Successful phage-antibiotic therapy of P. aeruginosa implant-associated infection in a Siamese cat.

Antibiotic-resistant pathogens are a growing global issue, leading to untreatable infectious diseases in both humans and animals. Personalized bacteriophage (phage) therapy, the use of specific anti-bacterial viruses, is currently a leading approach to combat antibiotic-resistant infections. The implementation of phage therapy has primarily been focused on humans, almost neglecting the impact of such infections on the health and welfare of companion animals. Pets also have the potential to spread resistant infections to their owners or the veterinary staff through zoonotic transmission. Here, we showcase personalized phage-antibiotic treatment of a cat with a multidrug-resistant Pseudomonas aeruginosa implant-associated infection post-arthrodesis surgery. The treatment encompassed a tailored combination of an anti-P. aeruginosa phage and ceftazidime, precisely matched to the pathogen. The phage was topically applied to the surgical wound while the antibiotic was administered intramuscularly. After two treatment courses spanning 7 and 3 weeks, the surgical wound, which had previously remained open for five months, fully closed. To the best of our knowledge, this is the first case of personalized phage therapy application in felines, which provides further evidence of the effectiveness of this approach. The successful outcome paves the way for personalized phage-antibiotic treatments against persistent infections therapy in veterinary practice.

Unraveling the immune functions of large yellow croaker Tmem208 in response to Pseudomonas plecoglossicida: Insights from cloning, expression profiling, and transcriptome analysis.

Pseudomonas plecoglossicida, the causative agent of Visceral White Spot Disease, poses substantial risks to large yellow croaker (Larimichthys crocea) aquaculture. Previous genome-wide association studies (GWAS), directed towards elucidating the resistance mechanisms of large yellow croaker against this affliction, suggested that the transmembrane protein 208 (named Lctmem208) may confer a potential advantage. TMEM proteins, particularly TMEM208 located in the endoplasmic reticulum, plays significant roles in autophagy, ER stress, and dynamics of cancer cell. However, research on TMEM's function in teleost fish immunity remains sparse, highlighting a need for further study. This study embarks on a comprehensive examination of LcTmem208, encompassing cloning, molecular characterization, and its dynamics in immune function in response to Pseudomonas plecoglossicida infection. Our findings reveal that LcTmem208 is highly conserved across teleost species, exhibiting pronounced expression in immune-relevant tissues, which escalates significantly upon pathogenic challenge. Transcriptome analysis subsequent to LcTmem208 overexpression in kidney cells unveiled its pivotal role in modulating immune-responsive processes, notably the p53 signaling pathway and cytokine-mediated interactions. Enhanced phagocytic activity in macrophages overexpressing LcTmem208 underscores its importance in innate immunity. Taken together, this is the first time reported the critical involvement of LcTmem208 in regulating innate immune responses of defensing P. plecoglossicida, thereby offering valuable insights into teleost fish immunity and potential strategies for the selective breeding of disease-resistant strains of large yellow croaker in aquaculture practices.

Sustained release of alginate hydrogel containing antimicrobial peptide Chol-37(F34-R) in vitro and its effect on wound healing in murine model of Pseudomonas aeruginosa infection.

BACKGROUND: Antibiotic resistance is a significant public health concern around the globe. Antimicrobial peptides exhibit broad-spectrum and efficient antibacterial activity with an added advantage of low drug resistance. The higher water content and 3D network structure of the hydrogels are beneficial for maintaining antimicrobial peptide activity and help to prevent degradation. The antimicrobial peptide released from hydrogels also hasten the local wound healing by promoting epithelial tissue regeneration and granulation tissue formation. OBJECTIVE: This study aimed at developing sodium alginate based hydrogel loaded with a novel antimicrobial peptide Chol-37(F34-R) and to investigate the characteristics in vitro and in vivo as an alternative antibacterial wound dressing to treat infectious wounds. METHODS: Hydrogels were developed and optimized by varying the concentrations of crosslinkers and subjected to various characterization tests like cross-sectional morphology, swelling index, percent water contents, water retention ratio, drug release and antibacterial activity in vitro, and Pseudomonas aeruginosa infected wound mice model in vivo. RESULTS: The results indicated that the hydrogel C proved superior in terms of cross-sectional morphology having uniformly sized interconnected pores, a good swelling index, with the capacity to retain a higher quantity of water. Furthermore, the optimized hydrogel has been found to exert a significant antimicrobial activity against bacteria and was also found to prevent bacterial infiltration into the wound site due to forming an impermeable barrier between the wound bed and external environment. The optimized hydrogel was found to significantly hasten skin regeneration in animal models when compared to other treatments in addition to strong inhibitory effect on the release of pro-inflammatory cytokines (interleukin-1ß and tumor necrosis factor-α). CONCLUSIONS: Our results suggest that sodium alginate -based hydrogels loaded with Chol-37(F34-R) hold the potential to be used as an alternative to conventional antibiotics in treating infectious skin wounds.

Characterization of 4 deletion mutants of Pseudomonas plecoglossicida and their potential for live attenuated vaccines in large yellow croaker (Larimichthys crocea).

To search for live attenuated vaccines (LAV) candidates against Pseudomonas plecoglossicida, the causative agent of the visceral granulomas disease in farmed large yellow croaker (Larimichthys crocea), two type Ⅵ secretion systems (T6SS) and a predicted α/ß fold family hydrolase encoding gene, ORF4885 were targeted to construct deletion mutants. The biological profiles of 4 mutants were characterized; LD50 to the croakers detected, in vivo survival post-infection investigated， relative percent of survival (RPS) of the croakers 28d post-vaccination determined, and transcription of five immunity-related genes of the treated fish was quantified. On comparison to the WT, the mutants revealed similar growth curves in 11h; swarming motility of Δ4885 declined significantly at 72h post-incubation (P < 0.05); ΔS1Δ4885 showed significantly poor biofilm formation and weak resistance to fish serum bactericidal activity (P < 0.05). LD50 of the mutants were much higher than the WT, indication of strong virulence attenuation; in vivo survival test showed the mutant ΔS1Δ4885 and ΔS1ΔS3 were eliminated by the host 10d post-infection, demonstration of the safety and potentiality to be LAV candidates. Immunization with the mutant ΔS1Δ4885 provided higher RPS than ΔS1ΔS3. Transcription of IgT was significant in all immunized groups while IgM increased only in intraperitoneally injected groups. This study successfully searched a quite safe and strong immunogenic LAV candidate to defeat P. plecoglossicida infection.

Polyactin A and CpG enhance inactivated Pseudomonas plecoglossicida vaccine potency in large yellow croaker (Larimichthys crocea).

Pseudomonas plecoglossicida is the causative agent of visceral granulomas disease (VGD) in large yellow croaker (LYC, Larimichthys crocea) farming. However, multi-antibiotic resistant of P. plecoglossicida creates an urgent need of an efficient vaccine to combat this pathogen. In this study, an inactivated vaccine added polyactin (PA), CpG-riched plasmid (pCpG) and aluminum adjuvant (Al) was developed. As a result, its relative percentage survival (RPS) against P. plecoglossicida were up to 64%. Comparatively, RPS of groups that vaccinated with vaccines adjuvanted with PA and Al or CpG and Al were 49% and 39%. However, an interesting result that the vaccine combined with PA, CpG and Al did not show the strongest activation of total serum protein and antibody levels in serum among three vaccinated groups. According to expressions of some cellular immune related genes, we found that the inactivated vaccine combined with PA, CpG and Al was more likely to induce a cellular immune response rather than humoral immune response. Totally, our study demonstrated that the mixture of PA, CpG and aluminum adjuvant is a potential adjuvant system for LYC vaccine development.

Immune responses and inorganic ion transport regulations of Epinephelus coioides in response to L321_RS13075 gene of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is a well-known pathogen of viscera granulomas disease in fish, which has led to severe economic losses. In our previous study, L321_RS13075 was predicted to be a key virulence gene of P. plecoglossicida during the host-pathogen interaction with Epinephelus coioides. To investigate the role of L321_RS13075 in the regulation of virulence in P. plecoglossicida, a L321_RS13075 knock-down strain was constructed. And a significant reduction in the ability of colonization, intracellular survival, motility, biofilm formation, and adhesion was detected in the L321_RS13075 knock-down strain. Compared with the wild-type strain, the silence of L321_RS13075 in P. plecoglossicida resulted in a significant change in the transcriptome of infected Epinephelus coioides (E. coioides). Results of COG and GO analysis on E. coioides showed that genes related to immune responses and inorganic ion transport were significantly affected by L321_RS13075 of P. plecoglossicida. Meanwhile, the interactions of the genes related to immune responses and inorganic ion transport were predicted, and the important hub genes were identified. Taken together, the results indicated that L321_RS13075 was a virulent gene of P. plecoglossicida, which significantly affected the immune responses and inorganic ion transport in E. coioides.

Dietary plant pigment on blood-digestive physiology, antioxidant-immune response, and inflammatory gene transcriptional regulation in spotted snakehead (Channa punctata) infected with Pseudomonas aeruginosa.

The current study addressed to investigate the effect of lycopene (LYC) on blood physiology, digestive-antioxidant enzyme activity, specific-nonspecific immune response, and inflammatory gene transcriptional regulation (cytokines, heat shock proteins, vitellogenins) in spotted snakehead (Channa punctata) against Pseudomonas aeruginosa. In unchallenged and challenged fish treated with 200 mg LYC enriched diet the growth performance and digestive-antioxidant enzymes increased after 30 days, whereas with inclusion of 100 or 400 mg LYC in the diets, the increase manifested on or after 45 days. No mortality in fish treated with any LYC diet against P. aeruginosa was revealed. In the unchallenged and challenged fish the phagocytic (PC) activity in head kidney (HK) and spleen were significantly enhanced when fed the control diet or other LYC diets, whereas the respiratory burst (RB) activity and nitric oxide (NO) production significantly increased when fed the 200 mg diet for 45 and 60 days. Similarly, the lysozyme (Lyz) activity in the HK and spleen, and total Ig content in serum were significantly higher in both groups fed the 200 mg LYC diet for 15, 45, and 60 days. Heat shock protein (Hsp 70) was significantly improved in the uninfected group fed the 200 mg LYC diet for 45 and 60 days, but Hsp27 did not significantly change among the experimental groups at any time points. TNF-α and IL-6 mRNA pro-inflammatory cytokine expression significantly increased in both groups fed the 200 mg LYC diet after 45 and 60 days, while the IL-12 mRNA expression was moderate in both groups fed the same diet for 60 days. The IL-10 did not significant mRNA expression between groups at any sampling. The iNOS and NF-κB mRNA expression was pointedly high in both groups fed the 200 mg LYC diet on day 45 and 60. Vitellogenin A (VgA) mRNA was significantly higher in the uninfected fish fed the 100 and 200 mg LYC diets for 45 and 60 days, but VgB did not reveal significant difference between the treatment groups at any time points. The present results suggest that supplementation of LYC at 200 mg significantly modulate the blood physiology, digestive-antioxidant enzymes, specific-nonspecific immune parameters, and cytokines, Hsp, and vitellogenins in spotted snakehead against P. aeruginosa.

The contributions of fliG gene to the pathogenicity of Pseudomonas plecoglossicida and pathogen-host interactions with Epinephelus coioides.

Pseudomonas plecoglossicida is a Gram-negative aerobic rod-shaped bacterium with polar flagella. It is the causative agent of visceral white spot disease in cultured fish, resulting in serious economic losses. In our previous study, RNA sequencing showed that the expression of the fliG gene in P. plecoglossicida is significantly up-regulated during infection of orange-spotted grouper (Epinephelus coioides). In this study, four P. plecoglossicida RNA interference (RNAi) mutants were successfully constructed by linking four short hairpin RNAs (shRNAs), which target different sites of the fliG gene, to pCM130/tac, respectively. The mRNA expression levels of the fliG gene in P. plecoglossicida were significantly decreased in four mutants. The shRNA-335 mutant (fliG-RNAi strain) showed the best silencing efficiency (88.2%) and was thus chosen for further analysis. Electron microscopy indicated that the flagella of the fliG-RNAi strain of P. plecoglossicida were shorter and finer than those of the wild type strain. The fliG-RNAi strain also showed significantly decreased mobility, chemotaxis, adhesion, and biofilm formation. Furthermore, compared with wild type strain infection, E. coioides infected with the fliG-RNAi strain exhibited a 0.5-d delay in the time of first death and 55% reduction in accumulated mortality, as well as milder splenic symptoms. RNAi of the fliG gene significantly affected the transcriptomes of both pathogen and host in the infected spleens of E. coioides. KEGG analysis revealed that the flagellar assembly pathway, bacterial chemotaxis pathway, and starch and sucrose metabolism pathway were significantly enriched in the pathogen at 3 days post infection (dpi). In contrast, the complement and coagulation cascade pathway and antigen processing and presentation pathway were significantly enriched in the host at 3 dpi. More immune-related pathways were enriched at 5 dpi and more differentially expressed genes were found in the complement and coagulation cascade and antigen processing and presentation pathways. Cytokine-cytokine receptor interaction, hematopoietic cell lineage, and IgA-producing intestinal immune network pathways were significantly enriched in the host at 5 dpi. These results indicate that fliG is an important virulence gene of P. plecoglossicida and contributes to the pathogenicity of P. plecoglossicida as well as pathogen-host interactions with E. coioides.

Preliminary studies on the different roles of T6SSs in pathogenicity of Pseudomonas plecoglossicida NB2011.

Pseudomonas plecoglossicida, the causative agent of visceral granulomas in the large yellow croaker (Larimichthys crocea) in China, encodes three sets of type Ⅵ secretion systems (T6SS1-3). The purpose of this study was to characterize the different roles of T6SSs involved in infection. In-frame deletion of T6SSs was constructed, which resulted in 8 mutants. Competition against E. coli DH5α, virulence against the croaker and in vivo survival ability of the mutants were tested. The expression and secretion of Hcp by P. plecoglossicida NB2011 were investigated. The results showed T6SS2 mutant failed to inhibit the growth of E. coli, which is an indication of T6SS2 acting against environmental bacteria. The LD50 value of T6SS1 mutant strongly increased; T6SS2 and T6SS3 mutants were similar to that of the wild type; and the virulence of double deletion or triple deletion mutant was drastically alleviated, indicating that T6SS1 being one of the major virulence factors, and T6SS2 and T6SS3 directly or indirectly being involved in the pathogenicity. T6SS1 mutant disappeared in the fish spleen in 3 days, while other strains kept increasing, indicating the T6SS1 stimulation bacteria replication in vivo. Hcp1 secreted at 12-28°C and Hcp2 secreted at 12-35°C, while Hcp3 secretion not detected in vitro. This study has thrown some insights on the understanding of pathogenicity mechanisms of this pathogen.

The Zinc Nutritional Immunity of Epinephelus coioides Contributes to the Importance of znuC During Pseudomonas plecoglossicida Infection.

Previously, the dual RNA-seq was carried out in a Pseudomonas plecoglossicida- Epinephelus coioides infection model to investigate the dynamics of pathogen-host interplay in vivo. ZnuC, a member of ZnuCBA Zn importer, was found transcriptionally up-regulated during infection. Thus, this study aimed to assess its role during the trade-off for Zn between host and P. plecoglossicida. ICP-MS analysis and fluorescent staining showed that Zn was withheld from serum and accumulated in the spleen, with increased Zn uptake in the Golgi apparatus of macrophages after infection. Additionally, growth assay, macrophage infection and animal infection after gene knockout / silencing revealed that znuC was necessary for growth in Zn-limiting conditions, colonization, intracellular viability, immune escape and virulence of P. plecoglossicida. Further analysis with dual RNA-seq revealed associations of host's Zn nutritional immunity genes with bacterial Zn assimilation genes. IL6 and ZIP4 played key roles in this network, and markedly affected znuB expression, intracellular viability and immune escape, as revealed by gene silencing. Moreover, EMSA and GFP reporter gene analysis showed that Fur sensed changes in Fe concentration to regulate znuCBA in P. plecoglossicida. Jointly, these findings suggest a trade-off for Zn between host and P. plecoglossicida, while ZnuC is important for P. plecoglossicida Zn acquisition.

An outbreak of visceral white nodules disease caused by Pseudomonas plecoglossicida at a water temperature of 12°C in cultured large yellow croaker (Larimichthys crocea) in China.

Visceral white nodules disease (VWND) caused by Pseudomonas plecoglossicida is a common disease in cage-farmed large yellow croaker (Larimichthys crocea) in China. VWND usually occurred at water temperature of 16-19℃, resulting in high mortality in farmed large yellow croaker. Now, P. plecoglossicida as its pathogen has been considered nonpathogenic at 7-12℃. During February 2019, an infectious disease outbreak was observed in cage-farmed large yellow croaker at a water temperature of 12℃ in Ningde, China. This disease is characterized by white granulomatous lesions in internal organs of the diseased fish, which was similar with the symptoms of the VWND in large yellow croaker. Then, we isolated a bacterial strain named PQLYC4 from visceral lesions of the diseased fish. The experimental infection studies demonstrated that the strain PQLYC4 was the pathogen of the disease, which was further identified as P. plecoglossicida by the analysis of morphology, 16s rRNA gene homology and average nucleotide identity based on the whole genome sequence. Our results revealed that P. plecoglossicida strain PQLYC4 could cause the outbreak of the VWND at 12℃, a water temperature lower than that reported previously, thus providing new knowledges of prevalence and prevention of the VWND in large yellow croaker.

Equine pastern vasculitis in a horse associated with a multidrug-resistant Pseudomonas aeruginosa isolate.

BACKGROUND: Equine pastern vasculitis is an uncommon disorder in horses. Underlying causes are difficult to assess, especially bacterial infections. CLINICAL SUMMARY: A 13-year-old French saddle gelding horse presented for evaluation of a six weeks history of pastern dermatitis. Histopathological examination of skin biopsy samples revealed small vessel vasculitis. A pure growth of a multidrug-resistant Pseudomonas aeruginosa (MRPA) was obtained from a deep skin biopsy. Clinical remission was observed after a six week course of enrofloxacin and lesions did not recur. CONCLUSIONS AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first report of a pastern vasculitis associated with MRPA and successfully treated with a six week course of enrofloxacin.

Aryl hydrocarbon receptor is required for immune response in Epinephelus coioides and Danio rerio infected by Pseudomonas plecoglossicida.

Aryl hydrocarbon receptor (AhR), a ligand-dependent transcriptional factor that responds to environmental chemicals, has been recently found to be closely associated with immune response in mammals. Pseudomonas plecoglossicida (P. plecoglossicida) is a temperature-dependent bacterial pathogen of visceral white spot disease in fish. Using dual RNA-seq, we previously evaluated the expression levels of ahr1a, ahr1b, ahr2 and cyp1a in the spleen of Epinephelus coioides at different time points after infection with P. plecoglossicida. In the present study, the expression levels of ahr1a, ahr1b, ahr2 and cyp1a in different organs of E. coioides and Danio rerio showed similar trends after being infected by P. plecoglossicida. It also was noted that liver, intestine, spleen, and heart were the most obviously affected organs, and ahr2 particularly showed a dramatically increase in the spleen. Subsequently, macrophages of E. coioides were isolated, and then infected by P. plecoglossicida, followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay, which revealed that the expression level of ahr1a in macrophages was significantly down-regulated, while expression levels of ahr1b, ahr2 and cyp1a were noticeably up-regulated. Eventually, it was noted that ahr1b and ahr2 were knocked-down in macrophages, and intracellular survival rate and immune escape rate of P. plecoglossicida were markedly improved. Taken together, ahr1a, ahr1b, ahr2 and cyp1a participate in the immune response to P. plecoglossicida in different organs of fish, while ahr1b and ahr2 may play pivotal roles in the immune response of spleen and macrophages.

Patulous Eustachian tube and palatine defect in a Dachshund with chronic unilateral otitis externa and otitis media.

BACKGROUND: Patulous Eustachian tube (pET) is a rare dysfunction of the Eustachian tube described in humans. It is characterized by failure of the ET to close, resulting in unrestricted passage of air, sound and material between the nasopharynx and the middle ear. OBJECTIVE: To report a case of pET associated with otitis in a dog. ANIMAL: A 6-year old-female spayed Dachshund dog. METHODS AND MATERIALS: Otoscopic examination, cytological evaluation, culture and susceptibility, computerized tomography (CT), video-otoscopic flushing and surgery. RESULTS: Left ear otoscopic examination revealed erythema, purulent frothy discharge, ceruminous gland hyperplasia, stenosis and a partial tear of the tympanum. Cytological evaluation from the left external canal showed neutrophils, macrophages, rods and cocci. Aerobic culture showed predominantly multidrug-resistant Pseudomonas aeruginosa. The CT findings of the left ear included chronic changes in the external canal, marked lysis of the tympanic bulla and marked dilation of the ET. During video-otoscope flushing, saline drained through the mouth. Bilateral incomplete hypoplasia of the soft palate was noted. Total ear canal ablation and bulla osteotomy with ET dissection were curative. Histopathological findings were compatible with chronic otitis externa (OE) and media. CONCLUSION AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first case of pET described in animals. The ET dysfunction and palatine defect were likely the cause of the otitis in this dog. Clinicians should investigate pET in animals with signs of OE characterized by frothy liquid and food fragments in the ear canal in addition to sneezing after drinking water.

Mucoid Pseudomonas aeruginosa infection in a cat with severe chronic rhinosinusitis.

A 6-year-old male neutered Bengal cat was presented to the University of Wisconsin Veterinary Care Hospital with a history of severe chronic rhinitis that was unresolved from kittenhood. In weeks prior to presentation, the cat's upper respiratory signs had significantly worsened and a left-sided facial swelling overlying the left frontal sinus was noted. Skull computed tomography, rhinoscopy, bilateral nasal biopsies, bacterial and fungal cultures of fluid from the left frontal sinus, and cryptococcal fungal antigen testing were performed. The cat was diagnosed with severe chronic rhinosinusitis and determined to have an infection with a mucoid variant of Pseudomonas aeruginosa (P aeruginosa). This case highlights an atypical cytomorphologic appearance of the well-known bacterial pathogen, P aeruginosa, an appearance that could be confused cytologically with other microorganisms, such as septate fungi. Mucoid variants of P aeruginosa are often associated with progressive lung or airway disease in people with cystic fibrosis and have not been previously documented in feline respiratory tract disease. This report also presents a brief review of chronic rhinosinusitis (CRS) in cats and describes a novel interventional treatment approach to feline CRS via sinusotomy and sinus flushing for severely affected cats.

Dual RNA-Seq uncovers the function of an ABC transporter gene in the host-pathogen interaction between Epinephelus coioides and Pseudomonas plecoglossicida.

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. The expression of an ABC transporter gene-L321_23611 of P. plecoglossicida at 18 °C was found significant higher than those at 28 °C by RNA-seq and qRT-PCR. RNAi significantly reduced the content of L321_23611 mRNA in P. plecoglossicida with a maximal decrease of 89.2%. Compared with the wild type strain, the infection of L321_23611-RNAi strain resulted in the reduction in mortality and the onset time delay of a kind of marine teleosts, Epinephelus coioides. The results of dual RNA-seq showed that the RNAi of L321_23611 resulted in a significant change in both pathogen and host transcriptome in the spleens of infected E. coioides. The result of GO and KEGG analysis from dual RNA-seq data showed both host genes of chemokine signaling pathway, coagulation and complement system, hematopoietic cell lineage pathway as well as hemoglobin complex GO term and pathogenic genes of bacterial-type flagellum-dependent cell mortality GO term and flagellar assembly, biosynthesis of amino acids and lysine biosynthesis systems pathways were mainly affected by L321_23611 gene of P. plecoglossicida. The results indicated that: 1. ABC transporter gene-L321_23611 was a virulent gene of P. plecoglossicida. 2. Both the activation of the host immune pathways and depression of pathogenic virulence-related pathways facilitated E. coioides to remove L321_23611-RNAi strain than the wild type strain of P. plecoglossicida.

Protection against Pseudomonas plecoglossicida in Epinephelus coioides immunized with a cspA1-knock-down live attenuated vaccine.

Pseudomonas plecoglossicida is well-known as the cause of viscera granulomas disease in fish. In this study, a cspA1 knock-down strain was constructed and tested in Epinephelus coioides to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the cspA1 knock-down strain caused a significant reduction in the ability of biofilm formation, motility, adhesion and virulence. E. coioides vaccinated with cspA1 knock-down strain were more tolerant of the infection by wild-type P. plecoglossicida. The relative percent survival value of E. coioides vaccinated with cspA1 knock-down strain reached 80% after challenging with wild-type P. plecoglossicida. In the meanwhile, the expression level of genes associated with immunity, including IL-1ß, IgM, MHC-I and MHC-II, was up-regulated after vaccination, indicating that the cspA1 knock-down strain can induce effective and durable immune response in E. coioides and it may be an effective attenuated live vaccine candidate for the prevention of infections by P. plecoglossicida.

Characterization, mechanism of action and optimization of activity of a novel peptide-peptoid hybrid against bacterial pathogens involved in canine skin infections.

Integumentary infections like pyoderma represent the main reason for antimicrobial prescription in dogs. Staphylococcus pseudintermedius and Pseudomonas aeruginosa are frequently identified in these infections, and both bacteria are challenging to combat due to resistance. To avoid use of important human antibiotics for treatment of animal infections there is a pressing need for novel narrow-spectrum antimicrobial agents in veterinary medicine. Herein, we characterize the in vitro activity of the novel peptide-peptoid hybrid B1 against canine isolates of S. pseudintermedius and P. aeruginosa. B1 showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius and P. aeruginosa isolates as well rapid killing kinetics. B1 was found to disrupt the membrane integrity and affect cell-wall synthesis in methicillin-resistant S. pseudintermedius (MRSP). We generated 28 analogues of B1, showing comparable haemolysis and MICs against MRSP and P. aeruginosa. The most active analogues (23, 26) and B1 were tested against a collection of clinical isolates from canine, of which only B1 showed potent activity. Our best compound 26, displayed activity against P. aeruginosa and S. pseudintermedius, but not the closely related S. aureus. This work shows that design of target-specific veterinary antimicrobial agents is possible, even species within a genus, and deserves further exploration.

Grape pomace flour alleviates Pseudomonas aeruginosa-induced hepatic oxidative stress in grass carp by improving antioxidant defense.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen in aquaculture systems being associated to extensive liver damage caused by oxidative stress in both marine and freshwater fish. Dietary supplementation with natural antioxidants is considered a rational strategy to prevent hepatic diseases involved with oxidative stress. Bio-residues resulting from the wine industry, such as grape pomace, are potential sources of bioactive phenolic compounds that can be applied as supplement for animal production. Thus, the aim of this study was to evaluate whether dietary supplementation with grape pomace flour (GPF) was able to prevent or reduce the hepatic oxidative damage of grass carp, Ctenopharyngodon idella, experimentally infected by P. aeruginosa. Hepatic reactive oxygen species (ROS), metabolites of nitric oxide (NOx), thiobarbituric acid reactive substances, and protein carbonylation levels were higher in fish experimentally infected by P. aeruginosa compared to the control group. Hepatic superoxide dismutase and catalase activities and antioxidant capacity against peroxyl radical levels were also higher in fish experimentally infected by P. aeruginosa compared to the control group. Dietary supplementation with 300 mg/kg GPF prevented all alterations elicited by P. aeruginosa, with the exception of protein carbonylation levels. The dietary supplementation with 150 mg/kg GPF was not able to avoid alteration of the analyzed variables, being results similar to those infected (positive control). Based on these results, dietary supplementation with 300 mg/kg GPF prevented P. aeruginosa-induced liver damage in grass carp, and this protective effect occurred through prevention on excessive ROS and NOx production, as well as via prevention of lipid damage. Moreover, 300 mg/kg GPF exerted its hepatoprotective effects by improving enzymatic and non-enzymatic antioxidant defense system. In summary, this supplementation can be an interesting approach to prevent P. aeruginosa-induced liver damage.

Attenuation of quorum sensing controlled virulence factors and biofilm formation in Pseudomonas aeruginosa by pentacyclic triterpenes, betulin and betulinic acid.

The production of virulence determinants and biofilm formation in numerous pathogens is regulated by the cell-density-dependent phenomenon, Quorum sensing (QS). The QS system in multidrug resistant opportunistic pathogen, P. aeruginosa constitutes of three main regulatory circuits namely Las, Rhl, and Pqs which are closely linked to its pathogenicity and establishment of chronic infections. In spite intensive antibiotic therapy, P. aeruginosa continue to be an important cause of nosocomial infections and also the major cause of mortality in Cystic Fibrosis patients with 80% of the adults suffering from chronic P. aeruginosa infection. Hence, targeting QS circuit offers an effective intervention to the ever increasing problem of drug resistant pathogens. In the present study, the pentacyclic triterpenes i.e. Betulin (BT) and Betulinic acid (BA) exhibited significant attenuation in production of QS-regulated virulence factors and biofilm formation in P. aeruginosa, at the sub-lethal concentration. The test compound remarkably interfered in initial stages of biofilm development by decreasing the exopolysaccharide production and cell surface hydrophobicity. Based on the in vivo studies, the test compounds notably enhanced the survival of Caenorhabditis elegans infected with P. aeruginosa. Furthermore, molecular docking analysis revealed that BT and BA can act as a strong competitive inhibitor for QS receptors, LasR and RhlR. The findings suggest that BT and BA can serve as potential anti-infectives in the controlling chronic infection of P. aeruginosa.