Xiaoli Chen

Accurate diagnostic techniques and effective therapeutic methods are required to treat H. pylori. The application of chicken single-chain variable fragment (scFv) antibodies may diagnose and treat H. pylori. This study used the phage display technique to construct a chicken-derived immune scFv antibody library against H. pylori. Total RNA was extracted from the spleens of five immunized chickens and reverse transcribed into cDNA. A fragment of scFv was produced by overlap extension PCR and cloned into a pHEN2 phagemid vector. After the package with the M13KO7 helper phage, the recombinant HpaA protein was used as a target antigen to validate the screening ability of our antibody library by bio-panning. The dilution counting results showed that the size of the primary antibody library was estimated to be 1 × 10⁹ cfu/mL. PCR analysis of 47 clones from the library revealed that about 100% of the clones were positive with scFv fragments, and there were no identical sequences, indicating the good diversity of the antibody library. After three rounds of bio-panning, high-affinity antibodies against recombinant HpaA protein were successfully obtained. The selected antibody specifically recognized HpaA protein in nine different H. pylori strains, confirming the screening ability of our library. The chicken immune scFv antibody library against H. pylori was successfully constructed, and the antibody library’s screening ability was validated by selecting specific scFv antibodies against recombinant HpaA and clinical strains. It provided a simple and rapid method to obtain antibodies against H. pylori for diagnosis or treatment.

Campylobacter is among the four main causes of gastroenteritis worldwide. Most reported Campylobacter infections are caused by C. jejuni and C. coli. However, other emerging Campylobacter pathogens have been recognized as important pathogens in humans and animals. A novel bacterial strain, PS10^T, was isolated from the gastric mucous of pigs in 2022 in Beijing, China. The cell was Gram-negative, microaerobic, motile, and negative for catalase, oxidase, and urease. Phylogenetic and phylogenomic analyses based on the 16S rRNA gene and core genome indicated that this isolate belongs to the genus Campylobacter. There were low dDDH relatedness and ANI values shared within this strain and its closest species C. mucosalis below the cut-off values generally recognized for isolates of the same species. The draft genome size of PS10^T is 2,240,910 bp in length with a percentage of DNA G+C contents of 37.72%. Comparing the phenotypic and phylogenetic features among this isolate and its related organisms, strain PS10^T represents a novel species within the genus Campylobacter, for which the name Campylobacter gastrosuis sp. nov. (Type strain PS10^T = GDMCC 1.3686^T = JCM 35849^T) is proposed.

Nine novel bacterial strains were isolated from the feces of cats and sheep in 2019 and 2020 in Beijing, China. Cells were 1–3 μm long and ≤0.5 μm wide, Gram-stain negative, microaerobic, motile, oxidase positive, and urease negative. Phylogenetic analyses based on 16S rRNA gene sequences indicated that these nine isolates belong to the genus Campylobacter but formed two robust clades that were clearly separate from the currently recognized species and, respectively, isolated from the cat and sheep. Both these strains shared low 16S rRNA gene sequence similarity, dDDH relatedness, and ANI values with their closest species C. upsaliensis CCUG 14913^T and C. lanienae NCTC 13004^T, and against each other, which are below the cut-off values generally recognized for isolates of the same species. The genomic DNA G + C contents of type strains XJK22-1^T and SYS25-1^T were 34.99 mol% and 32.43 mol%, respectively. Electron microscopy showed that these cells were spiral shaped, with bipolar single flagella. Based on results from genotypic, phenotypic, phylogenetic, and phylogenomic analyses, these nine strains represent two novel species within the genus Campylobacter, for which the names Campylobacter felis sp. nov. (Type strain XJK22-1^T = GDMCC 1.3684^T = JCM 35847^T) and Campylobacter ovis sp. nov. (Type strain SYS25-1^T = GDMCC 1.3685^T) are proposed.

Some amoxicillin-resistant strains of H. pylori show a sharp decrease in amoxicillin resistance after freezing. In China, most clinical gastric mucosal specimens are frozen and transported for isolation and drug susceptibility testing for H. pylori, which may lead to an underestimation of the amoxicillin resistance. The objective of this study is to investigated reasons for the decreased amoxicillin resistance after cryopreservation. A high-level amoxicillin-resistant clone (NX24r) was obtained through amoxicillin pressure screening. After cryopreservation at −80 °C for 3 months, the minimum inhibitory concentration (MIC) of NX24r was reduced sharply. Mutations and changes of transcriptome were analyzed after amoxicillin screening and cryopreservation. Mutations in PBP1 (I370T, E428K, T556S) and HefC (M337K, L378F, D976V) were detected in NX24r, which may be the main reason for the induced amoxicillin resistance. No mutations were found in PBP1 or HefC after cryopreservation. However, transcriptome analysis showed that down-regulated genes in the cryopreserved clone were significantly enriched in plasma membrane (GO:0005886), including lepB, secD, gluP, hp0871 and hp1071. These plasma membrane genes are involved in the biosynthesis and transport function of the membrane. The decreased amoxicillin resistance after cryopreservation may be related to the down-regulation of genes involved in membrane structure and transport function.