Preliminary snapshot reveals a relationship between multidrug-resistance and biofilm production among enterobacteriaceae isolated from fecal samples of farm-raised poultry in ceará, Brazil.

Antimicrobial resistance and biofilm formation by microbial pathogens pose a significant challenge to poultry production systems due to the persistent risk of dissemination and compromise of bird health and productivity. In this context, the study aimed to investigate the occurrence of different multiresistance phenotypes and the biofilm-forming ability of Enterobacteriaceae isolated from broiler chicken excreta in poultry production units in Ceará, Brazil. Samples were collected from three distinct broiler breeding facilities and subjected to isolation, identification, antibiotic susceptibility testing, phenotypic screening for ß-lactamases enzymes, and biofilm formation evaluation. Seventy-one strains were identified, being Escherichia coli (37 %) and Proteus mirabilis (32 %), followed by Klebsiella pneumoniae (11 %), Providencia stuartii (9 %), Klebsiella aerogenes (6 %), Alcaligenes faecalis (4 %), and Salmonella sp. (1 %). A significant proportion (87 %) of multiresistant strains were detected. For the phenotypic evaluation of ß-lactamases production, strains with resistance to second and third-generation cephalosporins and carbapenems were tested. About 4 of 6 and 10 of 26 were positive for inducible chromosomal AmpC ß-lactamase and extended-spectrum ß-lactamase (ESBL), respectively. Regarding biofilm formation, it was observed that all MDR strains were capable of forming biofilm. In this sense the potential of these MDR bacteria to develop biofilms becomes a significant concern, representing a real threat to both human and animal health, as biofilms offer stability, antimicrobial protection, and facilitate genetic transfer.

Adjuvant effects of ß-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus).

ß-defensin of flounder plays an important role in immunomodulation by recruiting immune cells and has a potential vaccine adjuvant effect in addition to its bactericidal activity. In this study, adjuvant effects of ß-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus) were investigated. The bicistronic eukaryotic expression plasmid pBudCE4.1 plasmid vector with two independent coding regions was selected to construct DNA vaccine of p-OmpC which express only the gene for the outer membrane protein of Edwardsiella tarda and the vaccine of p-OmpC-ßdefensin which express both the outer membrane protein of the bacterium and ß-defensin of flounder. In vitro and in vivo studies have shown that the constructed plasmids can be expressed in flounder embryonic cell lines and injection sites of muscles. After vaccination by intramuscular injection, both p-OmpC and p-OmpC-ßdefensin groups showed significant upregulation of immune-response. Compared to the pBbudCE4.1 and the p-OmpC vaccinated groups, the p-OmpC-ßdefensin vaccinated group showed significantly more cell aggregation at the injection site and intense immune response. The proportion of sIgM+ cells, as well as the CD4-1+ and CD4-2+ cells in both spleen and kidney was significantly higher in the p-OmpC-ßdefensin vaccinated group at peak time point than in the control groups. The relative survival rate of the p-OmpC-ßdefensin vaccine was 74.17%, which was significantly higher than that of the p-OmpC vaccinated group 48.33%. The results in this study determined that ß-defensin enhances the responses in cellular and humoral immunity and evokes a high degree of protection against E. tarda, which is a promising candidate for vaccine adjuvant.

Effects of Citrobacter freundii on sturgeon: Insights from skin mucosal immunology and microbiota.

Skin mucus analysis has recently been used as a non-invasive method to evaluate for fish welfare. The present research study was conducted to examine the skin mucosal immunity and skin microbiota profiles of sturgeons infected with Citrobacter freundii. Our histology results showed that the thickness of the epidermal layer of skin remained thinner, and the number of mucous cells was significantly decreased in sturgeons after infection (p < 0.05). Total protein, alanine aminotransferase, aspartate aminotransferase, superoxide dismutase, and creatine kinase levels in the mucus showed biphasic pattern (decrease and then increase). Lactate dehydrogenase, lysozyme, and acid phosphatase activities in the mucus showed an increasing trend after infection. Furthermore, 16S rRNA sequencing also revealed that C. freundii infection also affected the diversity and community structure of the skin mucus microbiota. An increase in microbial diversity (p > 0.05) and a decrease in microbial abundance (p < 0.05) after infection were noted. The predominant bacterial phyla in the skin mucus were Proteobacteria, Fusobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Specifically, the relative abundance of Fusobacteria increased after infection. The predominant bacterial genera in the skin mucus were Cetobacterium, Pelomonas, Bradyrhizobium, Flavobacterium, and Pseudomonas. The relative abundance of Cetobacterium, Pseudomonas, and Flavobacterium increased after infection. Our current research findings will provide new insights into the theoretical basis for future research studies exploring the mechanism of sturgeon infection with C. freundii.

Edwardsiella piscicida causes iron storage disorders by an autophagy pathway in fish monocytes/macrophages.

Edwardsiella piscicida (E. piscicida) is a gram-negative pathogen that survives in intracellular environment. Currently, the interplay between E. piscicida and host cells has not been completely explored. In this study, we found that E. piscicida disturbed iron homeostasis in grass carp monocytes/macrophages to maintain its own growth. Further investigation revealed the bacteria induced an increase of intracellular iron, which was subjected to the degradation of ferritin. Moreover, the autophagy inhibitor impeded the degradation of ferritin and increase of intracellular iron in E. piscicida-infected monocytes/macrophages, implying possible involvement of autophagy response in the process of E. piscicida-broken iron homeostasis. Along this line, confocal microscopy observed that E. piscicida elicited the colocalization of ferritin with LC3-positive autophagosome in the monocytes/macrophages, indicating that E. piscicida mediated the degradation of ferritin possibly through the autophagic pathway. These results deepened our understanding of the interaction between E. piscicida and fish cells, hinting that the disruption of iron homeostasis was an important factor for pathogenicity of E. piscicida. They also indicated that autophagy was a possible mechanism governing intracellular iron metabolism in response to E. piscicida infection and might offer a new avenue for anti-E. piscicida strategies in the future.

Granzyme B secreted by T cells is involved in anti-bacterial immune response of tilapia.

Secreted by natural killer cells and cytotoxic T lymphocytes, Granzyme B is involved in regulating the adaptive immune response in vertebrates and plays a pivotal role in resisting virus invasion and removing pathogens. Although it had been extensively studied in mammals, the involvement of Granzyme B in adaptive immune response of early vertebrates remained elusive. In this study, we investigated the Granzyme B in Oreochromis niloticus (OnGrB), found that its function domain was conserved. Additionally, OnGrB was widely expressed in various tissues and could respond to T-cell activation in vitro at the transcriptional level. Furthermore, we prepared the recombinant OnGrB (rOnGrB) as an immunogen to develop a mouse anti-OnGrB monoclonal antibody (mAb). Using this anti-OnGrB mAb as a tool, we explored the expression of OnGrB in the adaptive immune response of tilapia. Our findings revealed that T cell was a significant source of OnGrB production, the expression of OnGrB at the protein level and the proportion of OnGrB + T cells increased after both T cell activation in vitro and infection with Edwardsiella piscicida in vivo. More importantly, our findings also preliminarily illuminated that p65 could regulate the transcriptional activity of OnGrB. These results indicated that OnGrB was involved in the adaptive immunity of tilapia and played a critical role in T cell function in teleost. Our study provided theoretical support and new perspectives for understanding adaptive immunity in teleost.

CD122 is an activation marker ensuring proper proliferation of T cells in teleost.

As one of subunits for interleukin-2 receptor (IL-2R), CD122 can bind to IL-2 and then activate downstream signal transduction to participate in adaptive immune response. Although CD122 has been identified and investigated from several teleost species, studies on its function at T-cell level are still scarce for lack of specific antibodies. In this study, a typical CD122 in Nile tilapia (Oreochromis niloticus) was characterized by bioinformatics analysis, cloned to produce retrovirus infected NIH/3T3 cells for mouse immunization. After cell fusion and screening, we successfully developed a mouse anti-tilapia CD122 monoclonal antibody (mAb), which could specifically recognize CD122 and identify CD122-producing T cells of tilapia. Using the mAb to detect, CD122 was found to widely distribute in immune-related tissues, and significantly elevate post Edwardsiella piscicida infection or T-cell activation. More importantly, the expansion of CD122+ T cells and up-regulation of CD122 occurred both in total T cells and T-cell subsets during T-cell activation upon in vitro stimulation or in vivo infection. These results indicate that CD122 can be used as a T-cell activation marker in tilapia. Notably, CD122 mAb blocking blunted the activation of MAPK/Erk and mTORC1 pathways, and inhibited T-cell proliferation, suggesting a critical role of CD122 in ensuring proper proliferation of tilapia T cells. Therefore, this study enriches the knowledge of T-cell responses in fish and provides new evidence for understanding the evolution of lymphocyte-mediated adaptive immunity.

PoIL8-L, a teleost interleukin-8 like, enhances leukocyte cellular vitality and host defense against bacterial infections in Japanese flounder (Paralichthys olivaceus).

Interleukin-8 (IL-8), a CXC chemokine, exerts pivotal effect on cell migration, inflammatory response, and immune regulation. In this study, we examined the immunological characteristics of an IL-8 like homologue (PoIL8-L) in Japanese flounder (Paralichthys olivaceus). PoIL8-L contains a conserved chemokine CXC domain and 105 amino acid residues. PoIL8-L expression in tissues was constitutive, and significantly regulated by V. havieri or E. tarda infection. In vitro, rPoIL8-L could bind to eight tested bacteria, exhibited bacteriostatic and bactericidal effects against certain bacteria, and could bind to the targeted bacterial Ⅳ pilin protein rPilA of E. tarda. Furthermore, rPoIL8-L could attach to peripheral blood leukocytes, and enhance their immune genes expression, respiratory burst, chemotaxis, proliferation, acid phosphatase activity, and phagocytic activity. Additionally, rPoIL8-L induce neutrophils to extrude neutrophil extracellular traps. In vivo, rPoIL8-L could promote host resistance to E. tarda infection. In summary, these findings provide fresh perspectives on the immunological antibacterial properties of IL-8 in teleost.

Characterization and expression profiles of toll-like receptor genes (TLR2 and TLR5) in immune tissues of hybrid yellow catfish under bacterial infection.

The yellow catfish (Pelteobagrus fulvidraco) is one of the most economically important freshwater species in Asia. However, pathogenic bacterial infections often cause high rates of mortality and economic losses in practical aquaculture. Previous studies in mammals have shown that Toll-like receptor 2 (TLR2) and Toll-like receptor 5 (TLR5) are involved in the recognition of cell wall components such as lipopolysaccharides and flagella of various bacteria, thereby acting as key regulators in the innate immunity response. However, TLR2 and TLR5 in yellow catfish have not been characterized. In the present study, TLR2 and TLR5 were examined through comparative genomic approaches. The gene structure, collinearity, protein spatial structure, and phylogenetic relationships were compared with those in multiple representative vertebrates. Meanwhile, quantitative real-time PCR was conducted to explore transcriptional changes in TLR2 and TLR5 in immune tissues after infection with exogenous A. hydrophila and E. tarda. The results demonstrated the presence of TLR2 and TLR5 in yellow catfish. However, a systematic analysis showed that TLR2 was not associated with the arrangement of diverse neighboring genes. The expression of hybrid yellow catfish TLR2 transcripts in multiple tissues (including liver, spleen, kidney, and intestine) was significantly up-regulated after infection with A. hydrophila and E. tarda, suggesting that hybrid yellow catfish TLR2 and TLR5 may participate in the immune process. Taken together, the results indicate that TLR2 and TLR5 are conserved in terms of evolution and possess significant antibacterial activity as well as regulatory properties in immune-related tissues and thus play key roles in host defense against pathogen invasion.

Effects of adding Bacillus subtilis natto NTU-18 in paste feed on growth, intestinal morphology, gastrointestinal microbiota diversity, immunity, and disease resistance of Anguilla japonica glass eels.

Japanese eel, Anguilla japonica, holds significant importance in Taiwanese aquaculture. With the intensification of eel farming, the impact of Edwardsiella tarda has become increasingly severe. Consequently, the abusive use of antibiotics has risen. Bacillus subtilis natto NTU-18, a strain of Bacillus with a high survival rate in feed processing, plays a crucial role in promoting intestinal health through competitive rejection, enhancing immune responses against bacterial pathogens, and improving intestinal health by modulating gastrointestinal microbiota to produce beneficial metabolites of mice and grass carp, Ctenopharyngodon idella. This study investigated the effects of different proportions (control, 0.25 %, 0.5 %, 1 %, and 2 %) of B. subtilis natto NTU-18 added to paste feed on the growth performance, intestinal morphology, and microbiota, expression of immune-related genes, and resistance to E. tarda in Japanese glass eel. The results indicated that the growth performance of all groups with B. subtilis natto NTU-18 added was significantly higher than that of the control group and did not impact the villi morphology. The expression of immune-related genes in the kidney, specifically HSP70 and SOD, was significantly higher from 0.5 % and above than the control; however, no significant differences were observed in CAT, POD, and HSP90. In the liver, significant differences were found in HSP70 and IgM above 0.25 % compared to the control group, with no significant differences in SOD, CAT, POD, and HSP90 among all groups. Additionally, intestinal microbiota analysis revealed that the 2 % additional group had significantly lower diversity than other groups, with Cetobacterium as the dominant species. The challenge test observed that the survival rates of the 0.5 % and 1 % groups were significantly higher. This research suggests that adding 0.5 % and 1 % of B. subtilis natto NTU-18 to the diet is beneficial for Japanese glass eel's immunity, growth performance, and disease resistance.

Molecular characterization, expression analysis and function identification of Pf_IL-12p35, Pf_IL-23p19 and Pf_IL-12p40 genes in yellow catfish (Pelteobagrus fulvidraco).

The interleukin-12 (IL-12) family is a class of heterodimeric cytokines that play crucial roles in pro-inflammatory and pro-stimulatory responses. Although some IL-12 and IL-23 paralogues have been found in fish, their functional activity in fish remains poorly understood. In this study, Pf_IL-12p35a/b, Pf_IL-23p19 and Pf_IL-12p40a/b/c genes were cloned from yellow catfish (Pelteobagrus fulvidraco), four α-helices were found in Pf_IL-12p35a/b and Pf_IL-23p19. The transcripts of these six genes were relatively high in mucus and immune tissues of healthy individuals, and in gill leukocytes. Following Edwardsiella ictaluri infection, Pf_IL-12p35a/b and Pf_IL-23p19 mRNAs were induced in brain and kidney (or head kidney), Pf_IL-12p40a mRNA was induced in gill, and Pf_IL-12p40b/c mRNAs were induced in brain and liver (or skin). The mRNA expression of these genes in PBLs was induced by phytohaemagglutinin (PHA) and polyinosinic-polycytidylic acid (poly I:C), while lipopolysaccharides (LPS) induced the mRNA expression of Pf_IL-12p35a and Pf_IL-12p40b/c in PBLs. After stimulation with recombinant (r) Pf_IL-12 and rPf_IL-23 subunit proteins, either alone or in combination, mRNA expression patterns of genes related to T helper cell development exhibited distinct differences. The results suggest that Pf_IL-12 and Pf_IL-23 subunits may play important roles in regulating immune responses to pathogens and T helper cell development.

The TAK1/JNK axis participates in adaptive immunity by promoting lymphocyte activation in Nile tilapia.

The transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P + I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity.

Antioxidant and antibacterial efficiency of the ethanolic leaf extract of Kratom (Mitragyna speciosa (Korth.) Havil) and its effects on growth, health, and disease resistance against Edwardsiella tarda infection in Nile tilapia (Oreochromis niloticus).

The research examined the impact of an ethanolic extract from the leaves of Kratom (Mitragyna speciosa (Korth.) Havil.) on the growth, antioxidant capacity, immune-related gene expression, and resistance to disease caused by Edwardsiella tarda in Nile tilapia (Oreochromis niloticus). The findings revealed that the extract had the important phytochemical content in the extract included total phenolics content, total flavonoids content, vitamin C, and total antioxidant capacity and 5.42 % of the crude extract was mitragynine. The extract demonstrated antioxidant activity, as evidenced by its IC50 values against ABTS and DPPH radicals and its ferric reducing power in vitro. Moreover, the MIC-IC50 value of 0.625 mg/mL indicated that the growth of the bacteria was reduced by approximately 50 %, and the MBC was 2.50 mg/mL against E. tarda. Furthermore, the orally administered Kratom leaf extract to fingerling tilapia for 8 weeks exhibited a noticeable increase in oxidative stress, as demonstrated by the increase in MDA production in the 10 and 25 g/kg groups. It also exhibited an increase in acetylcholinesterase (AChE) activity in muscle tissue at the 50 g/kg group. However, when administered at a feeding rate of 5-10 g/kg feed, the extract showed an increase in the expression of immune-related genes (IL1, IL6, IL8, NF-kB, IFNγ, TNFα, Mx, CC-chemokine, CD4, TCRß, MHC-IIß, IgM, IgT, IgD) and enhanced resistance to E. tarda infection in fish. Conversely, administering the extract at 25-50 g/kg feed resulted in contrasting effects, suppressing and reducing the observed parameters. Nevertheless, feeding the extract at all concentrations for 8 weeks did not produce any changes in the histology or systemic functioning of the liver and intestines, as indicated by blood biochemistry. These findings suggest that the ethanolic leaf extract from Kratom has the potential to be used as a substitute for antibiotics in the management of bacterial infections in Nile tilapia culture, with a recommended dosage of 5-10 g/kg feed/day for a maximum of 8 weeks.

Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Overview on the role of dietary Spirulina platensis on immune responses against Edwardsiellosis among Oreochromis niloticus fish farms.

Edwardsiellosis is a bacterial fish disease that mostly occurs in freshwater farms and is characterized by a high mortality rate. Edwardsiella tarda strain was recovered from 17 fish out of 50 Nile tilapia, which were harboring clinical signs of systemic septicemia. The level of un-ionized ammonia (NH3) in the fish farm's water was 0.11-0.15 mg/L, which was stressful for the Nile tilapia.Sequencing of the gyrB1 gene confirmed that the isolate was E. tarda JALO4, and it was submitted to NCBI under the accession number PP449014. The isolated E. tarda harbored the virulence gene edw1 AHL-synthase (quorum sensing). In addition, the isolate was sensitive to trimethoprim and sulfamethoxazole mean while it was intermediate to florfenicol. The median lethal dose (LD50) of E. tarda JALO4 was determined to be 1.7 × 105 CFU/mL in Nile tilapia.In the indoor experiment, Nile tilapia (45.05 ± 0.4 g), which received dietary Spirulina platensis (5 and 10 g/kg fish feed), showed optimum growth and feed utilization. Meanwhile, after receiving dietary S. platensis, the fish's feed conversion ratio (FCR) was significantly enhanced compared to the control, which was 1.94, 1.99, and 2.88, respectively. The expression of immune-related genes interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were upsurged in E. tarda-challenged fish with higher intensity in S. platensis groups. Dietary S. platensis at a dose of 10 g/kg fish feed could provide a relative protection level (RPL) of 22.2% Nile tilapia challenged against E. tarda. Nile tilapia experimentally infected E. tarda, drastically altering their behavior: higher operculum movement, low food apprehension, and abnormal swimming dietary S. platensis (10 g/kg fish feed) could rapidly restore normal status.It was concluded that Edwardsiellosis could alter Nile tilapia behavior with a high loss in fish population. Fish received dietary-S. platensis could rapidly restore normal behavior after E. tarda infection. It is recommended the incorporation of S. platensis at doses of 10 g/kg into the Nile tilapia diet to boost their immunity and counteract E. tarda infection.

Prevalence, risk factors, and characterisation of extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) in horses entering an equine hospital and description of longitudinal excretion.

BACKGROUND: Extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) are important zoonotic pathogens that can cause serious clinical infections, also in horses. Preventing the spread of ESBL-E, especially in the equine hospital environment, is key to reducing the number of difficult-to-treat infections. Estimating the local prevalence of ESBL-E in horses is crucial to establish targeted infection control programs at equine hospitals. We conducted a prevalence and risk factor study in equine patients on admission to an equine teaching hospital in Finland through a rectal ESBL-E screening specimen of the horse and a questionnaire. RESULTS: The prevalence of ESBL-E in admitted horses was 3% (5/161, 95% CI 1-7%); none of the tested factors remained statistically significant in multivariate analysis, although antimicrobial treatment within three months was borderline significant (p = 0.052). Extended-spectrum ß-lactamase -producing Klebsiella pneumoniae ST6179:CTX-M-15 was detected in three horses using whole-genome sequencing, which in combination with patient records suggested nosocomial transmission. Escherichia coli isolates were ST1250:CTX-M-1 (n = 1), ST1079:CTX-M-1 (n = 1), and ST1245:CTX-M-14 (n = 1). Multiple virulence genes were detected in the ESBL-E isolates. In the ESBL-E positive horses enrolled in a one-year follow-up study, ESBL-E were unlikely to be isolated in rectal screening specimens after the initial positive specimen. CONCLUSIONS: The prevalence of ESBL-E in horses visiting a veterinary teaching hospital in Finland is low, indicating an overall low prevalence estimate in the country's equine population. No statistically significant risk factors were identified, likely due to the low number of cases. The duration of ESBL-E carriage is likely to be very short in horses.

Emergence of NDM-producing Enterobacterales infections in companion animals from Argentina.

Antimicrobial resistance is considered one of the most critical threat for both human and animal health. Recently, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals had been described. This study report the first molecular characterization of NDM-producing Enterobacterales causing infections in companion animals from Argentina. Nineteen out of 3662 Enterobacterales isolates analyzed between October 2021 and July 2022 were resistant to carbapenemes by VITEK2C and disk diffusion method, and suspected to be carbapenemase-producers. Ten isolates were recovered from canine and nine from feline animals. Isolates were identified as K. pneumoniae (n = 9), E. coli (n = 6) and E. cloacae complex (n = 4), and all of them presented positive synergy among EDTA and carbapenems disks, mCIM/eCIM indicative of metallo-carbapenemase production and were also positive by PCR for blaNDM gene. NDM variants were determined by Sanger sequencing method. All 19 isolates were resistant to ß-lactams and aminoglycosides but remained susceptible to colistin (100%), tigecycline (95%), fosfomycin (84%), nitrofurantoin (63%), minocycline (58%), chloramphenicol (42%), doxycycline (21%), enrofloxacin (5%), ciprofloxacin (5%) and trimethoprim/sulfamethoxazole (5%). Almost all isolates (17/19) co-harbored blaCTX-M plus blaCMY, one harbored blaCTX-M alone and the remaining blaCMY. E. coli and E. cloacae complex isolates harbored blaCTX-M-1/15 or blaCTX-M-2 groups, while all K. pneumoniae harbored only blaCTX-M-1/15 genes. All E. coli and E. cloacae complex isolates harbored blaNDM-1, while in K. pneumoniae blaNDM-1 (n = 6), blaNDM-5 (n = 2), and blaNDM-1 plus blaNDM-5 (n = 1) were confirmed. MLST analysis revealed the following sequence types by species, K. pneumoniae: ST15 (n = 5), ST273 (n = 2), ST11, and ST29; E. coli: ST162 (n = 3), ST457, ST224, and ST1196; E. cloacae complex: ST171, ST286, ST544 and ST61. To the best of our knowledge, this is the first description of NDM-producing E. cloacae complex isolates recovered from cats. Even though different species and clones were observed, it is remarkable the finding of some major clones among K. pneumoniae and E. coli, as well as the circulation of NDM as the main carbapenemase. Surveillance in companion pets is needed to detect the spread of carbapenem-resistant Enterobacterales and to alert about the dissemination of these pathogens among pets and humans.

Virulent and Multi-drug-Resistant Edwardsiella tarda Infection in Oscar Fish: Unveiling the Threat of Mass Mortality and AMR Dissemination.

The Oscar fish (Astronotus ocellatus) is among the most commonly domesticated and exported ornamental fish species from Kerala. The ornamental fish industry faces a significant challenge with the emergence of diseases caused by multi-drug-resistant bacteria. In the present study, six isolates were resolved from the diseased Oscar fish showing haemorrhages, necrosis, and loss of pigmentation. After phenotypic and genotypic characterization, the bacteria were identified as Edwardsiella tarda, Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, Brevibacillus borstelensis, and Staphylococcus hominis. Experimental challenge studies in healthy Oscar fish showed that E. tarda caused 100% mortality within 240 h with 6.99 × 106 CFU/fish as LD50 and histopathology revealed the typical signs of infection. The pathogen was re-recovered from the moribund fish thereby confirming Koch's postulates. E. tarda was confirmed through the positive amplification of tarda-specific gene and virulence genes viz., etfD and escB were also detected using PCR. Antibiotic susceptibility tests using disc diffusion displayed that the pathogen is multi-drug-resistant towards antibiotics belonging to aminoglycosides, tetracyclines, and quinolones categories with a MAR index of 0.32, which implicated the antibiotic pressure in the farm. Plasmid curing studies showed a paradigm shift in the resistance pattern with MAR index of 0.04, highlighting the resistance genes are plasmid-borne except for the chromosome-borne tetracycline resistance gene (tetA). This study is the first of its kind in detecting mass mortality caused by E. tarda in Oscar fish. Vigilant surveillance and strategic actions are crucial for the precise detection of pathogens and AMR in aquaculture.

Iron supplementation in the diets of hybrid catfish (Ictalurus punctatus × I. furcatus) juveniles affected haematocrit levels and potentially decreased disease resistance to Edwardsiella ictaluri.

To prevent catfish idiopathic anaemia, diets fortified with iron have been adopted as a regular practice on commercial catfish farms to promote erythropoiesis. However, the effects of prolonged exposure of excess dietary iron on production performance and disease resistance for hybrid catfish (Ictalurus punctatus × I. furcatus) remains unknown. Four experimental diets were supplemented with ferrous monosulphate to provide 0, 500, 1000, and 1500 mg of iron per kg of diet. Groups of 16 hybrid catfish juveniles (~22.4 g) were stocked in each of 20, 110-L aquaria (n = 5), and experimental diets were offered to the fish to apparent satiation for 12 weeks. At the end of the study, production performance, survival, condition indices, as well as protein and iron retention were unaffected by the dietary treatments. Blood haematocrit and the iron concentration in the whole-body presented a linear increase with the increasing the dietary iron. The remaining fish from the feeding trial was challenged with Edwardsiella ictaluri. Mortality was mainly observed for the dietary groups treated with iron supplemented diets. The results for this study suggest that iron supplementation beyond the required levels does affect the blood production, and it may increase their susceptibility to E. ictaluri infection.

Comparative pathogenicity and histopathological analysis of Edwardsiella anguillarum intraperitoneal infection in milkfish (Chanos chanos), Nile tilapia (Oreochromis niloticus) and Asian seabass (Lates calcarifer).

Edwardsiella anguillarum, a highly virulent species within the Edwardsiella genus, causes significant mortality in milkfish farms in Taiwan. This study aimed to investigate the comparison of milkfish susceptibility, a newly identified host species in Taiwanese aquaculture, with other species Nile tilapia (Oreochromis niloticus) and Asian seabass (Lates calcarifer), to E. anguillarum, elucidating its pathogenicity across both seawater and freshwater aquaculture environments. The results showed milkfish exhibited the highest mortality rate of 85% within 48 h of infection, whereas Nile tilapia exhibited a mortality rate of 70% between the second- and tenth-day post challenge, and seabass exhibited a mortality rate of 25% between the second- and sixth-day post challenge. Gross lesions observed in milkfish included splenomegaly and haemorrhage, whereas Nile tilapia exhibited signs of ascites, exophthalmia and brain haemorrhage. Seabass displayed spleen granulomas and haemorrhage at the injection site. Histopathological analysis revealed common features across all three species, including multifocal necrosis, bacterial presence in the necrotic areas, serositis and oedema. Asian seabass also exhibited chronic lesions in the form of splenic granulomas. This study highlights the high susceptibility of milkfish and Nile tilapia to E. anguillarum, emphasizing the urgent need for further investigation into targeted vaccine development for these fish species. These results not only deepen our understanding of the differing levels of pathogenicity among the three species but also offer valuable insights for improving disease prevention and management strategies in aquaculture, including those applied within polyculture systems and for the maintenance of aquaculture water environments.

Influence of probiotic and prebiotic supplementation on intestinal microbiota and resistance to Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus) following florfenicol administration.

Enteric septicemia of catfish (ESC), caused by the gram-negative enteric bacteria Edwardsiella ictaluri, is a significant threat to catfish aquaculture in the southeastern United States. Antibiotic intervention can reduce mortality; however, antibiotic use results in an imbalance, or dysbiosis, of the gut microbiota, which may increase susceptibility of otherwise healthy fish to enteric infections. Herein, recovery of the intestinal microbiota and survivability of channel catfish in response to ESC challenge was evaluated following a 10-day course of florfenicol and subsequent probiotic or prebiotic supplementation. Following completion of florfenicol therapy, fish were transitioned to a basal diet or diets supplemented with a probiotic or prebiotic for the remainder of the study. Digesta was collected on Days 0, 4, 8 and 12, beginning on the first day after cessation of antibiotic treatment, and gut microbiota was characterized by Illumina sequencing of the 16S rRNA gene (V4 region). Remaining fish were challenged with E. ictaluri and monitored for 32 days post-challenge. Florfenicol administration resulted in dysbiosis characterized by inflated microbial diversity, which began to recover in terms of diversity and composition 4 days after cessation of florfenicol administration. Fish fed the probiotic diet had higher survival in response to ESC challenge than the prebiotic (p = .019) and negative control (p = .029) groups.