Epsilon toxin (ETX) is an exotoxin produced by type B and D Clostridium perfringens that causes enterotoxemia or necrotic enteritis in animals such as goats, sheep, and cattle. Vaccination is a key method in preventing such diseases. In this study, we developed a new type of dissolving microneedle patch (dMN) with a nanoparticle adjuvant for enhanced immune response to deliver the rETX^Y196E-C protein vaccine. We chose FDA-approved poly(lactic-co-glycolic acid) (PLGA) to prepare nanospheres as the vaccine adjuvant and introduced dimethyldioctadecylammonium bromide (DDAB) to make the surface of PLGA nanoparticles (PLGA NPs) positively charged for antigen adsorption. PLGA NPs with a diameter of 100~200 nm, a surface ZETA potential of approximately +40 mV, and good safety were successfully prepared and could effectively adsorb rETX^Y196E-C protein. Using non-toxic and antibacterial fish gelatin as the microneedle (MN) matrix, we prepared a PLGA-DDAB dMN vaccine with good mechanical properties that successfully penetrated the skin. After immunization of subcutaneous (SC) and dMN, antibody titers of the PLGA and Al adjuvant groups were similar in both two immune ways. However, in vivo neutralization experiments showed that the dMN vaccines had a better protective effect. When challenged with 100 × LD₅₀ GST-ETX, the survival rate of the MN group was 100%, while that of the SC Al group was 80%. However, a 100% protective effect was achieved in both immunization methods using PLGA NPs. In vitro neutralization experiments showed that the serum antibodies from the dMN and SC PLGA NPs groups both protect naive mice from 10 × LD₅₀ GST-ETX attack after being diluted 20 times and could also protect MDCK cells from 20 × CT₅₀ GST-ETX attack. In conclusion, the PLGA-DDAB dMN vaccine we prepared has good mechanical properties, immunogenicity, and protection, and can effectively prevent ETX poisoning. This provides a better way of delivering protein vaccines.

Vibrio parahaemolyticus is as an important food-borne pathogen circulating in China. Since 1996, the core serotype has become O3:K6, which has specific genetic markers. This serotype causes the majority of outbreaks worldwide. Until now, nearly 21 serotypes were considered as serovariants of O3:K6. Among these, O4:K68, O1:K25 and O1:KUT have caused pandemic outbreaks. O4:K8, a serovariant of O3:K6, has become the second most dominant serotype circulating in China after O3:K6. In this study, we report the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze and characterize 146 V. parahaemolyticus isolates belonging to 23 serotypes. Upon mass spectral analysis, isolates belonging to O4:K8 formed a distinct group among the five main pandemic groups (O3:K6, O4:K8, O4:K68, O1:K25 and O1:KUT). Two major protein peaks (m/z 4383 and 4397) were significantly different between serotype O4:K8 and the four other pandemic strains. Both of these peaks were present in 32 out of 36 O4:K8 isolates, but were absent in 105 out of 110 non-O4:K8 isolates. These peaks were also absent in all 74 pandemic serotypes (O3:K6, O4:K68, O1:K25 and O1:KUT). Our results highlight the threat of O4:K8 forming a distinct group, which differs significantly from pandemic serotypes on the proteomic level. The use of MALDI-TOF MS has not been reported before in a study of this nature. Mass spectrum peaks at m/z 4383 and 4397 may be specific for O4:K8. However, we cannot conclude that MALDI-TOF MS can be used to serotype V. parahaemolyticus.

Background: As a Class A bioterrorism agent, botulinum neurotoxin serotype A (BoNT/A) carries the risk of being used by terrorists to cause mass poisoning. The microneedle (MN) patch has a great potential for application as a novel vaccine delivery method. The aim of this study is to develop a thermally stable, dissolving microneedle patch for the delivery of a recombinant protein vaccine using a recombinant C-terminal heavy chain of BoNT/A (Hc of BoNT/A, AHc) to prevent botulism. Methods: Fish gelatin, a natural non-toxic and bacteriostatic material, was selected as the microneedle matrix for the preparation of the dissolving microneedle vaccine. Subsequently, the mechanical performance, bacteriostatic properties, vaccination effect, and stability of the microneedle patches were evaluated using instruments such as the displacement-force test station and optical coherence tomography (OCT) scanner. Results: Fish gelatin matrix at high concentrations has good bacteriostatic properties, and excellent mechanical performance and vaccination effect, meeting the necessities of a vaccine. In both in vivo and in vitro neutralization experiments, MN vaccines containing different antigen doses achieved the same protective efficacy as subcutaneous vaccinations, protecting mice against 10⁶ LD₅₀ of BoNT/A injected intraperitoneally. Thermal stability analysis of the MN vaccines revealed that the fish gelatin matrix protected the AHc vaccine from protein denaturation even after 7 days of storage at 37 °C and enabled the vaccine patches to maintain good immunogenicity and protective efficacy even after 6 months of storage at room temperature. Conclusion: In this study, we successfully prepared a bacteriostatic MN patch using a fish gelatin matrix that not only has a good vaccination effect, but also obviates the need for a cold chain for the AHc vaccine, providing the possibility of rapid, painless, and large-scale vaccination.

Objective. The detection of Helicobacter pylori mutations that result in antimicrobial resistance can serve as a guideline of antimicrobial therapeutics and probably prevent the failure of clinical treatments. Evaluating the potential of Sanger sequencing to identify genetically resistant determinants in Helicobacter pylori clinical isolates will be important. Methods. 180 cultured strains have been tested using agar dilution for antibiotic susceptibility. NCBI BLAST was used to perform genotypic analysis on the sequencing data. Sanger sequencing was evaluated as an alternative method to detect resistant genotypes and susceptibility. Results. By the conventional E-test, resistance to levofloxacin, amoxicillin, metronidazole, and clarithromycin was 67.3, 15.1, 96.4, and 25.5, respectively. In contrast, tetracycline had no resistance. Resistance to multiple drugs was observed in 8.12 of the strains. The genetic determinants of resistance to CLA was 23s rRNA, the determinants of resistance to amoxicillin was Pbp1, the determinants of resistance to metronidazole was rdxA, and the determinants of resistance to levofloxacin were GyrA and GyrB. However, there was no association of resistance in tetracycline. Conclusion. We found increased rates of metronidazole antibiotic resistance, highlighting the necessity for alternative therapies and periodic evaluation. Sanger sequencing has proved to be highly effective and holds the potential to be implemented in policies catering to local treatments.

Human coronaviruses (HCoVs) are associated with a range of respiratory symptoms. The discovery of severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome, and SARS-CoV-2 pose a significant threat to human health. In this study, we developed a method (HCoV-MS) that combines multiplex PCR with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), to detect and differentiate seven HCoVs simultaneously. The HCoV-MS method had high specificity and sensitivity, with a 1–5 copies/reaction detection limit. To validate the HCoV-MS method, we tested 163 clinical samples, and the results showed good concordance with real-time PCR. Additionally, the detection sensitivity of HCoV-MS and real-time PCR was comparable. The HCoV-MS method is a sensitive assay, requiring only 1 μL of a sample. Moreover, it is a high-throughput method, allowing 384 samples to be processed simultaneously in 30 min. We propose that this method be used to complement real-time PCR for large-scale screening studies.

Ricin (RT) and abrin (AT) are plant toxins extracted from Ricinus communis and Abrus precatorius, respectively, and both have N-glycosidase activity. The detection of these toxins is vital because of their accessibility and bioterrorism potential. While ricin can be effectively detected based on its depurination activity, only a few tests are available for detecting the depurination activity of abrin. Therefore, it is unclear whether they share the same optimal reaction substrate and conditions. Here, we established optimum depurination conditions for ricin and abrin, facilitating the in vitro detection of their depurination activity using high-performance liquid chromatography–tandem mass spectrometry. The parameters optimized were the reaction substrate, bovine serum albumin (BSA), buffer, pH, temperature, time, antibodies, and magnetic beads. Both toxins showed better depurination with single-stranded DNA. However, substrate length, adenine content, BSA concentration, buffer concentration, reaction temperature, and reaction time differed between the two toxins. The optimal conditions for ricin depurination involved a reaction in 1 mM ammonium acetate solution (0.5 μM DNA15A, 20 μg/mL BSA, and 1 mM Zn²⁺, with pH 4.0) at 55 °C for 1 h. The optimal conditions for abrin depurination involved a reaction in 1 mM ammonium citrate solution (0.2 μM DNA20A, 10 μg/mL BSA, 1 mM Mg²⁺, and 0.5 mM EDTA, with pH 4.0) at 45 °C for 2 h. After optimization, the limits of detection (LOD) for ricin and abrin were 0.506 ng/mL and 0.168 ng/mL, respectively. The detection time was also significantly reduced.