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The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda. These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

is a Gram-negative bacterium that can infect a broad range of hosts including humans and fish. Accumulating evidences have indicated that is able to survive and replicate in host phagocytes. However, the pathways involved in the intracellular infection of are unclear. In this study, we examined the entry and endocytic trafficking of in the mouse macrophage cell line RAW264.7. We found that entered RAW264.7 and multiplied intracellularly in a robust manner. Cellular invasion of was significantly impaired by inhibition of clathrin- and caveolin-mediated endocytic pathways and by inhibition of endosome acidification, but not by inhibition of macropinocytosis. Consistently, RAW264.7-infecting was co-localized with clathrin, caveolin, and hallmarks of early and late endosomes, and intracellular was found to exist in acid organelles. In addition, in RAW264.7 was associated with actin and microtubule, and blocking of the functions of these cytoskeletons by inhibitors significantly decreased infection. Furthermore, formaldehyde-killed exhibited routes of cellular uptake and intracellular trafficking similar to that of live . Together these results provide the first evidence that entry of live into macrophages is probably a passive, virulence-independent process of phagocytosis effected by clathrin- and caveolin-mediated endocytosis and cytoskeletons, and that the intracellular traffic of involves endosomes and endolysosomes.

The production of striped catfish (Pangasianodon hypothalamus) has increased worldwide; recently, it was farmed with Nile tilapia in polyculture farms. Polyculture systems and water temperature (25℃ and 33℃) could affect Edwardsiella tarda infection, antibiotic efficacy, and residues. Moribund fishes were collected from three Farms 1–3: Farm 1 (monoculture, Nile tilapia), Farm 2 (monoculture, striped catfish), and Farm 3 (polyculture). Four E. tarda , LAMSH1, and LAMAH2-4 were isolated, whereas LAMAH3 was isolated from both fish spp., where striped catfish were highly susceptible to infection. The obtained E. tarda , which was isolated from striped catfish, has a significantly lower LD 50 than those retrieved from Nile tilapia, and co-infection occurred only in striped catfish on Farm 3. The infection was screened and confirmed by gyrB1 gene presence while detecting the cds1 , pvsA , and qseC genes indicated virulence. All isolates were sensitive to ciprofloxacin and florfenicol but showed resistance to a high number of other antibiotics, resulting in high multi-drug resistant (MDR) indices exceeding 0.2, except for strain LAMAH4, which had an index of 0.18. Analyses of farms water revealed high ammonia compounds total ammonia nitrogen (TAN), unionized ammonia (NH 3 ), nitrite (NO 2 ), and nitrate (NO 3 ) in Farm 2 (monoculture, striped catfish), and the recorded significantly higher concentrations were 2.75, 0.29, 0.24, and 2.01 mg/L, respectively, which were compared with Farm 1 and Farm 3. In the indoor experiment, at high water temperatures (33 °C), Nile tilapia and striped catfish had a high mortality rate and re-isolation of E. tarda (10–20%) compared to those exposed to low water temperatures (25 °C). These observations were concurrent with low antibiotic residues in their hepatic tissues. Despite water temperature, Nile tilapia showed higher ciprofloxacin residues than striped catfish. The study concluded that striped catfish are more susceptible to the bacteria E. tarda compared to Nile tilapia, particularly in polyculture farms, which resulted in a higher infection rate. Both Nile tilapia and striped catfish exposed to elevated water temperatures exhibited increased vulnerability to bacterial infections. Additionally, these fish showed a high re-isolation rate of E. tarda while having low ciprofloxacin residues in their hepatic tissues.

The overuse of antibiotics in the aquaculture sector to prevent and treat the pathogen Edwardsiella tarda in striped catfish Pangasianodon hypophthalmus has led to the rapid emergence of multidrug-resistant (MDR) E. tarda isolates. Alternative strategies, such as phage therapy have been considered to control this MDR bacteria pathogen. We present here three lytic bacteriophages vB_EtA_WAG25P1, vB_EtA_WCT72P1, and vB_EtA_DT115P1 infecting E. tarda isolates, with their geographical origins from Mekong Delta, Vietnam. Morphological analyses combined with their genomic data indicate that the bacteriophages are classified to Autosignataviridae family in Autographivirales order. In the latent period of 35 min, the phage WCT72P1 and WAG25P1 had the burst size of about 45 virions and 61 virions per infected cell, respectively, while 51 virions were released in the shorter latent period of 25 min for the phage DT115P1. They also owned several prominent biological properties as the high species-specificity of infection, the effective in vitro repression of the MDR E. tarda growth for approximately four hours, pH resistance, and thermal stability. The phage WAG25P1 and DT115P1 exhibited the highest nucleotide identity to Klebsiella phage RCIP0053 with the sequence identity of 67.4% and 68.1%, respectively, while the phage WCT72P1 was closely related to Klebsiella phage RCIP0089 with the similarity of 66.6%. The phages were in a cluster of new species belonging to the new unassigned genus. The genome analysis also showed their safety due to the absence of the recombination, integration and host virulence genes. To the best of our knowledge, this is the first report of novel lytic phages infecting E. tarda isolates in striped catfish Pangasianodon hypophthalmus and it will also pave way for their potential applications for phage biocontrol of the disease in striped catfish.

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.

Background Edwardsiella tarda ( E. tarda ) causes highly mortality, which is rare in septic patients. We herein reported a case of septic shock caused by drug-resistant E. tarda . Case presentation We herein describe a 32-year-old female with septic shock who had the medical history of abortion 1 month ago and “systemic lupus erythematosus and rheumatoid arthritis” presented abdominal pain, diarrhea, and dyspnea as the primary symptoms and rapidly deteriorated to MODS following breakfast (undercooked fish porridge) in the ICU. Sepsis surviving bundle was initiated by collecting pathogen culture (sputum, urine and blood samples), empirically broad-spectrum antibiotics administration (Meropenem), along with fluid resuscitation, vasopressor use. E. tarda was confirmed both in blood culture and mNGS (metagenomics next generation sequencing). Thus, the antibiotics were switched to piperacillin-tazobactam according to the susceptibility test that was susceptible to piperacillin-tazobactam and resistant to ampicillin, quinolones and gentamicin. The patient finally recovered and discharged after 18 days of ICU treatment. Conclusions Empiric antibiotics should be selected with piperacillin-tazobactam and amikacin, and avoid ampicillin, quinolones and gentamicin for suspecting E. tarda infection in southern China. Bacteremia complicated with septic shock caused by E. tarda requires intensive care to improve survival rates.

Edwardsiella tarda is a significant pathogen that causes edwardsiellosis in aquaculture, resulting in substantial economic losses while also posing a global public health threat. The increasing prevalence of antibiotic-resistant E. tarda strains has further exacerbated this challenge. The present study used a metabolic state-reprogramming approach to identify an ideal biomarker as an antibiotic adjuvant to increase conventional antibiotics that are already resistant. Exogenous aspartate most effectively potentiated tobramycin to lab-evolved and clinical isolated MDR E. tarda in vitro and in in vivo models (fish and mice). Mechanistically, aspartate enhanced tobramycin uptake in MDR E. tarda by overcoming efflux pump activity. This effect was mediated through aspartate-induced activation of the pyruvate cycle, which increased the proton motive force via NADH generation and enhanced membrane permeability. Our findings demonstrate that the aspartate-tobramycin combination represents a promising therapeutic strategy against MDR E. tarda infections.

Two new antimicrobial bisabolane-type sesquiterpenoid derivatives, ent-aspergoterpenin C (compound 1) and 7-O-methylhydroxysydonic acid (2), and two new butyrolactone-type monoterpenoids, pestalotiolactones C (3) and D (4), along with a known monoterpenoid pestalotiolactone A (5) and four known bisabolane sesquiterpenoids (6−9), were isolated and identified from the deep-sea sediment-derived fungus Aspergillus versicolor SD-330. The structures of these compounds were elucidated on the basis of spectroscopic analysis, and the absolute configurations of the new compounds 1−4 were determined by the combination of NOESY and TDDFT-ECD calculations and X-ray crystallographic analysis. Additionally, we first determined and reported the absolute configuration of the known monoterpenoid pestalotiolactone A (5) through the X-ray crystallographic experiment. All of these isolated compounds were evaluated for antimicrobial activities against human and aquatic pathogenic bacteria. Compounds 1, 2, 6 and 9 exhibited selective inhibitory activities against zoonotic pathogenic bacteria such as Escherichia coli, Edwardsiella tarda, Vibrio anguillarum and V. harveyi, with MIC values ranging from 1.0 to 8.0 μg/mL.

Toll-like receptors (TLRs) are an important class of pattern recognition receptors (PRRs) that recognize microbial and danger signals. Their downstream signaling upon ligand binding is vital for initiation of the innate immune response. In human and mammalian models, myeloid differentiation factor 88 (MYD88) is known for its central role as an adaptor molecule in interleukin 1 receptor (IL-1R) and TLR signaling. The zebrafish is increasingly used as a complementary model system for disease research and drug screening. Here, we describe a zebrafish line with a truncated version of MyD88 as the first zebrafish mutant for a TLR signaling component. We show that this immune-compromised mutant has a lower survival rate under standard rearing conditions and is more susceptible to challenge with the acute bacterial pathogens Edwardsiella tarda and Salmonella typhimurium. Microarray and quantitative PCR analysis revealed that expression of genes for transcription factors central to innate immunity (including NF-κB and AP-1) and the pro-inflammatory cytokine Il1b, is dependent on MyD88 signaling during these bacterial infections. Nevertheless, expression of immune genes independent of MyD88 in the myd88 mutant line was sufficient to limit growth of an attenuated S. typhimurium strain. In the case of infection with the chronic bacterial pathogen Mycobacterium marinum, we show that MyD88 signaling has an important protective role during early pathogenesis. During mycobacterial infection, the myd88 mutant shows accelerated formation of granuloma-like aggregates and increased bacterial burden, with associated lower induction of genes central to innate immunity. This zebrafish myd88 mutant will be a valuable tool for further study of the role of IL1R and TLR signaling in the innate immunity processes underlying infectious diseases, inflammatory disorders and cancer.

Edwardsiella tarda can cause fatal gastro-/extraintestinal diseases in fish and humans. Overuse of antibiotics has led to antibiotic resistance and contamination in the environment, which highlights the need to find new antimicrobial agents. In this study, the marine peptide-N6 was amidated at its C-terminus to generate N6NH2. The antibacterial activity of N6 and N6NH2 against E. tarda was evaluated in vitro and in vivo; their stability, toxicity and mode of action were also determined. Minimal inhibitory concentrations (MICs) of N6 and N6NH2 against E. tarda were 1.29–3.2 μM. Both N6 and N6NH2 killed bacteria by destroying the cell membrane of E. tarda and binding to lipopolysaccharide (LPS) and genomic DNA. In contrast with N6, N6NH2 improved the stability toward trypsin, reduced hemolysis (by 0.19% at a concentration of 256 μg/mL) and enhanced the ability to penetrate the bacterial outer and inner membrane. In the model of fish peritonitis caused by E. tarda, superior to norfloxacin, N6NH2 improved the survival rate of fish, reduced the bacterial load on the organs, alleviated the organ injury and regulated the immunity of the liver and kidney. These data suggest that the marine peptide N6NH2 may be a candidate for novel antimicrobial agents against E. tarda infections.