Modulating effects of heat-killed and live Limosilactobacillus reuteri PSC102 on the immune response and gut microbiota of cyclophosphamide-treated rats.

In the present study, we investigated the potential immunomodulatory effects of heat-killed (hLR) and live Limosilactobacillus reuteri PSC102 (LR; formerly Lactobacillus reuteri PSC102) in RAW264.7 macrophage cells and Sprague-Dawley rats. RAW264.7 murine macrophage cells were stimulated with hLR and LR for 24 h. Cyclophosphamide (CTX)-induced immunosuppressed Sprague-Dawley rats were orally administered with three doses of hLR (L-Low, M-Medium, and H-High) and LR for 3 weeks. The phagocytic capacity, production of nitric oxide (NO), and expression of cytokines in RAW264.7 cells were measured, and the different parameters of immunity in rats were determined. hLR and LR treatments promoted phagocytic activity and induced the production of NO and the expression of iNOS, TNF-α, IL-1ß, IL-6, and Cox-2 in macrophage cells. In the in vivo experiment, hLR and LR treatments significantly increased the immune organ indices, alleviated the spleen injury, and ameliorated the number of white blood cells, granulocytes, lymphocytes, and mid-range absolute counts in immunosuppressive rats. hLR and LR increased neutrophil migration and phagocytosis, splenocyte proliferation, and T lymphocyte subsets (CD4+, CD8+, CD45RA+, and CD28+). The levels of immune factors (IL-2, IL-4, IL-6, IL-10, IL-12A, TNF-α, and IFN-γ) in the hLR and LR groups were upregulated compared with those in the CTX-treatment group. hLR and LR treatments could also modulate the gut microbiota composition, thereby increasing the relative abundance of Bacteroidetes and Firmicutes but decreasing the level of Proteobacteria. hLR and LR protected against CTX-induced adverse reactions by modulating the immune response and gut microbiota composition. Therefore, they could be used as potential immunomodulatory agents.

The microglial innate immune protein PGLYRP1 mediates neuroinflammation and consequent behavioral changes.

Peptidoglycan recognition protein 1 (PGLYRP1) is a pattern-recognition protein that mediates antibacterial actions and innate immune responses. Its expression and role in neuroinflammatory conditions remain unclear. We observed the upregulation of PGLYRP1 in inflamed human and mouse spinal cord and brain, with microglia being the primary cellular source. Experiments using a recombinant PGLYRP1 protein show that PGLYRP1 potentiates reactive gliosis, neuroinflammation, and consequent behavioral changes in multiple animal models of neuroinflammation. Furthermore, shRNA-mediated knockdown of Pglyrp1 gene expression attenuates this inflammatory response. In addition, we identify triggering receptor expressed on myeloid cell-1 (TREM1) as an interaction partner of PGLYRP1 and demonstrate that PGLYRP1 promotes neuroinflammation through the TREM1-Syk-Erk1/2-Stat3 axis in cultured glial cells. Taken together, our results reveal a role for microglial PGLYRP1 as a neuroinflammation mediator. Finally, we propose that PGLYRP1 is a potential biomarker and therapeutic target in various neuroinflammatory diseases.

Antimicrobial Resistance Profiles of Enterococcus faecium and Enterococcus faecalis Isolated from Healthy Dogs and Cats in South Korea.

Enterococcus spp. are typically found in the gastrointestinal tracts of humans and animals. However, they have the potential to produce opportunistic infections that can be transmitted to humans or other animals, along with acquired antibiotic resistance. In this study, we aimed to investigate the antimicrobial resistance profiles of Enterococcus faecium and Enterococcus faecalis isolates obtained from companion animal dogs and cats in Korea during 2020-2022. The resistance rates in E. faecalis towards most of the tested antimicrobials were relatively higher than those in E. faecium isolated from dogs and cats. We found relatively higher resistance rates to tetracycline (65.2% vs. 75.2%) and erythromycin (39.5% vs. 49.6%) in E. faecalis isolated from cats compared to those from dogs. However, in E. faecium, the resistance rates towards tetracycline (35.6% vs. 31.5%) and erythromycin (40.3% vs. 35.2%) were comparatively higher for dog isolates than cats. No or very few E. faecium and E. faecalis isolates were found to be resistant to daptomycin, florfenicol, tigecycline, and quinupristin/dalfopristin. Multidrug resistance (MDR) was higher in E. faecalis recovered from cats (44%) and dogs (33.9%) than in E. faecium isolated from cats (24.1%) and dogs (20.5%). Moreover, MDR patterns in E. faecalis isolates from dogs (27.2%) and cats (35.2%) were shown to encompass five or more antimicrobials. However, E. faecium isolates from dogs (at 13.4%) and cats (at 14.8%) were resistant to five or more antimicrobials. Taken together, the prevalence of antimicrobial-resistant enterococci in companion animals presents a potential public health concern.

Antibiotic Resistance Profiles and Molecular Characteristics of blaCMY-2-Carrying Salmonella enterica Serovar Albany Isolated from Chickens During 2013-2020 in South Korea.

The production of ß-lactamase by nontyphoidal Salmonella has become a public health issue throughout the world. In this study, we aimed to investigate the antimicrobial resistance profiles and molecular characteristics of ß-lactamase-producing Salmonella enterica serovar Albany isolates. A total of 434 Salmonella Albany were obtained from feces and carcasses of healthy and diseased food-producing animals [cattle (n = 2), pigs (n = 3), chickens (n = 391), and ducks (n = 38)] during 2013-2020. Among the 434 Salmonella Albany isolates, 3.7% showed resistance to cefoxitin, and all the cefoxitin-resistant isolates were obtained from chickens. Moreover, Salmonella Albany isolates demonstrated high resistance to nalidixic acid (99.3%), trimethoprim/sulfamethoxazole (97.9%), ampicillin (86.6%), chloramphenicol (86.6%), and tetracycline (85.7%), as well as higher rates of multidrug resistance were detected in cefoxitin-resistant isolates compared to cefoxitin-susceptible isolates. All cefoxitin-resistant isolates harbored CMY-2-type ß-lactamase and belonged to seven different pulsotypes, with type IV-b (43.75%) and IV-a (25%) making up the majority. In addition, genes encoding cefoxitin resistant of all blaCMY-2-harboring Salmonella Albany isolates were horizontally transmitted to a recipient Escherichia coli J53 by conjugation. Furthermore, 93.75% (15/16) of conjugative plasmids harboring blaCMY-2 genes belong to ST12/CC12-IncI1. Genetic characteristics of transmitted blaCMY-2 genes were associated with ISEcp1, which can play an essential role in the effective mobilization and expression of these genes. Salmonella Albany containing blaCMY-2 in chickens can potentially be transferred to humans. Therefore, it is necessary to restrict antibiotic use and conduct continuous monitoring and analysis of resistant bacteria in the poultry industry.

Probiotics and Postbiotics as an Alternative to Antibiotics: An Emphasis on Pigs.

Probiotics are being used as feed/food supplements as an alternative to antibiotics. It has been demonstrated that probiotics provide several health benefits, including preventing diarrhea, irritable bowel syndrome, and immunomodulation. Alongside probiotic bacteria-fermented foods, the different structural components, such as lipoteichoic acids, teichoic acids, peptidoglycans, and surface-layer proteins, offer several advantages. Probiotics can produce different antimicrobial components, enzymes, peptides, vitamins, and exopolysaccharides. Besides live probiotics, there has been growing interest in consuming inactivated probiotics in farm animals, including pigs. Several reports have shown that live and killed probiotics can boost immunity, modulate intestinal microbiota, improve feed efficiency and growth performance, and decrease the incidence of diarrhea, positioning them as an interesting strategy as a potential feed supplement for pigs. Therefore, effective selection and approach to the use of probiotics might provide essential features of using probiotics as an important functional feed for pigs. This review aimed to systematically investigate the potential effects of lactic acid bacteria in their live and inactivated forms on pigs.

Prevalence and Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolated from Dogs and Cats in South Korea.

Overall, 836 Escherichia coli isolates (695 isolates from dogs and 141 from cats) were recovered from the diarrhea, skin/ear, urine, and genitals of dogs and cats between 2018 and 2019. Cefovecin and enrofloxacin resistance were noted in 17.1% and 21.2% of E. coli isolates, respectively. The cefovecin and enrofloxacin resistance rates were higher in dog isolates (18.1% and 22.9%) compared with the rates in cat isolates (12.1%, 12.8%). Interestingly, resistance to both antimicrobials was noted in 10.8% (90/836) of the isolates, predominantly in isolates from dogs. blaCTX-M-14, blaCTX-M-15, and blaCMY-2 were the most frequent extended-spectrum ß-lactamase/plasmid-mediated AmpC ß-lactamase (ESBL/AmpC)- gene types. The co-existence of blaCTX-M andblaCMY-2 was noted in six E. coli isolates from dogs. Sequencing analysis demonstrated that S83L and D87N in gyrA and S80I in parC were the most frequent point mutations in the quinolone resistance-determining regions of the cefovecin and enrofloxacin-resistant isolates. A total of 11 isolates from dogs carried the plasmid-mediated quinolone resistance genes (six aac(6')-Ib-cr, four qnrS, and one qnrB), while only two cat isolates carried the qnrS gene. Multilocus sequence typing of the cefovecin and enrofloxacin-resistant isolates revealed that sequence type (ST)131 E. coli carrying blaCTX-M-14 and blaCTX-M-15 genes and ST405 E. coli carrying blaCMY-2 gene were predominant among the isolated E. coli strains. The majority of the ESBL/AmpC-producing isolates displayed diverse pulsed-field gel electrophoresis profiles. This study demonstrated that third-generation cephalosporin- and fluoroquinolone-resistant E. coli were widely distributed in companion animals. The detection of the pandemic ST131 clone carrying blaCTX-M-14/15 in companion animals presented a public health threat.

Isolation and Identification of Limosilactobacillus reuteri PSC102 and Evaluation of Its Potential Probiotic, Antioxidant, and Antibacterial Properties.

We isolated and characterized Limosilactobacillus reuteri PSC102 and evaluated its probiotic, antioxidant, and antibacterial properties. We preliminarily isolated 154 candidates from pig feces and analyzed their Gram nature, morphology, and lactic acid production ability. Based on the results, we selected eight isolates and tested their ability to produce digestive enzymes. Finally, we identified one isolate using 16S rRNA gene sequencing, namely, L. reuteri PSC102. We tested its probiotic properties in vitro, including extracellular enzyme activities, low pH and bile salt tolerance, autoaggregation and coaggregation abilities, adhesion to Caco-2 cells, antibiotic susceptibility, and hemolytic and gelatinase activities. Antioxidant activity was determined using 1-diphenyl-2-picrylhydrazyl and 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical scavenging and reducing power assays. The antibacterial activity of this strain and its culture supernatant against enterotoxigenic Escherichia coli were evaluated using a time-kill assay and disk diffusion method, respectively. L. reuteri PSC102 exhibited tolerance toward low pH and bile salt and did not produce harmful enzymes or possess hemolytic and gelatinase activities. Its intact cells and cell-free extract exhibited potential antioxidant activities, and significantly inhibited the growth of enterotoxigenic E. coli. Our results demonstrate that L. reuteri PSC102 is a potential probiotic candidate for developing functional feed.

Antimicrobial Resistance in Escherichia coli Isolated from Healthy Dogs and Cats in South Korea, 2020-2022.

The occurrence of antimicrobial-resistant bacteria in companion animals poses public health hazards globally. This study aimed to evaluate the antimicrobial resistance profiles and patterns of commensal E. coli strains obtained from fecal samples of healthy dogs and cats in South Korea between 2020 and 2022. In total, 843 E. coli isolates (dogs, n = 637, and cats, n = 206) were assessed for susceptibility to 20 antimicrobials. The resistance rates of the most tested antimicrobials were significantly higher in dog than in cat isolates. Cefalexin (68.9%) demonstrated the highest resistance rates, followed by ampicillin (38.3%), tetracycline (23.1%), and cefazolin (18.7%). However, no or very low resistance (0-0.6%) to amikacin, imipenem, piperacillin, and colistin was found in both dog and cat isolates. Overall, 42.3% of the isolates exhibited multidrug resistance (MDR). MDR in isolates from dogs (34.9%) was significantly higher than in those from cats (20.9%). The main components of the resistance patterns were cefalexin and ampicillin in both dog and cat isolates. Additionally, MDR patterns in isolates from dogs (29.2%) and cats (16%) were shown to encompass five or more antimicrobials. Multidrug-resistant commensal E. coli could potentially be spread to humans or other animals through clonal or zoonotic transmission. Therefore, the incidence of antimicrobial resistance in companion animals highlights the urgent need to restrict antimicrobial resistance and ensure the prudent use of antimicrobials in Korea.

Heat-killed Limosilactobacillus reuteri PSC102 Ameliorates Impaired Immunity in Cyclophosphamide-induced Immunosuppressed Mice.

The immune functions of heat-killed Limosilactobacillus reuteri PSC102 (hLR) were investigated in cyclophosphamide (CP)-treated immunosuppressed mice. BALB/c mice were randomly divided into five groups: normal control group, CP group, CP treated with levamisole (positive control group), and CP treated with low- and high-dose hLR. After receiving the samples for 21 days, mice were sacrificed, and different parameters, such as immune organ index, immune blood cells, splenocyte proliferation, lymphocyte subpopulations, cytokines, and immunoglobulins, were analyzed. Results showed that the immune organ (thymus and spleen) indices of hLR treatment groups were significantly increased compared to the CP group (p < 0.05). hLR administration prevented CP-induced reduction in the numbers of white blood cells, lymphocytes, midrange absolute, and granulocytes, providing supporting evidence for hematopoietic activities. Splenocyte proliferation and T-lymphocyte (CD4+ and CD8+) subpopulations were also significantly augmented in mice treated with hLR compared to the CP group (p < 0.05). Moreover, Th1-type [interferon-γ, interleukin (IL)-2, and tumor necrosis factor-α] and Th2-type (IL-4 and IL-10) immune factors and immunoglobulin (IgG) showed significant increasing trends (p < 0.05). Additionally, the other proinflammatory cytokines (IL-1ß and IL-6) were also significantly elevated (p < 0.05). Taken together, this investigation suggested that orally administered hLR could recover immunosuppression caused by CP and be considered a potential immunostimulatory agent for the treatment of immunosuppressive disorders.

Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats.

BACKGROUND: Trifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Prolonged consumption of herbal extract has been shown to regulate the composition of gut microbiota. This study was designed to elucidate the influence of TPEE on the gut microbiota composition in the ovariectomized (OVX) rats. METHODS: OVX rats were treated with TPEE at 125, 250, 500 mg/kg/day, or controls (pomegranate extract, 500 mg/kg/day; estradiol, 25 µg/kg/day) for 12 weeks. Gut microbiota analysis was conducted by extracting the microbial DNA from fecal samples and microbiome taxonomic profiling was carried out by using next-generation sequencing. The levels of serum biomarkers were analyzed using enzyme-linked immunosorbent assay (ELISA) kit. The prediction of functional biomarker of microbiota was performed using PICRUSt to investigate the potential pathways associated with gut health and serum lipid profile regulation. To study the correlation between gut microbiota composition and serum lipid levels, Spearman's correlation coefficients were defined and analyzed. Additionally, gas chromatography-mass spectrometry analysis was conducted to uncover additional physiologically active ingredients. RESULTS: TPEE-treated OVX rats showed significant reduction in serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDL levels but increase in HDL level. The alteration in the pathways involve in metabolism was the most common among the other KEGG categories. Particularly, TPEE also significantly reduced the relative abundance of sequences read associated with inflammatory bowel disease (IBD) and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. TPEE intervention was seen to reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlation analysis at the phylum level revealed that Bacteriodetes and Proteobacteria were strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlate with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels. CONCLUSIONS: TPEE treatment showed therapeutic benefits by improving the intestinal microbiota composition which strongly correlated with the serum lipid and cholesterol levels in the OVX rats.

Ethanol-Induced Hepatotoxicity and Alcohol Metabolism Regulation by GABA-Enriched Fermented Smilax china Root Extract in Rats.

Chronic alcohol consumption can cause hepatic injury and alcohol-induced toxicities. Extracts from Smilax china root have been widely used in traditional medicine and for their potential pharmacological benefits. We aimed to determine if fermented Smilax china extract (FSC) regulates alcoholic fatty liver and liver injury using two in vivo experiments. Sprague-Dawley rats were administered ethanol (3 g/kg b.w.; po) with or without FSC pretreatment to induce an acute hangover. In another experiment, rats were fed either a normal or Lieber-DeCarli ethanol (6.7%) diet with or without FSC pretreatment (125, 250, and 500 mg/kg b.w.; po) for 28 days. Serum biomarkers, liver histopathology, and the mRNA levels of anti-inflammatory, antioxidant, lipogenic, and lipolytic genes were analyzed. FSC pretreatment significantly reduced blood alcohol and acetaldehyde concentrations, upregulated the mRNA expression of alcohol dehydrogenase, aldehyde dehydrogenase, and superoxide dismutase, and decreased the activities of liver enzymes in a dose-dependent manner. It also downregulated SERBP-1c and upregulated PPAR-α and reduced the gene expression of the anti-inflammatory cytokine IL-6 in the liver. The final extract after fermentation had increased GABA content. Furthermore, FSC was found to be safe with no acute oral toxicity in female rats. Thus, FSC increases alcohol metabolism and exhibits antioxidant and anti-inflammatory effects to induce hepatoprotection against alcohol-induced damage. It may be used as a functional food ingredient after excess alcohol consumption.

Aronia melanocarpa Extract Fermented by Lactobacillus plantarum EJ2014 Modulates Immune Response in Mice.

The present study aimed to assess the immunomodulatory effects of fermented Aronia melanocarpa extract (FAME) on RAW 264.7 cells and BALB/c mice. Aronia melanocarpa fruit was fermented with Lactobacillus plantarum EJ2014 by adding yeast extract and monosodium glutamate for 9 days at 30 °C to produce γ-aminobutyric acid (GABA). After fermentation, significant GABA production was noted, along with minerals, polyphenols, and flavonoids (p < 0.05). The polyphenol content was confirmed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. RAW 264.7 cells were stimulated with lipopolysaccharide (LPS, 1 µg/mL) in the presence or absence of FAME, and proinflammatory cytokine contents were measured by qPCR. In the in vivo experiment, female BALB/c mice were administered 125, 250, and 500 mg/kg of FAME for 21 days. FAME treatment increased neutrophil migration and phagocytosis (p < 0.05). It also increased splenocyte proliferation, CD4+ and CD8+ T-cell expression, and lymphocyte proliferation. Furthermore, it increased IFN-γ, IL-2, and IL-4 cytokine levels in a dose-dependent manner (p < 0.05). However, it decreased TNF-α and IL-6 levels (p < 0.05). These results indicate that FAME fortified with GABA including bioactive compounds exerts anti-inflammatory effects by inhibiting proinflammatory cytokines in RAW 264.7 cells and modulates immune response in mice. Thus, FAME could be a potential therapeutic agent for inflammatory disorders.

Investigation of Potential Antioxidant, Thrombolytic and Neuropharmacological Activities of Homalomena aromatica Leaves Using Experimental and In Silico Approaches.

The leaves of Homalomena aromatica are traditionally used in Bangladesh for the treatment of different chronic ailments. The purpose of this study was to explore in vitro antioxidant, thrombolytic activities, and in vivo neuropharmacological effects of methanolic extract of Homalomena aromatica (MEHA) leaves. Antioxidant activity of MEHA was assessed by a DPPH free radical scavenging assay and total phenolics content, total flavonoids content were also measured. The thrombolytic activity was determined by percentage of clot lysis and neuropharmacological activities by hole board, tail suspension, forced swimming and elevated plus maze tests. The results showed that the IC50 value of the extract against DPPH was 199.51 µg/mL. Quantitative analysis displayed higher contents of phenolics and flavonoids (147.71 mg gallic acid equivalent/g & 66.65 mg quercetin equivalent/g dried extract, respectively). The extract also showed a significant clot lysis (33.31%) activity. In case of anxiolytic activity, the elevate plus maze (EPM) test demonstrated an increase in time spent in open arms, and in case of hole board test, the number of head dipping was also significantly increased (p < 0.05). All the test compared with control (1% Tween in water) and standard (diazepam 1 mg/kg), significant dose (200 & 400 mg/kg) dependent anxiolytic activity was found. In antidepressant activity, there was a significant decrease in period of immobility in both test models (tail suspension and forced swimming) (p < 0.05). Moreover, 13 compounds were identified as bioactive, showed good binding affinities to xanthine oxidoreductase, tissue plasminogen activator receptor, potassium channel receptor, human serotonin receptor targets in molecular docking experiments. Furthermore, ADME/T analysis revealed their drug-likeness, likely pharmacological actions and non-toxic upon consumption. Taken together, our finding support the traditional medicinal use of this plant, which may provide a potential source for future drug discovery.

Cornus officinalis Ethanolic Extract with Potential Anti-Allergic, Anti-Inflammatory, and Antioxidant Activities.

Atopic dermatitis (AD) is an allergic and chronic inflammatory skin disease. The present study investigates the anti-allergic, antioxidant, and anti-inflammatory activities of the ethanolic extract of Cornus officinalis (COFE) for possible applications in the treatment of AD. COFE inhibits the release of ß-hexosaminidase from RBL-2H3 cells sensitized with the dinitrophenyl-immunoglobulin E (IgE-DNP) antibody after stimulation with dinitrophenyl-human serum albumin (DNP-HSA) in a concentration-dependent manner (IC50 = 0.178 mg/mL). Antioxidant activity determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power assay, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity, result in EC50 values of 1.82, 10.76, and 0.6 mg/mL, respectively. Moreover, the extract significantly inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production and the mRNA expression of iNOS and pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) through attenuation of NF-κB activation in RAW 264.7 cells. COFE significantly inhibits TNF-α-induced apoptosis in HaCaT cells without cytotoxic effects (p < 0.05). Furthermore, 2-furancarboxaldehyde and loganin are identified by gas chromatography/mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, respectively, as the major compounds. Molecular docking analysis shows that loganin, cornuside, and naringenin 7-O-ß-D-glucoside could potentially disrupt the binding of IgE to human high-affinity IgE receptors (FceRI). Our results suggest that COFE might possess potential inhibitory effects on allergic responses, oxidative stress, and inflammatory responses.

Antibacterial and cytotoxic activity of ethanol extract of mikania cordata (burm.f.) B.L. Robinson leaves.

The purpose of the present study to examine the antibacterial and cytotoxic properties of ethanol extract of leaves of Mikania cordata (Burm.f.) B.L. Robinson. To determine antibacterial activities, the extract was tested against four Gram positive and six Gram negative bacteria at three concentrations (500, 800, 1000 µg disc(-1)) through disc diffusion method. The extract showed moderate antibacterial actions and that was increased by increasing the concentration of the sample. The maximum antimicrobial potential was obtained against Shigella flexneri and no sen sitivity was found for Klebsiella sp. Comparatively gram-positive bacteria demonstrated more susceptibility to the extract than gram-negative bacteria. Cytotoxic property of the sample was done using Brine shrimp lethality bioassay where it did not show noticeable toxicity. So, our present study reveals that the leaves extract of M. cordata possess considerable antibacterial properties along with lesser amount of cytotoxicity.