Background and Aim: Helicobacter pylori is an important pathogen in humans and animals involved in chronic gastritis, leading to the development of gastric cancer. Urease produced by H. pylori is an enzyme that promotes bacterial colonization and can be used clinically as a biomarker of H. pylori infection as part of a rapid urease test (RUT). A test with high specificity (95-100%) would be more convenient and faster than histopathology, bacterial culture, and polymerase chain reaction (PCR). The aim of this study was to develop a simple, cheap, and fast kit for detecting H. pylori infection in the gastric mucosa of canines, which can be used in clinical practice for diagnosing infection with this bacterium. Materials and Methods: The RUT assays developed were prepared using 1% agar, 1% sodium phosphate monobasic, and 1% urea followed by the addition of 3% methyl red indicator. The cutoff value of sensitivity of the RUT assay was established using the urease of H. pylori ATCC 43504 and color change was monitored for 24 h. Comparisons of the sensitivity to H. pylori ATCC 43504 were made between the developed RUT assays and the Hp Fast™ commercial kit. Then, the limit of detection for H. pylori ATCC 43504 number was analyzed by the SYBR Green real-time PCR assay to measure the copy number of the ureC gene. Gastric biopsy samples from the antrum, body, and fundus of the stomach were collected from eight canines presenting with vomiting and gastroenteritis. Analyses were performed on fresh samples using the developed RUT assays and the Hp Fast™ commercial kit, which were read within 24 h; then, the results were confirmed with SYBR Green real-time PCR. The specificity of the RUT assays was tested with a number of different bacteria, including Staphylococcus pseudintermedius, Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus spp., Escherichia coli, and Salmonella spp.; H. pylori ATCC 43504 was used as a positive control. Results: The results showed that the developed assays were sensitive to the urease enzyme at 0.1 mg/mL. The lowest detection limit of this assay for H. pylori ATCC 43504 was found to be 102 copies at 30 min. The sensitivity of detection of H. pylori in gastric biopsies of canines occurred in a minimum of 30 min. The RUT showed similar results to the Hp Fast™ commercial kit. In the developed RUT, the color change of the test from red to yellow could be clearly distinguished between the color of the positive test and the negative one; however, in the commercial Hp Fast™, it was difficult to observe the gel color change in the negative pH range of 5.8 and the positive pH of 6.5. The developed RUT was specific for H. pylori and did not detect any of the other tested bacteria. The test kit can also be stored for 6 months at 4°C. Conclusion: The sensitivity of the developed assays allowed the detection of urease enzyme at a minimum concentration of 0.1 mg/mL. Our RUT could also detect H. pylori from one in eight canine specimens at a minimum of 102 copies within 30 min. This RUT is specific to H. pylori as it did not detect any of the other tested bacteria.

Methicillin-resistant Staphylococcus epidermidis (MRSE) contamination is commonly found on human skin and medical devices. Herein, we present a sensor utilizing molecularly imprinted polymer nanobodies (nanoMIP) for recognition and electrochemical impedance spectroscopy (EIS) to detect S. epidermidis. Sensor manufacturing involves synthesizing nanoMIP via solid-phase synthesis using whole bacteria as templates. Screen-printed gold electrode (AuSPE)-modified 16-mercaptohexadecanoic acid (MHDA) served to immobilize the nanoMIPs on the sensor surface through an amide bond, with the remaining functional groups blocked by ethanolamine (ETA). Scanning electron microscope (SEM) analysis of the modified AuSPE surface reveals immobilized spherical nanoMIP particles of 114–120 nm diameter, while atomic force microscope (AFM) analysis showed increased roughness and height compared to bare AuSPE. The sensor is selective for S. epidermidis, with a remarkable detection limit of 1 CFU/mL. This research demonstrates that the developed nanoMIP-based sensor effectively detects S. epidermidis. Further research will focus on developing protocols to integrate the nanoMIP-based EIS sensor into medical and industrial applications, ultimately contributing to improved safety for both humans and animals in the future.

Staphylococcus epidermidis (S. epidermidis) belongs to methicillin-resistant bacteria strains that cause severe disease in humans. Herein, molecularly imprinted polymer (MIP) nanoparticles resulting from solid-phase synthesis on entire cells were employed as a sensing material to identify the species. MIP nanoparticles revealed spherical shapes with diameters of approximately 70 nm to 200 nm in scanning electron microscopy (SEM), which atomic force microscopy (AFM) confirmed. The interaction between nanoparticles and bacteria was assessed using height image analysis in AFM. Selective binding between MIP nanoparticles and S. epidermidis leads to uneven surfaces on bacteria. The surface roughness of S. epidermidis cells was increased to approximately 6.3 ± 1.2 nm after binding to MIP nanoparticles from around 1 nm in the case of native cells. This binding behavior is selective: when exposing Escherichia coli and Bacillus subtilis to the same MIP nanoparticle solutions, one cannot observe binding. Fluorescence microscopy confirms both sensitivity and selectivity. Hence, the developed MIP nanoparticles are a promising approach to identify (pathogenic) bacteria species.

Feline infectious peritonitis (FIP) is a progressive and often fatal disease caused by a virulent biotype of feline coronavirus (FCoV). Although antiviral treatments are now available, relapse and resistance remain ongoing concerns. This study investigates the therapeutic potential of P. indusiatus, a medicinal mushroom, for its antiviral and anti-inflammatory activities against FIP. The main protease (FIPV M^pro) of feline infectious peritonitis virus (FIPV) was recombinantly expressed and purified to facilitate enzyme inhibition screening. P. indusiatus exhibited the strongest FIPV M^pro inhibitory activity among the 17 mushroom extracts tested (69.2%), showing a notable level of inhibition relative to standard antiviral agents such as lopinavir and ritonavir. To assess its anti-inflammatory potential, PBMCs derived from healthy cats and FIP-associated effusions (FIP fluid) were cultured and stimulated with LPS to induce inflammation. In healthy PBMCs, P. indusiatus significantly reduced nitrite levels, with effects similar to dexamethasone. However, PBMCs from FIP fluid, already in an activated state, showed no additional response. Notably, this study is the first to successfully isolate and culture PBMCs from FIP fluid, providing a new platform for future immunological research. These findings suggest that P. indusiatus possesses both antiviral and anti-inflammatory properties, positioning it as a potential dual-action therapeutic candidate for FIP. Further investigation into cytokine signaling pathways is warranted to clarify its mechanisms of action and advance future therapeutic development.

Feline infectious peritonitis (FIP) is a fatal disease driven by feline coronavirus induced immune dysregulation and excessive inflammatory cytokine production. Immunomodulatory agents capable of rebalancing this response are therefore of increasing interest. Phallus indusiatus (P. indusiatus), an edible mushroom containing diverse bioactive compounds, has previously demonstrated antiviral and anti-inflammatory potential. This study investigated the immunomodulatory effects of P. indusiatus extract on peripheral blood mononuclear cells (PBMCs) from healthy cats and FIP cats and characterized its chemical constituents using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). PBMCs were isolated from whole blood and FIP fluid. Cytotoxicity screening identified 19 µg/mL as a non-toxic concentration for subsequent assays. Cytokine responses (IL-1β, IFN-γ, and IL-10) were evaluated following LPS stimulation in PBMCs from whole blood and under basal conditions in PBMCs from FIP fluid after treatment with P. indusiatus extract and dexamethasone. LC–MS/MS profiling combined with STITCH analysis was used to identify bioactive metabolites and their predicted molecular targets. PBMCs derived from FIP fluid exhibited markedly elevated IL-1β and IFN-γ, indicating strong baseline immune activation. P. indusiatus significantly reduced IL-1β and IFN-γ in PBMCs from FIP fluid and suppressed LPS-induced IL-1β and IL-10 in whole-blood PBMCs, demonstrating immunomodulatory patterns comparable to dexamethasone. LC–MS/MS analysis identified compounds including adenosine, phenylalanine, tyrosine, cystathionine, arginine, and sialic acid, which were linked to inflammatory signaling. Overall, the extract exhibited context-dependent modulation of pro- and anti-inflammatory cytokines, suggesting that P. indusiatus may serve as a promising natural adjunctive candidate for managing immune imbalance in cats with FIP.