOR]

The diversity of O-polysaccharides (O-antigens) among 28 Aeromonas salmonicida strains isolated from ill fish has been determined by using high-resolution magic angle spinning (HR MAS) NMR spectroscopy. The new O-polysaccharide has been identified in two isolates. This new structure was investigated by 1H and 13C NMR spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The following structure of the linear hexasaccharide repeating unit of A. salmonicida O-antigen has been established: →3)-α-L-Rhap-(1→3)-α-D-ManpNAc-(1→2)-β-D-Glcp-(1→3)-α-L-Rhap2OAc4OAc-(1→3)-β-D-ManpNAc-(1→3)-α-D-Glcp-(1→. This new A. salmonicida O-polysaccharide was detected among two isolates collected from trout and turbot fish in 2010 and 2011, respectively. Further investigations should be conducted to evaluate the distribution of this new O-polysaccharide among a larger collection of isolates, depending on their geographic origin, the species of fish, and the health status of the fish. [ABSTRACT FROM AUTHOR]

Hsp40–Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein–protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40–Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot. [ABSTRACT FROM AUTHOR]

Infectious disease is a major challenge in fish and shrimp farming systems caused by microorganisms including bacteria, viruses and parasites. Physical detection has limitations in detecting diseases in cultivated animals earlier, because of the varied clinical symptoms. The aims of this study was to identify the physical and molecular presence of infectious pathogens in fish and shrimp cultivated in several ponds in West Java, Banten and Jakarta. Monitoring was carried out in two stages, namely the period March–April 2021 and September 2021 by taking samples from several ponds in Bogor, Tangerang, Depok and Jakarta. The sample criteria used were fish and shrimp showing normal and clinical symptoms of being attacked by a disease which were collected by purposive sampling. The collected samples were examined in two stages, namely physical examination of clinical symptoms and molecular examination using PCR. The results showed that of the 24 species samples collected, 17 species showed normal clinical symptoms and seven species showed clinical symptoms. Of the 24 fish and shrimp samples, four samples were infected (2 samples from normal species and 2 samples with clinical symptoms). The infected normal samples were Osphronemus goramy and Litopenaeus vannamei. The infected samples with clinical symptoms were Carassius auratus by Aeromonas salmonicida and red zebra cichlid (Metriaclima estherae) by Red Sea Bream Iridoviral Disease (RSBIV). Physical testing supported by molecular detection of aquatic animals can be an effort to manage aquaculture systems in Indonesia. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida is a common pathogen of salmonid fishes that poses a significant threat to the fresh water and marine culture industry, potentially resulting in huge economic losses. To prevent and control fish diseases caused by A. salmonicida, rapid and effective diagnostic approaches must be developed, and which are important for routine monitoring and clinical care. By combining recombinase polymerase amplification (RPA) technology with a visible lateral flow strip (RPA-LF), we have enhanced both the precision of RPA detection and the convenience of real-time monitoring. In this study, we introduce a robust method for detecting A. salmonicida using RPA-LF. This assay specifically targets the ASA_1441 gene of A. salmonicida, ensuring high specificity, without cross-reactivity with other prevalent fresh water or marine pathogens. The optimal amplification temperature of the RPA assay was 39 °C. Its sensitivity extends to as low as 100 fg of purified DNA, representing more than 1000-fold higher sensitivity than conventional PCR methods. Furthermore, to enhance the usability of the RPA-LF assay, we developed a rapid sample preparation method using cellulose dipsticks for nucleic acid extraction. This method achieves a limit of detection (LOD) as low as 1.67 CFU/µL and completes the entire process within 20 min. In conclusion, our findings present a rapid and precise tool for monitoring A. salmonicida infection in aquaculture and marine culture. This advancement offers valuable insights for effective disease prevention and control strategies. [ABSTRACT FROM AUTHOR]

The misuse of antibiotics and the emergence of antimicrobial resistance (AMR) is a concern in the aquaculture industry because it contributes to global health risks and impacts the environment. This study analyzed the AMR of sentinel bacteria associated with striped catfish (Pangasisanodon hypophthalmus) and giant snakehead (Channa micropeltes), the two main fish species reared in the pond culture in Cambodia. Phenotypic and genotypic characterization of the recovered isolates from fish, water, and sediment samples revealed the presence of bacteria, such as 22 species belonging to families Aeromonadaceae, Enterobacteriaceae, and Pseudomonadaceae. Among 48 isolates, Aeromonas caviae (n = 2), Aeromonas hydrophila (n = 2), Aeromonas ichthiosmia (n = 1), Aeromonas salmonicida (n = 4) were detected. A. salmonicida and A. hydrophilla are known as fish pathogens that occur worldwide in both fresh and marine water aquaculture. Antibiotic susceptibility testing revealed antibiotic resistance patterns of 24 (50 %) isolates among 48 isolates with higher multiple antibiotic resistance index (> 0.2). All the isolates of Enterobacteriaceae were susceptible to ciprofloxacin. Ciprofloxacin is a frontline antibiotic that is not recommended to use in aquaculture. Therefore, its use has to be strictly controlled. This study expands our knowledge of the AMR status in aquaculture farms which is very limited in Cambodia. [ABSTRACT FROM AUTHOR]

This study evaluates the effects of dietary Clostridium butyricum on growth performance and intestinal health in triploid rainbow trout (Oncorhynchus mykiss). Administered in a 12-week trial, five isonitrogenous and isolipidic feeds contained different C. butyricum levels [G1 (0), G2 (1.6 × 106 CFU/g), G3 (1.2 × 107 CFU/g), G4 (1.1 × 108 CFU/g), and G5 (1.3 × 109 CFU/g)]. Significant enhancements in growth performance, including improved feed conversion ratios and specific growth rates in the G4 group, were indicative of enhanced nutrient utilization, corroborated by optimal digestive enzyme activity levels. Antioxidant capabilities were also enhanced in the G4 group, indicated by increased serum superoxide dismutase and catalase activities, along with a significant decrease in malondialdehyde levels. Gut microbiota analysis indicated a probiotic concentration-dependent modulation of microbial communities, with a marked enrichment in beneficial bacterial phyla like Firmicutes in the G4 group. This microbial shift correlated with significant downregulations in immune-related gene expressions, including interleukins (IL-1β, IL-8), NF-κB, MyD88, and TNF-α, highlighting an activated immune response. Correspondingly, serum lysozyme and immunoglobulin M contents were significantly elevated in the G4 group. Challenge tests with Aeromonas salmonicida exhibited the higher disease resistance of fish fed the G4 diet. In conclusion, the study shows the potential of dietary C. butyricum, especially at 1.1 × 108 CFU/g, in enhancing the growth, health, and disease resistance of triploid rainbow trout through modulating gut microflora and stimulating immune responses. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria Ligilactobacillus salivarius has been shown to be a substitute for antibiotics in the treatment of bacterial disease in high animals. However, its beneficial mechanism in fish farming is still unclear. This study evaluated the antagonistic effects of the Ligilactobacillus salivarius GX118 strain on Aeromonas salmonicida and its regulation of rainbow trout growth in vivo and in vitro. The results found that GX118 produces an antibacterial substance that can directly destroy the cell wall of A. salmonicida. Whole-genome sequencing of GX118 revealed that Enterolysin A is a type III bacteriocin with antibacterial properties. An in vivo experiment showed that the supplementation of GX118 in diet competitively inhibited the colonization of A. salmonicida in the intestine. In addition, it was able to improve the growth performance of rainbow trout within a 21-day feeding experiment. The supplementation of GX118 increased the diversity of gut microbiota, in which the abundance of Bacteroidota, Blautia, and Rhodobacteraceae increased. In addition, the use of GX118 activated the expression of IFN-γ and NF-κB genes and reduced the expression level of IL-6 and IL-8, thus exhibiting a certain effect on activating the immunity of rainbow trout. This study provides a scientific basis for the development of antibacterial probiotics in the healthy farming of rainbow trout. [ABSTRACT FROM AUTHOR]

The Aeromonas salmonicida is responsible for causing furunculosis in various fish species. Furunculosis is a ubiquitous disease that affects the aquaculture industry and causes the mass mortality of turbot. Vibrio vulnificus is a pathogen that causes skin ulcers and hemorrhagic septicemia in fish, resulting in significant mortality in aquaculture. In this study, we have established a bivalent inactivated vaccine against A. salmonicida and V. vulnificus with Montanide™ ISA 763 AVG as an adjuvant. This bivalent inactivated vaccine was used to immunize turbot by intraperitoneal injection, and the relevant immune indexes were detected. The results demonstrate that the bivalent inactivated vaccine exhibited a relative percent survival (RPS) of 77% following A. salmonicida and V. vulnificus intraperitoneal challenge. The vaccinated group exhibited higher levels of acid phosphatase activity and lysozyme activity compared to the control group. ELISA results showed a significant increase in serum antibody levels in immunized turbot, which was positively correlated with immunity. In the kidney tissue, related immune genes (TLR5, CD4, MHCI and MHCII) were up-regulated significantly, showing that the vaccine can induce cellular and humoral immune responses in turbot. In conclusion, the bivalent inactivated vaccine against A. salmonicida and V. vulnificus was immunogenic, efficiently preventing turbot from infection, which has the potential to be applied in aquaculture. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida is an important pathogen that causes furunculosis in trout and salmon with high morbidity and mortality, resulting in significant economic losses in aquaculture. Overuse of antibiotics has led to the continuous emergence of drug-resistant strains. Hence, there is an urgent need to find an alternative environmentally friendly antimicrobial agent. In this study, we isolated a virulent phage of A. salmonicida, named ASG01, which belongs to the Myoviridae family and maintains lytic activity at a pH value range from 4 to 12 and in the temperature range from 30 °C to 60 °C. The whole genomic sequence of ASG01 showed 82% similarity to Aeromonas phage pAh6-C. The cell wall hydrolase (Cwh)-encoding gene from the genome of ASG01 was predicted and heterologously expressed. Notably, in the absence of additional phage genes, endogenous expression of Cwh could lyse E. coli cells and greatly inhibit the growth of tested fish pathogenic bacteria. The lytic activity of Cwh was eliminated when the predicted active site was mutated. These results indicate that Cwh of ASG01 possessed excellent lytic activity and a wide antibacterial spectrum, suggesting its potential as an effective enzybiotic. [ABSTRACT FROM AUTHOR]

Representatives of the bacterial genus Aeromonas are some of the most notorious aquaculture pathogens associated with a range of diseases in different fish species. As the world forges toward the post-antibiotic era, alternative options for combating bacterial pathogens are needed. One such alternative option is phage biocontrol. In this study, a novel podophage—JELG-KS1—infecting Aeromonas salmonicida was retrieved from wastewater along with its host strain. The genome of the JELG-KS1 phage is a 40,505 bp dsDNA molecule with a GC% of 53.42% and 185 bp direct terminal repeats and encodes 53 predicted proteins. Genomic analysis indicates that JELG-KS1 might represent a novel genus within the subfamily Studiervirinae. Podophage JELG-KS1 is a strictly lytic phage without any identifiable virulence or AMR genes that quickly adsorbs onto the surface of host cells to initiate a 48 min long infectious cycle, resulting in the release of 71 ± 12 JELG-KS1 progeny virions per infected cell. JELG-KS1 effectively lyses its host population in vitro, even at very low multiplicities of infection. However, when challenged against a panel of Aeromonas spp. strains associated with diseases in aquaculture, JELG-KS1 shows host-specificity that is confined only to its isolation strain, immediately compromising its potential for Aeromonas spp. biocontrol in aquaculture. [ABSTRACT FROM AUTHOR]

17α-ethinylestradiol (EE2), a synthetically derived analogue of endogenous estrogen, is widely employed as a hormonal contraceptive and is recognized as a highly hazardous emerging pollutant, causing acute and chronic toxic effects on both aquatic and terrestrial organisms. It has been included in the initial Water Watch List. The aim of this study was to isolate bacteria from consortia recovered from mine sediments and acid mine drainage samples, both considered extreme environments, with the ability to degrade EE2. From the most promising consortia, isolates affiliated with the Aeromonas, Rhizobium, and Paraburkholderia genera were obtained, demonstrating the capability of growing at 50 mg/L EE2. Subsequently, these isolates were tested with 9 mg/L of EE2 as the sole carbon source. Among the isolated strains, Aeromonas salmonicida MLN-TP7 exhibited the best performance, efficiently degrading EE2 (95 ± 8%) and reaching concentrations of this compound below the limits of detection within 7 and 9 days. The final metabolites obtained are in accordance with those of the TCA cycle; this may indicate EE2 mineralization. As far as is known, Aeromonas salmonicida was isolated for the first time and identified in acid mine drainage, demonstrating its capacity to degrade EE2, making it a promising candidate for bioaugmentation and suggesting its possible applicability in low pH environments. [ABSTRACT FROM AUTHOR]

Given the dual roles in immune function and metabolism, the liver emerge as a compelling candidate to bridge host defense and metabolic adjustments during pathogen infections in teleosts. In order to dissect the roles of the liver in the immune response of Sebastes schlegelii, we conducted an investigation into the activities of SOD, CAT and GPX4, performed a systematic analysis of circRNA, miRNA and mRNA expression profiles, as well as constructed circRNA-miRNA-mRNA regulatory networks in the liver of S. schlegelii following Aeromonas salmonicida infection were performed in the present study. The present results demonstrated the content of SOD, CAT and GPX4 increased significantly at early infection stage to protect the liver tissue from excessive damage. Additionally, 622 circRNA-miRNAs pairs, 78 miRNA-mRNA pairs and 327 circRNA-miRNA-mRNA pairs were identified in our study. These differently expressed circRNAs and miRNAs were found to be associated with the regulation of LMNB1, DMBT1, NAMPT, IFIT1, CELSRs, PYGL. GO and KEGG enrichment analysis revealed that these differently expressed genes were involved in TLR signal pathway, RLRs signal pathway, PPAR signal pathway, shedding light on antibacterial ceRNA networks within the liver of S. schlegelii post A. hydrophila post infection, which provides new insights into the immune mechanisms of teleost.

The diversity of O-polysaccharides (O-antigens) among 28 Aeromonas salmonicida strains isolated from ill fish has been determined by using high-resolution magic angle spinning (HR MAS) NMR spectroscopy. The new O-polysaccharide has been identified in two isolates. This new structure was investigated by 1H and 13C NMR spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The following structure of the linear hexasaccharide repeating unit of A. salmonicida O-antigen has been established: →3)-α-L-Rhap-(1→3)-α-D-ManpNAc-(1→2)-β-D-Glcp-(1→3)-α-L-Rhap2OAc4OAc-(1→3)-β-D-ManpNAc-(1→3)-α-D-Glcp-(1→. This new A. salmonicida O-polysaccharide was detected among two isolates collected from trout and turbot fish in 2010 and 2011, respectively. Further investigations should be conducted to evaluate the distribution of this new O-polysaccharide among a larger collection of isolates, depending on their geographic origin, the species of fish, and the health status of the fish.

Furunculosis, caused by Aeromonas salmonicida, poses a significant threat to both salmonid and non-salmonid fish in diverse aquatic environments. This study explores the genomic intricacies of re-emergent A. salmonicida outbreaks in Atlantic salmon (Salmo salar). Previous clinical cases have exhibited pathological characteristics, such as periorbital hemorrhages and gastrointestinal abnormalities. Genomic sequencing of three Chilean isolates (ASA04, ASA05, and CIBA_5017) and 25 previously described genomes determined the pan-genome, phylogenomics, insertion sequences, and restriction-modification systems. Unique gene families have contributed to an improved understanding of the psychrophilic and mesophilic clades, while phylogenomic analysis has been used to identify mesophilic and psychrophilic strains, thereby further differentiating between typical and atypical psychrophilic isolates. Diverse insertion sequences and restriction-modification patterns have highlighted genomic structural differences, and virulence factor predictions can emphasize exotoxin disparities, especially between psychrophilic and mesophilic strains. Thus, a novel plasmid was characterized which emphasized the role of plasmids in virulence and antibiotic resistance. The analysis of antibiotic resistance factors revealed resistance against various drug classes in Chilean strains. Overall, this study elucidates the genomic dynamics of re-emergent A. salmonicida and provides novel insights into their virulence, antibiotic resistance, and population structure. [ABSTRACT FROM AUTHOR]

Sustainable ecosystem is of essence to the river for its survival and maintenance of ecological balance. Gomti river, a groundwater-fed tributary of the Holy Ganges, is the lifeline of the middle Indo-Gangetic Plains. Screening of river pollutants for river cleanup is a challenging task. To this end, 20 km Lucknow stretch of Gomti was sampled at 5 locations for physicochemical–bacteriological analysis. Collected samples were analyzed for pH, TA, turbidity, DO, BOD, TH, COD, TDS, fecal coliform, and E. coli. Analytical instruments AAS and GC-ECD were used for heavy metal and organochlorine pesticide analysis. DO was 3.2 ± 0.3 to 7.4 ± 0.4 (avg 5 ± 1.8) mg/l, BOD was 10.6 ± 0.5 to 41 ± 1.7 (avg 22.9 ± 13.3) mg/l, while COD was 26.6 ± 4.6 to 93.5 ± 4.6 (avg 56 ± 31.3) mg/l. WQI values gradually increased from S1 (56.7) to S5 (181.8) which denotes a significant deterioration of water quality downstream of the river. Cd, Mn, Fe, Pb, and Cr had increment of 64, 18, 12, 6, and 2 fold, respectively, as compared to individual BIS:10500 standards. Among the OCPs, concentrations were in the order: aldrin > pp-DDT > β-HCH > γ-HCH > α-endosulfan > pp-DDE > α-HCH. Statistically strong positive correlation (r > 0.95 at 0.01) was observed among physicochemical parameters. Furthermore, bacterial species Aeromonas salmonicida subsp. Achromogenes, Aeromonas salmonicida strain A527, Aeromonas encheleia strain NCTC12917, and Aeromonas salmonicida strain ATCC33658 were identified by 16S rRNA gene amplification and molecular phylogenetic analysis. The study indicated heavy contamination of the river with organic and inorganic pollutants owing to mixing of untreated sewage, industrial effluents, and associated toxicants along with pathogenic bacteria which are a barrier to the harmony of aquatic life. [ABSTRACT FROM AUTHOR]

Rainbow trout, one of the most economically important aquaculture fish species worldwide, is affected by the pathogenic bacteria A. salmonicida, which causes furunculosis outbreaks, leading to huge economic losses. In this study, an oligonucleotide microarray was applied to identify transcriptional changes in the skin of rainbow trout individuals in response to a bacterial infection. Overall, 656 and 434 differentially expressed genes (DEGs) were identified at 2 and 6 days after a bacterial challenge (dpi), respectively. A comparison of moribund (2 dpi) and survivor fish (6 dpi) revealed 169 DEGs. Between these were many genes involved in immune response, including lysozymes, pattern recognition receptors (c-type lectins), antimicrobial peptides (cathelicidin and hepcidin), acute-phase proteins (serum amyloids and haptoglobin), complement cascade proteins (c3, c4, c6 and c7), interleukins (il11 and il1b) and chemokines (ccl19 and cxcl8). Alterations of leptin, eicosanoids and prostaglandins have been found, which suggest metabolic remodeling in conjunction with immune response. Further, the regulation of programmed cell death genes (caspase 8, bcl2 apoptosis regulator, nfkb inhibitor alpha and heme oxygenase) and structural proteins (collagens, myosins, keratins and metalloproteinases) was observed. This study provides, for the first time, a gene expression analysis of rainbow trout skin in response to A. salmonicida infection, revealing the complexity of defense strategies in response to furunculosis. [ABSTRACT FROM AUTHOR]

The plasmid known as pAsa5 is present in Aeromonas salmonicida subsp. salmonicida, a fish pathogen. The pAsa5 plasmid carries genes that are essential for the bacterium's virulence. Recombination events are known to occur in pAsa5, resulting in the loss of certain segments or the acquisition of additional genetic elements. For example, the transposon carried by the large pAsa8 plasmid was found to be inserted into the pAsa5 plasmid in the SHY16-3432 strain, enabling the addition of antibiotic resistance genes to this plasmid, which does not normally possess any. In this study, we present the isolation of additional strains carrying pAsa8. Further analyses of these strains revealed that a fusion between pAsa5 and the complete version of pAsa8 is possible. The pAsa8 transposon insertion in pAsa5 seen in the SHY16-3432 strain appears to be an aberrant event compared to the fusion of the two full-length plasmids. A 22-nucleotide sequence, present in both plasmids, serves as the site for the fusion of the two plasmids. Moreover, it is possible to introduce pAsa8 through conjugation into naive strains of A. salmonicida subsp. salmonicida and once the plasmid is within a new strain, the fusion with pAsa5 is detectable. This study reveals a previously unexplored aspect of pAsa5 plasmid biology, highlighting an additional risk for the spread of antibiotic resistance genes in A. salmonicida subsp. salmonicida. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida is the causative agent of septicemia in fish, and it is associated with significant economic losses in the aquaculture industry. While piscine Aeromonas infections are mainly treated with antibiotics, the emergence of resistance in bacterial populations requires the development of alternative methods of treatment. The use of phages can be one of them. A novel A. salmonicida jumbo phage, AerS_266, was isolated and characterized. This phage infects only mesophilic A. salmonicida strains and demonstrates a slow lytic life cycle. Its genome contains 243,674 bp and 253 putative genes: 84 encode proteins with predicted functions, and 3 correspond to tRNAs. Genes encoding two multisubunit RNA polymerases, chimallin and PhuZ, were identified, and AerS_266 was thus defined as a phiKZ-like phage. While similar phages with genomes >200 kb specific to Aeromonas hydrophila and Aeromonas veronii have been previously described, AerS_266 is the first phiKZ-like phage found to infect A. salmonicida. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hsp40–Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein–protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40–Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

Aeromonas salmonicida is an ancient fish pathogen. Lysozymes are important molecules in the innate immune system that fight bacterial infections. The expression characteristics of C-type lysozyme in crucian carp infected with A. salmonicida and its antibacterial effect against A. salmonicida had not been investigated. Thus, we used bioinformatics to analyze the gene and protein characteristics of C-type lysozymes in crucian carp. Changes in C-type lysozyme expression before and after crucian carp infection with A. salmonicida were detected, and the in vitro antibacterial effect of recombinant carp C-type lysozyme on A. salmonicida was validated. The results showed that the coding DNA sequence region of the lysozyme gene sequence was 438 bp long, encoding 145 amino acids and containing two conserved catalytic sites: Glu53 and Asp69. Phylogenetic analysis revealed that crucian carp C-type lysozymes clustered with Cyprinus carpio lysozyme C. After crucian carp were infected with A. salmonicida, the gene and protein expression of C-type lysozymes in the liver, spleen, kidney, and hindgut were significantly upregulated, with the liver showing the highest upregulation that was 15 times higher than that in the uninfected group. In addition, recombinant C-type lysozyme exhibited significant antibacterial activity against A. salmonicida, with an average inhibition zone radius of 0.92 cm when using 40 μg recombinant lysozyme. In conclusion, this study reveals the important role of C-type lysozymes in the innate immune response of crucian carp and provides a theoretical basis for preventing crucian carp infection with A. salmonicida.

Simple Summary: Aeromonas salmonicida represents an important cause of mortality in a broad diversity of fish species, in which it has been extensively reported. Nevertheless, aeromoniasis in sturgeons is far from being fully understood. Four juvenile sturgeons from a mortality outbreak in a farm located in the north of Spain were submitted to the VISAVET Health Surveillance Centre. Aeromonas salmonicida was isolated from the bacteriological culture. They were found to suffer from a hyperacute form of the disease (almost no clinical signs and non-specific gross or histological lesions), thus confirming the susceptibility of sturgeons to this pathogen. The findings described here hope to provide a better understanding of this potentially fatal disease in the industries of aquaculture and caviar. Aeromonas salmonicida is one of the major threats to world aquaculture, causing fish furunculosis and high mortality rates in cultured fish, particularly salmonids. Although Aeromonas spp. is a thoroughly studied pathogen, little is known regarding aeromoniasis in sturgeons. After a mortality outbreak, four juvenile sturgeons (Acipenser baerii) were submitted for autopsy and tissue samples were collected for histopathological and microbiological studies. The external examination revealed size heterogenicity, skin hyperpigmentation and reduced body condition of sturgeons. Within the abdominal cavity, mild hepatomegaly and splenomegaly were observed, as well as generalized organic congestion. Histology revealed severe multifocal haemorrhagic and ulcerative dermatitis, mainly localized in the dorsal and latero-ventral areas of fish. The histological study also showed moderate to severe inflammation of gills and organic lesions compatible with septicaemia. Bacterial isolates were identified as Aeromonas salmonicida subsp. salmonicida using MALDI-TOF MS and PCR. Overall, the lesions first described here are consistent with those previously reported in other cultured fish species and contribute to a better understanding of the pathogenesis of Aeromonas salmonicida subsp. salmonicida in the Siberian sturgeon, aside from providing new diagnostic tools for bacterial diseases impacting the fast-growing industry of caviar. [ABSTRACT FROM AUTHOR]

Turbot aquaculture production is an important economic activity in several countries around the world; nonetheless, the incidence of diseases, such furunculosis, caused by the etiological agent A. salmonicida subsp. salmonicida, is responsible for important losses to this industry worldwide. Given this perspective, this study aimed to evaluate early immune responses in turbot (S. maximus L.) following infection with A. salmonicida subsp. salmonicida. For this, 72 fish were individually weighed and randomly distributed into 6 tanks in a circulating seawater system. For the bacterial challenge, half of the individuals (3 tanks with 36 individuals) were infected using a peritoneal injection with the bacterial suspension, while the other half of individuals were injected with PBS and kept as a control group. Several factors linked to the innate immune response were studied, including not only haematological (white blood cells, red blood cells, haematocrit, haemoglobin, mean corpuscular volume, mean cell haemoglobin, mean corpuscular haemoglobin concentration, neutrophils, monocytes, lymphocytes, thrombocytes) and oxidative stress parameters, but also the analyses of the expression of 13 key immune-related genes (tnf-α, il-1β, il-8, pparα-1, acox1, tgf-β1, nf-kB p65, srebp-1, il-10, c3, cpt1a, pcna, il-22). No significant differences were recorded in blood or innate humoral parameters (lysozyme, anti-protease, peroxidase) at the selected sampling points. There was neither any evidence of significant changes in the activity levels of the oxidative stress indicators (catalase, glutathione S-transferase, lipid peroxidation, superoxide dismutase). In contrast, pro-inflammatory (tnf-α, il-1β), anti-inflammatory (il-10), and innate immune-related genes (c3) were up-regulated, while another gene linked with the lipid metabolism (acox1) was down-regulated. The results showed new insights about early responses of turbot following infection with A. salmonicida subsp. salmonicida. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria Ligilactobacillus salivarius has been shown to be a substitute for antibiotics in the treatment of bacterial disease in high animals. However, its beneficial mechanism in fish farming is still unclear. This study evaluated the antagonistic effects of the Ligilactobacillus salivarius GX118 strain on Aeromonas salmonicida and its regulation of rainbow trout growth in vivo and in vitro. The results found that GX118 produces an antibacterial substance that can directly destroy the cell wall of A. salmonicida. Whole-genome sequencing of GX118 revealed that Enterolysin A is a type III bacteriocin with antibacterial properties. An in vivo experiment showed that the supplementation of GX118 in diet competitively inhibited the colonization of A. salmonicida in the intestine. In addition, it was able to improve the growth performance of rainbow trout within a 21-day feeding experiment. The supplementation of GX118 increased the diversity of gut microbiota, in which the abundance of Bacteroidota, Blautia, and Rhodobacteraceae increased. In addition, the use of GX118 activated the expression of IFN-γ and NF-κB genes and reduced the expression level of IL-6 and IL-8, thus exhibiting a certain effect on activating the immunity of rainbow trout. This study provides a scientific basis for the development of antibacterial probiotics in the healthy farming of rainbow trout.

The Aeromonas salmonicida is responsible for causing furunculosis in various fish species. Furunculosis is a ubiquitous disease that affects the aquaculture industry and causes the mass mortality of turbot. Vibrio vulnificus is a pathogen that causes skin ulcers and hemorrhagic septicemia in fish, resulting in significant mortality in aquaculture. In this study, we have established a bivalent inactivated vaccine against A. salmonicida and V. vulnificus with Montanide™ ISA 763 AVG as an adjuvant. This bivalent inactivated vaccine was used to immunize turbot by intraperitoneal injection, and the relevant immune indexes were detected. The results demonstrate that the bivalent inactivated vaccine exhibited a relative percent survival (RPS) of 77% following A. salmonicida and V. vulnificus intraperitoneal challenge. The vaccinated group exhibited higher levels of acid phosphatase activity and lysozyme activity compared to the control group. ELISA results showed a significant increase in serum antibody levels in immunized turbot, which was positively correlated with immunity. In the kidney tissue, related immune genes (TLR5, CD4, MHCI and MHCII) were up-regulated significantly, showing that the vaccine can induce cellular and humoral immune responses in turbot. In conclusion, the bivalent inactivated vaccine against A. salmonicida and V. vulnificus was immunogenic, efficiently preventing turbot from infection, which has the potential to be applied in aquaculture.

Representatives of the bacterial genus Aeromonas are some of the most notorious aquaculture pathogens associated with a range of diseases in different fish species. As the world forges toward the post-antibiotic era, alternative options for combating bacterial pathogens are needed. One such alternative option is phage biocontrol. In this study, a novel podophage—JELG-KS1—infecting Aeromonas salmonicida was retrieved from wastewater along with its host strain. The genome of the JELG-KS1 phage is a 40,505 bp dsDNA molecule with a GC% of 53.42% and 185 bp direct terminal repeats and encodes 53 predicted proteins. Genomic analysis indicates that JELG-KS1 might represent a novel genus within the subfamily Studiervirinae. Podophage JELG-KS1 is a strictly lytic phage without any identifiable virulence or AMR genes that quickly adsorbs onto the surface of host cells to initiate a 48 min long infectious cycle, resulting in the release of 71 ± 12 JELG-KS1 progeny virions per infected cell. JELG-KS1 effectively lyses its host population in vitro, even at very low multiplicities of infection. However, when challenged against a panel of Aeromonas spp. strains associated with diseases in aquaculture, JELG-KS1 shows host-specificity that is confined only to its isolation strain, immediately compromising its potential for Aeromonas spp. biocontrol in aquaculture.

Aeromonas salmonicida is a conditional pathogen and abundant in water environment, which causes infection in various aquatic animals. However, there are few reports on A. salmonicida infections in prawns. In this study, the pathogenic A. salmonicida CS3 was first isolated from diseased Macrobrachium nipponense. Obvious severe lesions in diseased M. nipponense were observed by histopathological analysis. Gill filament cells were obviously enlarged, the tissue boundaries were incomplete, hepatopancreas vacuoles were ruptured, and basal lamina was damaged. The infection test confirmed that A. salmonicida CS3 had high virulence, and the LD50 was 4.43 × 106 CFU/mL at 7 d post infection. Furthermore, detection of virulence-related genes showed that A. salmonicida CS3 was positive for alt, dna, sp, aer, lip, act, and hly. The antibacterial tests demonstrated that B. velezensis CPA1–1 exhibited antibacterial activity against A. salmonicida. Following the challenge with A. salmonicida, the survival rate of M. nipponense was significantly higher in B. velezensis treated group compared to the control group, and the mRNA expression of CDSP, gC1qR, relish, MyD88, hemocyanin, and α2M were increased to varying degrees. This study indicated that A. salmonicida was a pathogen causing the mass mortalities in M. nipponense, and B. velezensis CPA1–1 holds promise as a potential probiotic for preventing A. salmonicida infections in M. nipponense.

The Gram-negative bacterium A. salmonicida is the causal agent of furunculosis and used to be one of the most loss-causing bacterial infections in the salmonid aquaculture industry with a mortality rate of about 90% until the 1990s, when an inactivated vaccine with mineral oil as adjuvant was successfully implemented to control the disease. However, the use of this vaccine is associated with inflammatory side effects in the peritoneal cavity as well as autoimmune reactions in Atlantic salmon, and incomplete protection has been reported in rainbow trout. We here aimed at developing and testing a recombinant alternative vaccine based on virus-like particles (VLPs) decorated with VapA, the key structural surface protein in the outer A-layer of A. salmonicida. The VLP carrier was based on either the capsid protein of a fish nodavirus, namely red grouper nervous necrotic virus (RGNNV) or the capsid protein of Acinetobacter phage AP205. The VapA and capsid proteins were expressed individually in E. coli and VapA was fused to auto-assembled VLPs using the SpyTag/SpyCatcher technology. Rainbow trout were vaccinated/immunized with the VapA-VLP vaccines by intraperitoneal injection and were challenged with A. salmonicida 7 weeks later. The VLP vaccines provided protection comparable to that of a bacterin-based vaccine and antibody response analysis demonstrated that vaccinated fish mounted a strong VapA-specific antibody response. To our knowledge, this is the first demonstration of the potential use of antigendecorated VLPs for vaccination against a bacterial disease in salmonids. [ABSTRACT FROM AUTHOR]

Aeromonas salmonicida subsp. salmonicida (A. salmonicida), a Gram-negative bacterium causing furunculosis in fish, produces the siderophores acinetobactin and amonabactins in order to extract iron from its hosts. While the synthesis and transport of both systems is well understood, the regulation pathways and conditions necessary for the production of each one of these siderophores are not clear. The acinetobactin gene cluster carries a gene (asbI) encoding a putative sigma factor belonging to group 4 σ factors, or, the ExtraCytoplasmic Function (ECF) group. By generating a null asbI mutant, we demonstrate that AsbI is a key regulator that controls acinetobactin acquisition in A. salmonicida, since it directly regulates the expression of the outer membrane transporter gene and other genes necessary for Fe-acinetobactin transport. Furthermore, AsbI regulatory functions are interconnected with other iron-dependent regulators, such as the Fur protein, as well as with other sigma factors in a complex regulatory network. [ABSTRACT FROM AUTHOR]

This study aimed to identify histamine-forming bacteria (HFB) and the sources of introduction of such bacteria to recommend control measures to mitigate histamine formation in yellowfin tuna (YFT). Field samples were collected from multi-day boats that landed at Dikkowita, Negombo, Trincomalee and Dondra fishery harbours. Ice from the fish holds (n=63) and chill transport vehicles (n=63), and swabs from the fish holds (n=63), the boat decks (n=63) and the skin of YFT (n=63) were collected. Fish loin samples (n=15), ice samples (n=36) and swabs from the skin of YFT (n=18), floor (n=18) and chill transport vehicles (n=18) were collected from fish processing plants. Presumptive HFB isolated from Nivens medium and Violet Red Bile Glucose (VRBG) agar were screened for histamine forming ability in Tripticase soy broth (TSB) supplemented with 1.0% L-histidine. HFB isolates were characterized by sequencing approximately 1400 bp of the 16S rDNA. Seven isolates that produced histamine in the range of 3000--4000 ppm in TSB isolated from ice samples, and a swab sample collected from the boat deck, were confirmed as Klebsiella aerogens (n=6) and Morganella morganii (n=1) respectively. Hafnia alvei (n=1), Serratia sp. (n=2), Citrobacter freundii (n=1), Rahnella sp. (n=1) and Aeromonas salmonicida (n=8) were also among the isolated histamine forming bacteria. Pseudomonas sp. (n=24) and Shewanella baltica (n=7), which are known as spoilage bacteria were also isolated and showed weak histamine formation. Hence, it is evident that histamineforming bacteria could be introduced into the fish from ice and contacting surfaces. This necessitates the practice of rigorous cleaning procedures and adaptation of proper postharvest handling procedures to minimize contamination of the fish. [ABSTRACT FROM AUTHOR]

All the 36 known species to date of the genus Aeromonas are mesophilic except the species Aeromonas salmonicida, which includes both psychrophilic and mesophilic subspecies. For 20 years, more and more mesophilic A. salmonicida strains have been discovered. Only A. salmonicida subsp. pectinolytica has officially been classified as a mesophilic subspecies. Most mesophiles have been isolated in hot countries. We present, for the first time, the characterization of two new mesophilic isolates from Quebec (Canada). Phenotypic and genomic characterizations were carried out on these strains, isolated from dead fish from a fish farm. Isolates 19-K304 and 19-K308 are clearly mesophiles, virulent to the amoeba Dictyostelium discoideum, a surrogate host, and close to strain Y577, isolated in India. To our knowledge, this is the first time that mesophilic strains isolated from different countries are so similar. The major difference between the isolates is the presence of plasmid pY47-3, a cryptic plasmid that sometimes presents in mesophilic strains. More importantly, our extensive phylogenetic analysis reveals two well-defined clades of mesophilic strains with psychrophiles associated with one of these clades. This helps to have a better understanding of the evolution of this species and the apparition of psychrophilic subspecies. [ABSTRACT FROM AUTHOR]

Fish culture accounts for about 40 percent of the total protein intake of households in Nigeria. However, disease outbreaks have negatively affected the growth of the aquaculture subsector in the absence of measures targeted at effective disease prevention and control. Between the 9th and 14th of March 2023, a suspected outbreak of furunculosis in cultured Clarias gariepinus was reported at a backyard fishpond in Ebonyi State, Nigeria. Clinical symptoms seen on affected fishes were furuncles on the skin, bleeding sores (ulcers) on the dorsal, anal fins, mouth of the fish, and exophthalmos. A retrospective and prospective disease investigation was conducted by reviewing case history, sources of fish, hatchery records, stocking density, water quality, sources of breeding stock and treatment regimen. The samples collected were whole fish (fresh carcasses) packaged in a Giostyle and a sample of pond water for laboratory analysis. Mortality was 2%, P.H of pond water was 7.3 (6.5-9) dissolved oxygen (D.O) in pond water was 8.2 ppm (5-20 ppm). The farmer introduced new stock of catfish into the pond from an adjoining farm without proper quarantine measures. This apparently healthy stock was suspected to be the source of the primary infection. Tissue samples from fish tested positive by fluorescent antibody test (FAT) for Aeromonas species and gave an apple green sheen on microscopy. We recommend that fish farmers should ensure proper quarantine of new stock before introducing the same into their fishponds. Prophylaxis with tetracycline may reduce the chances of disease outbreaks. Ensure pond hygiene and eliminate water lice, which could serve as intermediate hosts for Aeromonas salmonicida in the pond. [ABSTRACT FROM AUTHOR]

ABSTRACTCarbohydrates can both protect against infection and act as targets promoting infection. Mucins are major components of the slimy mucus layer covering the fish epithelia. Mucins can act as decoys for intimate pathogen interaction with the host afforded by binding to glycosphingolipids in the host cell membrane. We isolated and characterized glycosphingolipids from Atlantic salmon skin, gill, stomach, pyloric caeca, and intestine. We characterized the glycosphingolipids using liquid chromatography – mass spectrometry and tandem mass spectrometry and the glycan repertoire was compared with the glycan repertoire of mucins from the same epithelia. We also investigated Aeromonas salmonicida binding using chromatogram and microtiter well based binding assays. We identified 29 glycosphingolipids. All detected acid glycans were of the ganglio-series (unless shorter) and showed a high degree of polysialylation. The non-acid glycans were mostly composed of the neolacto, globo, and ganglio core structures. The glycosphingolipid repertoire differed between epithelia and the proportion of the terminal moieties of the glycosphingolipids did not reflect the terminal moieties on the mucins from the same epithelia. A. salmonicida did not bind the Atlantic salmon glycosphingolipids. Instead, we identified that A. salmonicida binding to sialic acid occurred to α2–6 Neu5Ac but not to α2–3 Neu5Ac. α2–6 Neu5Ac was present on mucins whereas mainly α2–3 Neu5Ac was found on the glycosphingolipids, explaining the difference in A. salmonicida binding ability between these host glycoconjugates. A. salmonicida´s ability to bind to Atlantic salmon mucins, but not the glycosphingolipids, is likely part of the host defence against this pathogen.

Oranda goldfish is an ornamental fish that developed worldwide. This industry faces serious problems such as Aeromonas salmonicida infection (furunculosis). Alternative therapy is required to be developed. One of them is using desert rose (Adenium obesum (Forssk.) Roem. & Schult.). This study aimed to elucidate the potency of desert rose flower extract (DRFE) against artificially induced furunculosis in oranda goldfish. The desert rose was extracted and its quercetin content was analyzed. Sixty oranda goldfish were used as a model. Fish were separated into I (healthy); II (infected); III (infected + 55 ppm ciprofloxacin); IV (infected + 1,000 ppm DRFE); V (infected + 2,000 ppm DRFE); VI (infected + 4,000 ppm DRFE). The therapy was given 5-days. On day six, the fish's blood and organs were collected. The samples were tested against haematology, histopathology, and immunohistochemistry. The data were analyzed by SPSS26. The results showed that DRFE contained quercetin (5.32±0.18%). The DRFE has potential benefits as healing promoters against furunculosis in oranda goldfish. It is indicated by significant differences in parameters: erythrocytes, haemoglobin, PCV, erythrocyte indices, leucocytes, differential leucocytes, histopathology, serology, and immune-expression of CD4+, CD8+, and ratio CD4+/CD8+ compared to the infected group (P=0.05). The most significant concentration of DRFE was 4,000 ppm and it has no differences compared to 55 ppm ciprofloxacin (P=0.05). The DRFE can be used as the alternative treatment against artificially induced furunculosis in oranda goldfish and it must be explored further against another type of bacterial infection. [ABSTRACT FROM AUTHOR]

Widespread and inappropriate use of antibiotics has been shown to increase the spread of antibiotics and antimicrobial resistance genes (ARGs) in aquatic environments and organisms. Antibiotic use for the treatment of human and animal diseases is increasing continuously globally. However, the effects of legal antibiotic concentrations on benthic consumers in freshwater environments remain unclear. In the present study, we tested the growth response of Bellamya aeruginosa to florfenicol (FF) for 84 days under high and low concentrations of sediment organic matter (carbon [C] and nitrogen [N]). We characterized FF and sediment organic matter impact on the bacterial community, ARGs, and metabolic pathways in the intestine using metagenomic sequencing and analysis. The high concentrations of organic matter in the sediment impacted the growth, intestinal bacterial community, intestinal ARGs, and microbiome metabolic pathways of B. aeruginosa. B. aeruginosa growth increased significantly following exposure to high organic matter content sediment. Proteobacteria, at the phylum level, and Aeromonas at the genus level, were enriched in the intestines. In particular, fragments of four opportunistic pathogens enriched in the intestine of high organic matter content sediment groups, Aeromonas hydrophila, Aeromonas caviae, Aeromonas veronii, and Aeromonas salmonicida, carried 14 ARGs. The metabolic pathways of the B. aeruginosa intestine microbiome were activated and showed a significant positive correlation with sediment organic matter concentrations. In addition, genetic information processing and metabolic functions may be inhibited by the combined exposure to sediment C, N, and FF. The findings of the present study suggest that antibiotic resistance dissemination from benthic animals to the upper trophic levels in freshwater lakes should be studied further. [ABSTRACT FROM AUTHOR]

The use of antibiotics in aquaculture leads to the proliferation of multidrug-resistant bacteria, and an urgent need for developing new alternatives to prevent and control disease has, thus, arisen. In this scenario, postbiotics represent a promising tool to achieve this purpose; thus, in this study, isolation and selection of bacteria to further produce and evaluate their postbiotics antibacterial activity against fish pathogens was executed. In this respect, bacterial isolates from rainbow trout and Nile tilapia were obtained and tested in vitro against Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida. From 369 obtained isolates, 69 were selected after initial evaluation. Afterwards, additional screening was carried out by spot-on-lawn assay to finally select twelve isolates; four were identified as Pediococcus acidilactici, seven as Weissella cibaria, and one as Weissella paramesenteroides by matrix assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS). Selected bacteria were used to obtain postbiotic products to test their antagonistic activity through coculture challenge and broth microdilution assays. The influence of incubation time prior to postbiotic production on antagonistic behavior was also recorded. Two isolates identified as W. cibaria were able to significantly reduce (p < 0.05) A. salmonicida subsp. salmonicida's growth in the coculture challenge up to 4.49 ± 0.05 Log CFU/mL, and even though the reduction in Y. ruckeri was not as effective, some inhibition on the pathogen's growth was reported; at the same time, most of the postbiotic products obtained showed more antibacterial activity when obtained from broth cultures incubated for 72 h. Based on the results obtained, the preliminary identification of the isolates that expressed the highest inhibitory activity was confirmed by partial sequencing as W. cibaria. Through our study, it can be concluded that postbiotics produced by these strains are useful to inhibit the growth of the pathogens and could, thereby, be applicable in further research to develop suitable tools as feed additives for disease control and prevention in aquaculture. [ABSTRACT FROM AUTHOR]

Environmental stressors can disrupt the relationship between the microbiota and the host and lead to the loss of its functions. Among them, bacterial infection caused by Aeromonas salmonicida, the causative agent of furunculosis, results in high mortality in salmonid aquaculture. Here, rainbow trout were exposed to A. salmonicida achromogenes and its effects on the taxonomic composition and structure of the microbiota was assessed on different epithelia (gills, skin, and caudal fin) at 6 and 72 h post-infection (hpi) using the V1–V3 region of the 16S rRNA sequencing. Moreover, the infection by the pathogen and immune gene responses were evaluated in the head kidney by qPCR. Our results suggested that α-diversity was highly diverse but predominated by a few taxa while β-diversity was affected very early by infection in the gills after 6 h, subsequently affecting the microbiota of the skin and caudal fin. A dysbiosis of the microbiota and an increase in genera known to be opportunistic pathogens (Aeromonas, Pseudomonas) were also identified. Furthermore, an increase in pro-inflammatory cytokines and virulence protein array (vapa) was observed in trout head kidney as soon as 6 hpi and remained elevated until 72 hpi, while the anti-inflammatory genes seemed repressed. This study suggests that the infection by A. salmonicida achromogenes can alter fish microbiota of gills in the few hours post-infection. This result can be useful to develop a non-invasive technique to prevent disease outbreak in aquaculture. [ABSTRACT FROM AUTHOR]

Plasmids that carry antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen with severe consequences in salmonid farming. Here, we describe a 67 kb plasmid found in the A. salmonicida subsp. salmonicida Strain SHY15-2939 from Quebec, Canada. This new plasmid, named pAsa-2939 and identified by high throughput sequencing, displays features never found before in this bacterial species. It contains a transposon related to the Tn21 family, but with an unusual organization. This transposon bears a catB3 gene (chloramphenicol resistance) that has not been detected yet in A. salmonicida subsp. salmonicida. The plasmid is transferable by conjugation into Aeromonas hydrophila, but not into Escherichia coli. Based on PCR analysis and genomic sequencing (Illumina and PacBio), we determined that the transposon is unstable in A. salmonicida subsp. salmonicida Strain SHY15-2939, but it is stable in A. hydrophila trans-conjugants, which explains the chloramphenicol resistance variability observed in SHY15-2939. These results suggest that this bacterium is likely not the most appropriate host for this plasmid. The presence of pAsa-2939 in A. salmonicida subsp. salmonicida also strengthens the reservoir role of this bacterium for antibiotic resistance genes, even those that resist antibiotics not used in aquaculture in Québec, such as chloramphenicol. [ABSTRACT FROM AUTHOR]

Acipenser baerii (Siberian sturgeon) is native to Kazakhstan and is currently endangered and is listed within the first class of protected animals. Sturgeon aquaculture is becoming an important tool for the recovery of this endangered species. Nonetheless, diseases involving typical symptoms of skin ulceration and systemic bacterial hemorrhagic septicemia have occurred in cultured A. baerii on a fish farm located in Western Kazakhstan. In this study, an infectious strain of bacteria isolated from an ulcer of diseased A. baerii was identified as Aeromonas salmonicida (strain AB001). This identification involved analyses of 16S rRNA, gyrB, rpoD, and flaA genes’ sequences. Even though strain AB001 belongs to A. salmonicida, it exhibited noticeable mobility and growth at temperatures of ≥37 °C. Profiling of virulence genes uncovered the presence of seven such genes related to pathogenicity. Antibiotic sensitivity testing showed that the strain is sensitive to aminoglycosides, amphenicols, nitrofurans, quinolones, and tetracyclines. Half-lethal doses (LD50) of strain AB001 for Oreochromis mossambicus and A. baerii were determined: respectively 1.7 × 108 and 7.2 × 107 colony-forming units per mL. The experimentally induced infection revealed that strain AB001 causes considerable histological lesions in O. mossambicus, including tissue degeneration, necrosis, and hemorrhages of varied severity.

Furunculosis, caused by Aeromonas salmonicida, poses a significant threat to both salmonid and non-salmonid fish in diverse aquatic environments. This study explores the genomic intricacies of re-emergent A. salmonicida outbreaks in Atlantic salmon (Salmo salar). Previous clinical cases have exhibited pathological characteristics, such as periorbital hemorrhages and gastrointestinal abnormalities. Genomic sequencing of three Chilean isolates (ASA04, ASA05, and CIBA_5017) and 25 previously described genomes determined the pan-genome, phylogenomics, insertion sequences, and restriction-modification systems. Unique gene families have contributed to an improved understanding of the psychrophilic and mesophilic clades, while phylogenomic analysis has been used to identify mesophilic and psychrophilic strains, thereby further differentiating between typical and atypical psychrophilic isolates. Diverse insertion sequences and restriction-modification patterns have highlighted genomic structural differences, and virulence factor predictions can emphasize exotoxin disparities, especially between psychrophilic and mesophilic strains. Thus, a novel plasmid was characterized which emphasized the role of plasmids in virulence and antibiotic resistance. The analysis of antibiotic resistance factors revealed resistance against various drug classes in Chilean strains. Overall, this study elucidates the genomic dynamics of re-emergent A. salmonicida and provides novel insights into their virulence, antibiotic resistance, and population structure.

Rainbow trout, one of the most economically important aquaculture fish species worldwide, is affected by the pathogenic bacteria A. salmonicida, which causes furunculosis outbreaks, leading to huge economic losses. In this study, an oligonucleotide microarray was applied to identify transcriptional changes in the skin of rainbow trout individuals in response to a bacterial infection. Overall, 656 and 434 differentially expressed genes (DEGs) were identified at 2 and 6 days after a bacterial challenge (dpi), respectively. A comparison of moribund (2 dpi) and survivor fish (6 dpi) revealed 169 DEGs. Between these were many genes involved in immune response, including lysozymes, pattern recognition receptors (c-type lectins), antimicrobial peptides (cathelicidin and hepcidin), acute-phase proteins (serum amyloids and haptoglobin), complement cascade proteins (c3, c4, c6 and c7), interleukins (il11 and il1b) and chemokines (ccl19 and cxcl8). Alterations of leptin, eicosanoids and prostaglandins have been found, which suggest metabolic remodeling in conjunction with immune response. Further, the regulation of programmed cell death genes (caspase 8, bcl2 apoptosis regulator, nfkb inhibitor alpha and heme oxygenase) and structural proteins (collagens, myosins, keratins and metalloproteinases) was observed. This study provides, for the first time, a gene expression analysis of rainbow trout skin in response to A. salmonicida infection, revealing the complexity of defense strategies in response to furunculosis.

Psychrophilic Aeromonas salmonicida could not grow above 25°C and therefore thought unable to infect mammals and humans. In our previous study, a mesophilic A. salmonicida SRW-OG1 was isolated from Epinephelus coioides with furunculosis. Through the analysis of preliminary RNA-seq, it was found that the Zn2+ uptake related genes znuA, znuB and znuC might be involved in the virulence regulation of A. salmonicida SRW-OG1. Therefore, the purpose of this study was to explore the effect of znuABC silencing on the virulence regulation of A. salmonicida SRW-OG1. The results showed that the growth of the znuA-RNAi, znuB-RNAi, and znuC-RNAi strains was severely restricted under the Fe2+ starvation, but surprisingly there was no significant difference under the Zn2+ restriction. In the absence of Zn2+ and Fe2+, the expression level of znuABC was significantly increased. The motility, biofilm formation, adhesion and hemolysis of the znuA-RNAi, znuB-RNAi, and znuC-RNAi strains were significantly reduced. We also detected the expression of znuABC under different growth periods, temperatures, pH, as well as Cu2+ and Pb2+ stresses. The results showed that znuABC was significantly up-regulated in the logarithmic phase and the decline phase of A. salmonicida. Interestingly, the trend of expression levels of the znuABC at 18, 28, and 37°C was reversed to another Zn2+ uptake related gene zupT. Taken together, these indicated that the znuABC was necessary for A. salmonicida SRW-OG1 pathogenicity and environmental adaptability, and was cross regulated by iron starvation, but it was not irreplaceable for A. salmonicida SRW-OG1 Zn2+ uptake in the host.

Turbot (Scophthalmus maximus) is an important commercial fish in China that can be infected by a series of bacterial pathogens, leading to great economic losses. In this study we focused on the epidemiology of turbot bacterial diseases in the major farming areas in China for three years. A total of 155 cases with 446 diseased turbots were investigated, and dominant bacterial pathogens were isolated from 137 cases (344 turbots). Thus, bacteria are the major threat to farming turbot in China. Edwardsiella piscicida was the major pathogen, which isolated as the dominant colony in 62 cases (40.00%) with 151 turbots (33.85%). Aeromonas salmonicida was isolated in 57 cases (36.77%) with 116 turbots (26.01%). Vibrio anguillarum was isolated in nine cases (5.81%), and Streptococcus parauberis in five cases (3.23%). Photobacterium damselae and Mycobacterium marinum were also isolated from one or two diseased fish. Other Vibrio spp. were isolated in 15 cases (9.68%). Two species of pathogen were isolated in 13 cases, and three species (E. piscicida, A. salmonicida, and S. parauberis) in one case. In 19 cases, no bacteria were isolated. Based on the annual disease analysis, we found that the E. piscicida infection proportion of total cases was greatly decreased, which may be caused by the attenuated vaccine inoculated in 2018. The antibiotic resistance of E. piscicida strains isolated in Weifang city was also determined. We found that the resistance to ceftriaxone, doxycycline, and SMZ/TMP were significantly increased from October 2016 to June 2018, and all the E. piscicida isolates exhibited resistance to SMZ/TMP in June 2018. These results indicated that E. piscicida is the major threat to turbot farming in China, and the attenuated E. piscicida vaccine exhibits effective protection. The usage of antibiotics may induce resistance quickly. Thus, development of vaccines is an important work for sustainable development of turbot farming in the future.

IntroductionDNA methylation was one of the most important modification in epigenetics and played an important role in immune response. Since the introduction of Scophthalmus maximus, the scale of breeding has continued to expand, during which diseases caused by various bacteria, viruses and parasites have become increasingly serious. Therefore, the inactivated vaccines have been widely researched and used in the field of aquatic products with its unique advantages. However, the immune mechanism that occurred in turbot after immunization with inactivated vaccine of Aeromonas salmonicida was not clear.MethodsIn this study, differentially methylated regions (DMRs) were screened by Whole Genome Bisulfite Sequencing (WGBS) and significantly differentially expressed genes (DEGs) were screened by Transcriptome sequencing. Double luciferase report assay and DNA pull-down assay were further verified the DNA methylation state of the gene promoter region affected genes transcriptional activity after immunization with inactivated vaccine of Aeromonas salmonicida.ResultsA total of 8149 differentially methylated regions (DMRs) were screened, in which there were many immune-related genes with altered DNA methylation status. Meanwhile, 386 significantly differentially expressed genes (DEGs) were identified, many of which were significantly enriched in Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and C-type lectin receptor signaling pathway. Combined analysis of WGBS results and RNA-seq results, a total of 9 DMRs of negatively regulated genes are located in the promoter region, including 2 hypermethylated genes with lower expression and 7 hypomethylated genes with higher expression. Then, two immune-related genes C5a anaphylatoxin chemotactic receptor 1-like (C5ar1-Like) and Eosinophil peroxidase-like (EPX-Like), were screened to explore the regulation mechanism of DNA methylation modification on their expression level. Moreover, the DNA methylation state of the gene promoter region affected genes transcriptional activity by inhibiting the binding of transcription factors, which lead to changes in the expression level of the gene.DiscussionWe jointly analyzed WGBS and RNA-seq results and revealed the immune mechanism that occurred in turbot after immunized with inactivated vaccine of A. salmonicida from the perspective of DNA methylation.

Aeromonas salmonicida, a psychrophilic bacterial pathogen, is widely distributed in marine freshwater, causing serious economic losses to major salmon farming areas in the world. At present, it is still one of the most important pathogens threatening salmon farming. Hcp (haemolysin-coregulated protein) is an effector protein in the type-VI secretion system (T6SS), which is secreted by T6SS and functions as its structural component. The results of our previous genomic sequencing showed that hcp existed in the mesophilic A. salmonicida SRW-OG1 isolated from naturally infected Epinephelus coioides. To further explore the role of Hcp in A. salmonicida SRW-OG1, we constructed an hcp-RNAi strain and verified its effect on the virulence of A. salmonicida. The results showed that compared with the wild strain, the hcp-RNAi strain suffered from different degrees of decreased adhesion, growth, biofilm formation, extracellular product secretion, and virulence. It was suggested that hcp may be an important virulence gene of A. salmonicida SRW-OG1. [ABSTRACT FROM AUTHOR]

Bacterial exotoxins with ADP-ribosyltransferase activity can be divided into distinct clades based on their domain organization. Exotoxins from several clades are known to modify actin at Arg177; but of the 14-3-3 dependent exotoxins only Aeromonas salmonicida exoenzyme T (AexT) has been reported to ADP-ribosylate actin. Given the extensive similarity among the 14-3-3 dependent exotoxins, we initiated a structural and biochemical comparison of these proteins. Structural modeling of AexT indicated a target binding site that shared homology with Pseudomonas aeruginosa Exoenzyme T (ExoT) but not with Exoenzyme S (ExoS). Biochemical analyses confirmed that the catalytic activities of both exotoxins were stimulated by agmatine, indicating that they ADP-ribosylate arginine residues in their targets. Side-by-side comparison of target protein modification showed that AexT had activity toward the SH2 domain of the Crk-like protein (CRKL), a known target for ExoT. We found that both AexT and ExoT ADP-ribosylated actin and in both cases, the modification compromised actin polymerization. Our results indicate that AexT and ExoT are functional homologs that affect cytoskeletal integrity via actin and signaling pathways to the cytoskeleton. [ABSTRACT FROM AUTHOR]

Bacterial communities, microbial populations, and antibiotic resistance of potential pathogens in the water and fish (Cyprinus carpio, flesh and gut) from different areas (A1, A2 and A3—A1 was linked with river water, A2 with cattle activity, and A3 with waters of a spring after heavy rains) of Lake Karla (Thessaly, Central Greece) were investigated. The isolated bacteria were identified using Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and were tested for resistance in 21 antibiotics. The microbiota composition of fish flesh was also studied using 16S amplicon-based sequencing Serratia fonticola and several species of Aeromonas (e.g., Aeromonas salmonicida, Aeromonas bestiarium, Aeromonas veronii, etc.) exhibited the highest abundances in all studied samples, while the microbiota profile between the three studied areas was similar, according to the culture-dependent analysis. Of them, S. fonticola was found to be resistant in the majority of the antibiotics for the water and fish (gut and flesh), mainly of the areas A1 and A2. Regarding 16S metabarcoding, the presence of Serratia and Aeromonas at genus level was confirmed, but they found at very lower abundances than those reported using the culture-dependent analysis. Finally, the TVC and the rest of the studied microbiological parameters were found at acceptable levels (4 log cfu/mL or cfu/g and 2–4 log cfu/mL or cfu/g, extremely low levels of E. coli/coliforms) in both water and fish flesh. Based on our findings, the water of Lake Karla would be used for activities such as irrigation, recreation and fishing, however, the development and implementation of a quality management tool for Lake Karla, to ensure environmental hygiene and prevention of zoonosis during the whole year, is imperative. [ABSTRACT FROM AUTHOR]