The poor stability of antibacterial peptide to protease limits its clinical application. Among these limitations, trypsin mainly exists in digestive tract, which is an insurmountable obstacle to orally delivered peptides. OM19R is a random curly polyproline cationic antimicrobial peptide, which has high antibacterial activity against some gram-negative bacteria, but its stability against pancreatin is poor. According to the structure-activity relationship of OM19R, all cationic amino acid residues (l-arginine and l-lysine) at the trypsin cleavage sites were replaced with corresponding d-amino acid residues to obtain the designed peptide OM19D, which not only maintained its antibacterial activity but also enhanced the stability of trypsin. Proceeding high concentrations of trypsin and long-time (such as 10 mg/mL, 8 h) treatment, it still had high antibacterial activity (MIC = 16–32 µg/mL). In addition, OM19D also showed high stability to serum, plasma and other environmental factors. It is similar to its parent peptide in secondary structure and mechanism of action. Therefore, this strategy is beneficial to improve the protease stability of antibacterial peptides.

Antibiotic tolerance has become an increasingly serious crisis that has seriously threatened global public health. However, little is known about the exogenous factors that can trigger the development of antibiotic tolerance, both in vivo and in vitro. Herein, we found that the addition of citric acid, which is used in many fields, obviously weakened the bactericidal activity of antibiotics against various bacterial pathogens. This mechanistic study shows that citric acid activated the glyoxylate cycle by inhibiting ATP production in bacteria, reduced cell respiration levels, and inhibited the bacterial tricarboxylic acid cycle (TCA cycle). In addition, citric acid reduced the oxidative stress ability of bacteria, which led to an imbalance in the bacterial oxidation–antioxidant system. These effects together induced the bacteria to produce antibiotic tolerance. Surprisingly, the addition of succinic acid and xanthine could reverse the antibiotic tolerance induced by citric acid in vitro and in animal infection models. In conclusion, these findings provide new insights into the potential risks of citric acid usage and the relationship between antibiotic tolerance and bacterial metabolism.

Antimicrobial peptides (AMPs) have a unique action mechanism that can help to solve global problems in antibiotic resistance. However, their low therapeutic index and poor stability seriously hamper their development as therapeutic agents. In order to overcome these problems, we designed peptides based on the sequence template XXRXXRRzzRRXXRXX-NH₂, where X represents a hydrophobic amino acid like Phe (F), Ile (I), and Leu (L), while zz represents Gly–Gly (GG) or d-Pro–Gly (pG). Showing effective antimicrobial activity against Gram-negative bacteria and low toxicity, designed peptides had a tendency to form an α-helical structure in membrane-mimetic environments. Among them, peptide LR_pG (X: L, zz: pG) showed the highest geometric mean average treatment index (GM_TI = 73.1), better salt, temperature and pH stability, and an additive effect with conventional antibiotics. Peptide LR_pG played the role of anti-Gram-negative bacteria through destroying the cell membrane. In addition, peptide LR_pG also exhibited an anti-inflammatory activity by effectively neutralizing endotoxin. Briefly, peptide LR_pG has the potential to serve as a therapeutic agent to reduce antibiotic resistance owing to its high therapeutic index and great stability.

Pasteurella multocida (Pm) is one of the major pathogens of bovine respiratory disease (BRD), which can develop drug resistance to many of the commonly used antibiotics. Our earlier research group found that with clinical use of enrofloxacin, Pm was more likely to develop drug resistance to enrofloxacin. In order to better understand the resistance mechanism of Pm to enrofloxacin, we isolated PmS and PmR strains with the same PFGE typing in vitro, and artificially induced PmR to obtain the highly resistant phenotype, PmHR. Then transcriptome sequencing of clinically isolated sensitive strains, resistant and highly drug-resistant strains, treated with enrofloxacin at sub-inhibitory concentrations, were performed. The satP gene, of which the expression changed significantly with the increase in drug resistance, was screened. In order to further confirm the function of this gene, we constructed a satP deletion (ΔPm) strain using suicide vector plasmid pRE112, and constructed the C-Pm strain using pBBR1-MCS, and further analyzed the function of the satP gene. Through a continuously induced resistance test, it was found that the resistance rate of ΔPm was obviously lower than that of Pm in vitro. MDK₉₉, agar diffusion and mutation frequency experiments showed significantly lower tolerance of ΔPm than the wild-type strains. The pathogenicity of ΔPm and Pm was measured by an acute pathogenicity test in mice, and it was found that the pathogenicity of ΔPm was reduced by about 400 times. Therefore, this study found that the satP gene was related to the tolerance and pathogenicity of Pm, and may be used as a target of enrofloxacin synergistic effect.

It is well known that Particulate Matter2.5 (PM_2.5) has a major adverse effect on the organism. However, the health hazards of livestock farm PM_2.5 to humans and animals are not yet known, and the role of miRNAs in the cellular damage induced by livestock farm PM_2.5 is also unclear. Therefore, our study used cowshed PM_2.5 to stimulate rat alveolar macrophage NR8383 to construct an in vitro injury model to investigate the effect of miR–122–5p on PM_2.5-induced apoptosis in the NR8383. The level of apoptosis was quantified by flow cytometry and Hoechst 33342/PI double staining. Furthermore, the potential target gene Collagen type IV alpha (COL4A1) of miR–122–5p was identified through the use of bioinformatics methods. The results demonstrated a decline in cell viability and an increase in apoptosis with rising PM_2.5 concentrations and exposure durations. The transfection of miR–122–5p mimics resulted in an upregulation of the pro-apoptotic protein Bcl–xL/Bcl–2 and activation of cleaved caspase–3 while inhibiting the anti-apoptotic protein B–cell lymphoma–2. The experimental data indicate that miR–122–5p is involved in the apoptotic process by targeting COL4A1. Furthermore, the overexpression of COL4A1 was observed to enhance the PM_2.5-activated PI3K/AKT/NF–κB signaling pathway, which contributed to the inhibition of apoptosis. This finding offers a promising avenue for the development of therapeutic strategies aimed at mitigating cellular damage induced by PM_2.5 exposure.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.