An unnatural amino acid dependent, conditional Pseudomonas vaccine prevents bacterial infection.

Live vaccines are ideal for inducing immunity but suffer from the need to attenuate their pathogenicity or replication to preclude the possibility of escape. Unnatural amino acids (UAAs) provide a strategy to engineer stringent auxotrophies, yielding conditionally replication incompetent live bacteria with excellent safety profiles. Here, we engineer Pseudomonas aeruginosa to maintain auxotrophy for the UAA p-benzoyl-L-phenylalanine (BzF) through its incorporation into the essential protein DnaN. In vivo evolution using an Escherichia coli-based two-hybrid selection system enabled engineering of a mutant DnaN homodimeric interface completely dependent on a BzF-specific interaction. This engineered strain, Pa Vaccine, exhibits undetectable escape frequency (<10-11) and shows excellent safety in naïve mice. Animals vaccinated via intranasal or intraperitoneal routes are protected from lethal challenge with pathogenic P. aeruginosa PA14. These results establish UAA-auxotrophic bacteria as promising candidates for bacterial vaccine therapy and outline a platform for expanding this technology to diverse bacterial pathogens.

A computational approach to developing a multi-epitope vaccine for combating Pseudomonas aeruginosa-induced pneumonia and sepsis.

Pseudomonas aeruginosa is a complex nosocomial infectious agent responsible for numerous illnesses, with its growing resistance variations complicating treatment development. Studies have emphasized the importance of virulence factors OprE and OprF in pathogenesis, highlighting their potential as vaccine candidates. In this study, B-cell, MHC-I, and MHC-II epitopes were identified, and molecular linkers were active to join these epitopes with an appropriate adjuvant to construct a vaccine. Computational tools were employed to forecast the tertiary framework, characteristics, and also to confirm the vaccine's composition. The potency was weighed through population coverage analysis and immune simulation. This project aims to create a multi-epitope vaccine to reduce P. aeruginosa-related illness and mortality using immunoinformatics resources. The ultimate complex has been determined to be stable, soluble, antigenic, and non-allergenic upon inspection of its physicochemical and immunological properties. Additionally, the protein exhibited acidic and hydrophilic characteristics. The Ramachandran plot, ProSA-web, ERRAT, and Verify3D were employed to ensure the final model's authenticity once the protein's three-dimensional structure had been established and refined. The vaccine model showed a significant binding score and stability when interacting with MHC receptors. Population coverage analysis indicated a global coverage rate of 83.40%, with the USA having the highest coverage rate, exceeding 90%. Moreover, the vaccine sequence underwent codon optimization before being cloned into the Escherichia coli plasmid vector pET-28a (+) at the EcoRI and EcoRV restriction sites. Our research has developed a vaccine against P. aeruginosa that has strong binding affinity and worldwide coverage, offering an acceptable way to mitigate nosocomial infections.

Immunostimulatory chimeric protein encapsulated in gelatin nanoparticles elicits protective immunity against Pseudomonas aeruginosa respiratory tract infection.

This study presents the design and fabrication of an innovative vaccine candidate targeting Pseudomonas aeruginosa (P. aeruginosa). The vaccine consists of gelatin nanoparticles (GNPs) encapsulating a chimeric protein (CP) derived from the ExoS and OprI proteins from P. aeruginosa. The physicochemical properties of the GNPs were assessed using dynamic light scattering (DLS) and electron microscopy. The toxicity, encapsulation efficacy, release profile, and effectiveness of CP-encapsulated GNPs (CP-GNPs) in an animal model were investigated. The resulting nanovaccine demonstrated uniform spherical particles with an average size of 135 nm and an encapsulation efficiency of 85 %. The release assay revealed that 23 % of the antigen was released from the CP-GNPs after 20 days. The GNPs did not exhibit any toxic effects on L929 cells in vitro. The formulation induced both systemic and mucosal antibody responses. Additionally, CP-GNPs stimulated cytokine responses, including IFN-γ, IL-4, and IL-17, indicating the induction of both humoral (Th2) and cellular (Th1) responses. The CP-encapsulated GNPs formulation effectively protected the mice lungs against experimental respiratory tract infection, reducing colony count and inflammation. These findings suggest that CP-GNPs hold promise as a potential strategy for preventing respiratory tract infections caused by P. aeruginosa. Further research is needed to explore its clinical application.

Selection of vaccine candidates against Pseudomonas koreensis using reverse vaccinology and a preliminary efficacy trial in Empurau (Tor tambroides).

This study marks the first utilization of reverse vaccinology to develop recombinant subunit vaccines against Pseudomonas koreensis infection in Empurau (Tor tambroides). The proteome (5538 proteins) was screened against various filters to prioritize proteins based on features that are associated with virulence, subcellular localization, transmembrane helical structure, antigenicity, essentiality, non-homology with the host proteome, molecular weight, and stability, which led to the identification of eight potential vaccine candidates. These potential vaccine candidates were cloned and expressed, with six achieving successful expression and purification. The antigens were formulated into two distinct vaccine mixtures, Vac A and Vac B, and their protective efficacy was assessed through in vivo challenge experiments. Vac A and Vac B demonstrated high protective efficacies of 100 % and 81.2 %, respectively. Histological analyses revealed reduced tissue damage in vaccinated fish after experimental infection, with Vac A showing no adverse effects, whereas Vac B exhibited mild degenerative changes. Quantitative real-time PCR results showed a significant upregulation of TNF-α and downregulation of IL-1ß in the kidneys, spleen, gills, and intestine in both Vac A- and Vac B-immunized fish after challenged with P. koreensis. Additionally, IL-8 exhibits tissue-specific differential expression, with significant upregulation in the kidney, gills, and intestine, and downregulation in the spleen, particularly notable in Vac A-immunized fish. The research underscores the effectiveness of the reverse vaccinology approach in fish and demonstrates the promising potential of Vac A and Vac B as recombinant subunit vaccines.

Palmitic acid-capped MIL-101-Al as a nano-adjuvant to amplify immune responses against Pseudomonas aeruginosa.

As a highly contagious opportunistic pathogen, Pseudomonas aeruginosa (P. aeruginosa) is one of the main causes of healthcare-associated infections. The drug-resistant nature of P. aeruginosa can render antibiotic treatments ineffective, leading to a high morbidity and mortality. Higher specificity and reduced toxicity are features of immunotherapy, which can generate robust immune responses and preserve long-term immunological memory to completely eradicate infections. In this study, we developed a type of P. aeruginosa vaccine based on a metal-organic framework. Specifically, MIL-101-Al nanoparticles were synthesized to encapsulate antigens derived from the bacterial lysate (BL) of PAO1, a drug-resistant P. aeruginosa, and the adjuvant unmethylated cytosine-phosphate-guanine oligonucleotide (CpG), which were then modified with palmitic acid (PAA) to obtain MIL-BC@PAA. The stability and biocompatibility were significantly increased by capping with PAA. Moreover, MIL-BC@PAA showed significantly enhanced uptake by antigen presenting cells (APCs), and promoted their maturation. Importantly, immunity studies revealed the greatly elicited antigen-specific humoral and cellular responses, and a protection rate of about 70% was observed in P. aeruginosa-challenged mice. Overall, these results demonstrate the promising potential of MIL-BC@PAA as an ideal nanovaccine for P. aeruginosa vaccination.

Development of a nano-emulsion based multivalent protein subunit vaccine against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen responsible for severe hospital acquired infections in immunocompromised and elderly individuals. Emergence of increasingly drug resistant strains and the absence of a broad-spectrum prophylactic vaccine against both T3SA+ (type III secretion apparatus) and ExlA+/T3SA- Pa strains worsen the situation in a post-pandemic world. Thus, we formulated a candidate subunit vaccine (called ExlA/L-PaF/BECC/ME) against both Pa types. This bivalent vaccine was generated by combining the C-terminal active moiety of exolysin A (ExlA) produced by non-T3SA Pa strains with our T3SA-based vaccine platform, L-PaF, in an oil-in-water emulsion. The ExlA/L-PaF in ME (MedImmune emulsion) was then mixed with BECC438b, an engineered lipid A analogue and a TLR4 agonist. This formulation was administered intranasally (IN) to young and elderly mice to determine its potency across a diverse age-range. The elderly mice were used to mimic the infection seen in elderly humans, who are more susceptible to serious Pa disease compared to their young adult counterparts. After Pa infection, mice immunized with ExlA/L-PaF/BECC/ME displayed a T cell-mediated adaptive response while PBS-vaccinated mice experienced a rapid onset inflammatory response. Important genes and pathways were observed, which give rise to an anti-Pa immune response. Thus, this vaccine has the potential to protect aged individuals in our population from serious Pa infection.

Anti-Pseudomonal Effect of Nephrite-Impregnated Contact Lenses.

PURPOSE: This study compared the anti-pseudomonal effects between nephrite-impregnated contact lenses (CLs) and conventional and cosmetic CLs. METHODS: After inoculation with Pseudomonas aeruginosa (P.aeruginosa), we counted the number of bacteria on the CL surface and observed each surface using atomic force microscopy (AFM) and scanning electron microscopy (SEM). To estimate potential harm of nephrite-impregnated CLs, we conducted a safety test using a rabbit model, treated with all CL types. RESULTS: Both conventional and cosmetic CLs (n = 258 ± 2.9 × 104, 368 ± 2.2 × 104) showed significantly decreased number of attached bacteria when compared with those without nephrite impregnation (n = 134 ± 0.8 × 104, 238 ± 2.5 × 104, p < 0.0001, respectively). AFM and SEM revealed that P. aeruginosa was less attached to the nephrite-impregnated CLs than to the conventional and cosmetic CLs, although those with nephrite impregnation had rougher surface. In the safety test, there were no significant differences in the findings between four groups, and the clarity and stability of all corneas were preserved. CONCLUSIONS: Nephrite may be used as a next-generation substance to reduce infectious keratitis caused by P. aeruginosa when added to CLs.

AAV-vectored expression of monospecific or bispecific monoclonal antibodies protects mice from lethal Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.

Eye infection risks from Pseudomonas aeruginosa via hand soap and eye drops.

Eye infections from bacterial contamination of bulk-refillable liquid soap dispensers and artificial tear eye drops continue to occur, resulting in adverse health outcomes that include impaired vision or eye enucleation. Pseudomonas aeruginosa (P. aeruginosa), a common cause of eye infections, can grow in eye drop containers and refillable soap dispensers to high numbers. To assess the risk of eye infection, a quantitative microbial risk assessment for P. aeruginosa was conducted to predict the probability of an eye infection for two potential exposure scenarios: (i) individuals using bacteria-contaminated eye drops and (ii) contact lens wearers washing their hands with bacteria-contaminated liquid soap prior to placing the lens. The median risk of an eye infection using contaminated eye drops and hand soap for both single and multiple exposure events (per day) ranged from 10-1 to 10-4, with contaminated eye drops having the greater risk. The concentration of P. aeruginosa was identified as the parameter contributing the greatest variance on eye infection risk; therefore, the prevalence and level of bacterial contamination of the product would have the greatest influence on health risk. Using eye drops in a single-use container or with preservatives can mitigate bacterial growth, and using non-refillable soap dispensers is recommended to reduce contamination of hand soap. Given the opportunistic nature of P. aeruginosa and its ability to thrive in unique environments, additional safeguards to mitigate bacterial growth and exposure are warranted.IMPORTANCEPseudomonas aeruginosa (P. aeruginosa) is a pathogen that can persist in a variety of unusual environments and continues to pose a significant risk for public health. This quantitative microbial risk assessment (QMRA) estimates the potential human health risks, specifically for eye infections, associated with exposure to P. aeruginosa in bacteria-contaminated artificial tear eye drops and hand soap. This study applies the risk assessment framework of QMRA to evaluate eye infection risks through both consumer products. The study examines the prevalence of this pathogen in eye drops and soap, as well as the critical need to implement measures that will mitigate bacterial exposure (e.g., single-use soap dispensers and eye drops with preservatives). Additionally, limitations and challenges are discussed, including the need to incorporate data regarding consumer practices, which may improve exposure assessments and health risk estimates.

The bacterial pathogen Pseudomonas plecoglossicida, its epidemiology, virulence factors, vaccine development, and host-pathogen interactions.

OBJECTIVE: Pseudomoans plecoglossicida has been identified as a fish pathogen since 2000 and has caused serious infections in cultured Large Yellow Croakers Larimiththys crocea in coastal eastern China during recent years. METHODS: Published literatures of this pathogen have been reviewed. RESULT: Several strains with high genomic similarity have been isolated and identified; the bacteria induce natural infection at lower water temperatures (12.0-25.5°C) and induce numerous granulomas and nodules in the visceral organs of croakers. Researchers have investigated the epidemiology of P. plecoglossicida infection, identified major virulence factors, searched for pathogenic genes, analyzed host-pathogen interactions, and endeavored to develop efficient vaccines. CONCLUSION: This paper provides an overview of these research advances to elucidate the virulence mechanisms of the pathogen and to promote vaccine development against infection.

Enhanced protective efficacy of an OprF/PcrV bivalent DNA vaccine against Pseudomonas aeruginosa using a hydrogel delivery system.

Pseudomonas aeruginosa (PA) is one of the leading pathogens responsible for hospital-acquired infections. With the increasing antibiotic resistance of PA, clinical treatment has become increasingly challenging. DNA vaccines represent a promising approach for combating PA infection. However, the immune response induced by a single antigen is limited, and combination vaccines hold greater therapeutic potential. The highly conserved OprF and PcrV genes are attractive candidate antigens for vaccine development, but the poor delivery of such vaccines has limited their clinical application. In this study, we constructed an OprF/PcrV bivalent DNA vaccine, and a polyaspartamide/polyethylene glycol di-aldehyde (PSIH/PEG DA) hydrogel was formulated to improve DNA delivery. The OprF/PcrV DNA vaccine formulated with the PSIH/PEG DA hydrogel was carefully characterized in vitro and in vivo and showed suitable compatibility. The PSIH/PEG DA hydrogel formulation induced a mixed Th1/Th2/Th17 immune response in mice, leading to a significant increase in antibody titers, lymphocyte proliferation rates, and cytokine levels compared to those in mice treated with single or combined vaccines. The PSIH/PEG DA hydrogel delivery system significantly enhanced the immune protection of the DNA vaccine in a murine pneumonia model, as revealed by the reduced bacterial burden and inflammation in the mouse lungs and increased survival rate. In conclusion, the PSIH/PEG DA hydrogel delivery system can further enhance the immune efficacy of the combination OprF/PcrV DNA vaccine. This research provides a novel optimized strategy for the prevention and treatment of PA infection using DNA vaccines.

Protective Effect of Probiotics against Pseudomonas aeruginosa Infection of Human Corneal Epithelial Cells.

Probiotic therapy needs consideration as an alternative strategy to prevent and possibly treat corneal infection. This study aimed to assess the preventive effect of Lactobacillus reuteri and Bifidobacterium longum subsp. infantis on reducing the infection of human corneal epithelial (HCE) cells caused by Pseudomonas aeruginosa. The probiotics' preventive effect against infection was evaluated in cell monolayers pretreated with each probiotic 1 h and 24 h prior to P. aeruginosa challenge followed by 1 h and 24 h of growth in combination. Cell adhesion, cytotoxicity, anti-inflammatory, and antinitrosative activities were evaluated. L. reuteri and B. longum adhered to HCE cells, preserved occludin tight junctions' integrity, and increased mucin production on a SkinEthicTM HCE model. Pretreatment with L. reuteri or B. longum significantly protected HCE cells from infection at 24 h, increasing cell viability at 110% (110.51 ± 5.15; p ≤ 0.05) and 137% (137.55 ± 11.97; p ≤ 0.05), respectively. Each probiotic showed anti-inflammatory and antinitrosative activities, reducing TNF-α level (p ≤ 0.001) and NOx amount (p ≤ 0.001) and reestablishing IL-10 level (p ≤ 0.001). In conclusion, this study demonstrated that L. reuteri and B. longum exert protective effects in the context of corneal infection caused by P. aeruginosa by restoring cell viability and modulating inflammatory cytokine release.

Evaluation of immunogenicity and protective efficacy of the outer membrane porin F (OprF) against Pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) farming dominates the marine aquaculture industry in China. However, the epidemic outbreaks of visceral white nodules disease (VWND), caused by bacterial pathogen Pseudomonas plecoglossicida, have emerged as a significant concern within the large yellow croaker industry. Although vaccination is considered to be an effective method for preventing and controlling P. plecoglossicida infection, there is currently no commercially available vaccine targeting this bacterium. In the present study, the outer membrane porin F (OprF) of P. plecoglossicida was characterized and revealed a high sequence similarity with that of other Pseudomonas species. The recombinant OprF protein (rOprF) produced in Escherichia coli was then evaluated for its immunogenicity and protective role against P. plecoglossicida in large yellow croaker. The rOprF was identified to have immunogenicity by Western blot using large yellow croaker anti-P. plecoglossicida sera. Additionally, the indirect immunofluorescence assay (IIFA) provided evidence indicating the surface exposure of OprF in P. plecoglossicida. Fish vaccinated twice via intraperitoneal (IP) injection with the purified rOprF combined with commercial adjuvant ISA 763A VG exhibited a relative percent survival (RPS) of 70.60% after challenge with virulent P. plecoglossicida strain through immersion. The administration of rOprF resulted in a notable increase in specific serum antibody levels and serum lysozyme activity compared to the control groups. The immune-related genes in the spleen and head kidney of rOprF-vaccinated fish were remarkably upregulated compared with the PBS-vaccinated sham group after the P. plecoglossicida challenge. In summary, the findings of this study suggest that rOprF exhibits considerable potential in inducing a robust immune response, making it a viable candidate for vaccination against P. plecoglossicida infection in large yellow croaker.

Polyclonal anti-whole cell IgY passive immunotherapy shields against P. aeruginosa-induced acute pneumonia and burn wound infections in murine models.

Pseudomonas aeruginosa (PA) is a multidrug-resistant (MDR) opportunistic pathogen causing severe hospital-, and community-acquired infections worldwide. Thus, the development of effective immunotherapy-based treatments is essential to combat the MDR-PA infections. In the current study, we evaluated the protective efficacy of polyclonal avian antibodies raised against inactivated whole cells of the PAO1 strain in murine models of acute pneumonia and burn wound. The efficacy of generated antibodies was evaluated against different PA strains through several in vitro, ex vivo and in vivo experiments. The results showed that the anti-PAO1-IgY effectively reduced the motility, biofilm formation and cell internalization ability, and enhanced the opsonophagocytic killing of PA strains through the formation of immobilized bacteria and induction of increased cell surface hydrophobicity. Furthermore, immunotherapy with anti-PAO1-IgY completely protected mice against all PA strains in both acute pneumonia and burn wound murine models. It was found to reduce the bacterial loads in infected burned mice through interfering with virulence factors that play vital roles in the early stages of PA infection, such as colonization and cell internalization. The immunotherapy with anti-PAO1-IgYs could be instrumental in developing effective therapies aimed at reducing the morbidity and mortality associated with PA infections.

Whole-genome sequencing reveals two prolonged simultaneous outbreaks involving Pseudomonas aeruginosa high-risk strains ST111 and ST235 with resistance to quaternary ammonium compounds.

OBJECTIVE: Water-bearing systems are known as frequent Pseudomonas aeruginosa (PA) outbreak sources. However, many older buildings continue to have sanitary facilities in high-risk departments such as the ICU. We present two simultaneous prolonged multi-drug-resistant (MDR) PA outbreaks detected at the ICU of a pulmonology hospital, which were resolved by whole-genome sequencing (WGS). METHODS: Outbreak management and investigations were initiated in August 2019 after detecting two patients with nosocomial VIM-2-positive MDR PA. The investigations involved weekly patient screenings for four months and extensive environmental sampling for 15 months. All patient and environmental isolates were collected and analysed by WGS. RESULTS: From April to September 2019, we identified 10 patients with nosocomial MDR PA, including five VIM-2-positive strains. VIM-2-positive strains were also detected in nine sink drains, two toilets, and a cleaning bucket. WGS revealed that of 16 VIM-2-positive isolates, 14 were ST111 that carried qacE, or qacEΔ1 genes, whereas 13 isolates clustered (difference of ≤11 alleles by cgMLST). OXA-2 (two toilets), and OXA-2, OXA-74, PER-1 (two patients, three toilets) qacEΔ1-positive ST235 isolates dominated among VIM-2-negative isolates. The remaining seven PA strains were ST17, ST233, ST273, ST309 and ST446. Outbreak containment was achieved by replacing U-bends, and cleaning buckets, and switching from quaternary ammonium compounds (QUATs) to oxygen-releasing disinfectant products. CONCLUSION: Comprehension and management of two simultaneous MDR PA outbreaks involving the high-risk strains ST111 and ST235 were facilitated by precise control due to identification of different outbreak sources per strain, and by the in-silico detection of high-level QUATs resistance in all isolates.

An aluminium adjuvant compound with ginseng stem leaf saponins enhances the potency of inactivated Pseudomonas plecoglossicida vaccine in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) farm industry in China suffered from huge economic loss caused by Pseudomonas plecoglossicida infection. Due to multi-antibiotic resistance, efficient vaccines are urgent to be developed to combat this pathogen. In this study, an inactivated vaccine was developed with an aluminium adjuvant (Alum) plus ginseng stem and leaf saponins (GSLS). As a result, the relative percentage survival (RPS) against P. plecoglossicida was up to 67.8 %. Comparatively, RPS of groups that vaccinated with only inactivated vaccine and vaccine containing Alum or Montanide™ 763A as adjuvant were 21.8 %, 32.2 % and 62.1 %, respectively. Assays for total serum protein and serum lysozyme activity in group vaccinated with inactivated vaccine plus Alum + GSLS adjuvant were significantly higher than that in control group. Moreover, specific antibody in serum elicited a rapid and persistent level. According to the expression of some immune related genes, inactivated vaccine plus Alum + GSLS adjuvant induced a stronger cellular immune response which was vital to defend against P. plecoglossicida. In conclusion, our study demonstrated that the compound Alum and GSLS adjuvant is a potential adjuvant system to develop LYC vaccine.

Lactobacillus plantarum E2 regulates intestinal microbiota and alleviates Pseudomonas plecoglossicida induced inflammation and apoptosis in zebrafish (Danio rerio).

Pseudomonas plecoglossicida infection is a highly contagious epidemic in aquaculture, causing significant mortality among teleost. Our previous research has demonstrated that Lactobacillus plantarum E2 is beneficial for large yellow croaker in resisting infections caused by P. plecoglossicida. However, the relevant mechanisms remain largely unclear. In the present study, we used zebrafish (Danio rerio) to further explore the function of L. plantarum E2 and its mechanisms for resisting P. plecoglossicida infection. E2 supplementation diet significantly improved the growth rates and α-amylase and trypsin activities of the liver in zebrafish. After challenge with P. plecoglossicida strain PQLYC4, the survival rates of zebrafish were improved, and immune-related genes expression (IL-1ß, TNF-α, IL-8, Ig-Z, TLR-22 and IL-12α) were down-regulated. Histological analysis showed that E2 group had a longer intestinal villus and thicker intestinal walls after 30 days of feeding and healthier intestinal structure after challenge with P. plecoglossicida strain PQLYC4. Furthermore, co-incubation of zebrafish embryo fibroblast (ZF-4 cells) with L. plantarum E2 reduced apoptosis of ZF-4 cells after exposed to P. plecoglossicida. Intestinal microbiota analysis showed that E2 strain significantly increased the relative abundance of Lactobacillus and Pseudomonas, and PCoA analysis revealed a noticeable divergence in the intestinal microbial communities after E2 supplement. Together, our results suggested that E2 strain may promote zebrafish survival against P. plecoglossicida infection by regulating the intestinal microbiota and alleviating inflammatory response and apoptosis, thus exhibiting the potential as a probiotic.

Pulling the plug on a pseudomonas outbreak: ancillary equipment as vectors of infection.

OBJECTIVES: Outbreaks of infection related to flexible endoscopes are well described. However, flexible endoscopy also requires the use of ancillary equipment such as irrigation plugs. These are potential vectors of infection but are infrequently highlighted in the literature. This paper reports a cystoscopy-associated outbreak of Pseudomonas aeruginosa from contaminated irrigation plugs in a UK tertiary care centre. METHODS: Laboratory, clinical and decontamination unit records were reviewed, and audits of the decontamination unit were performed. Flexible cystoscopes and irrigation plugs were assessed for contamination. Retrospective and prospective case finding was performed utilizing the microbiology laboratory information management system. Available P. aeruginosa isolates underwent variable nucleotide tandem repeat (VNTR) typing. Confirmed cases were defined as P. aeruginosa infection with an identical VNTR profile to an outbreak strain. RESULTS: Three strains of P. aeruginosa were isolated from five irrigation plugs but none of the flexible cystoscopes. No acquired resistance mechanisms were detected. Fifteen confirmed infections occurred, including bacteraemia, septic arthritis and urinary tract infection. While failure of decontamination likely occurred because the plugs were not dismantled prior to reprocessing, the manufacturer's reprocessing instructions were also incompatible with standard UK practice. The Medicines and Healthcare Products Regulatory Agency was informed. A field safety notice was issued, and the manufacturer issued updated reprocessing instructions. CONCLUSIONS: Ancillary equipment can represent an important vector for infection, and should be considered during outbreak investigations. Users should review the manufacturer's instructions for reprocessing ancillary equipment to ensure that they are compatible with available procedures.

Monoclonal antibodies against lipopolysaccharide protect against Pseudomonas aeruginosa challenge in mice.

Pseudomonas aeruginosa is a common cause of hospital-acquired infections, including central line-associated bloodstream infections and ventilator-associated pneumonia. Unfortunately, effective control of these infections can be difficult, in part due to the prevalence of multi-drug resistant strains of P. aeruginosa. There remains a need for novel therapeutic interventions against P. aeruginosa, and the use of monoclonal antibodies (mAb) is a promising alternative strategy to current standard of care treatments such as antibiotics. To develop mAbs against P. aeruginosa, we utilized ammonium metavanadate, which induces cell envelope stress responses and upregulates polysaccharide expression. Mice were immunized with P. aeruginosa grown with ammonium metavanadate and we developed two IgG2b mAbs, WVDC-0357 and WVDC-0496, directed against the O-antigen lipopolysaccharide of P. aeruginosa. Functional assays revealed that WVDC-0357 and WVDC-0496 directly reduced the viability of P. aeruginosa and mediated bacterial agglutination. In a lethal sepsis model of infection, prophylactic treatment of mice with WVDC-0357 and WVDC-0496 at doses as low as 15 mg/kg conferred 100% survival against challenge. In both sepsis and acute pneumonia models of infection, treatment with WVDC-0357 and WVDC-0496 significantly reduced bacterial burden and inflammatory cytokine production post-challenge. Furthermore, histopathological examination of the lungs revealed that WVDC-0357 and WVDC-0496 reduced inflammatory cell infiltration. Overall, our results indicate that mAbs directed against lipopolysaccharide are a promising therapy for the treatment and prevention of P. aeruginosa infections.

An X-ray inactivated vaccine against Pseudomonas aeruginosa Keratitis in mice.

Pseudomonas aeruginosa (P. aeruginosa) is one of the most prevalent pathogens of bacterial keratitis. Bacterial keratitis is a major cause of blindness worldwide. The rising incidence of multidrug resistance of P. aeruginosa precludes treatment with conventional antibiotics. Herein, we evaluated the protective efficiency and explored the possible underlying mechanism of an X-ray inactivated vaccine (XPa) using a murine P. aeruginosa keratitis model. Mice immunized with XPa exhibit reduced corneal bacterial loads and pathology scores. XPa vaccination induced corneal macrophage polarization toward M2, averting an excessive inflammatory reaction. Furthermore, histological observations indicated that XPa vaccination suppressed corneal fibroblast activation and prevented irreversible visual impairment. The potency of XPa against keratitis highlights its potential utility as an effective and promising vaccine candidate for P. aeruginosa.