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Akkermansia muciniphila, a member of the Verrucomicrobiota phylum, is recognized as a key gut microbe and has emerged as a potential next-generation probiotic. Assessment of antibiotic resistance in probiotics is a prerequisite for their application, while very few is studied in Akkermansia species. To address this, eight representative class A β-lactamases (36.90 %-41.30 % identity with known β-lactamases) from the Akkermansia species were screened and found to increase the minimum inhibitory concentration (MIC) of Escherichia coli to β-lactams (2-1024 fold). Secondly, four β-lactamases were successfully purified and identified as extended-spectrum β-lactamase because they exhibited hydrolase activity against β-lactams from penicillin, cephalosporins, and monobactam classes. Based on predicted three-dimensional structure, we hypothesized and validated that serine at 51 position was catalytic amino acid. Thirdly, the genomic context analysis revealed the absence of mobile genetic elements or other antibiotic resistance genes surrounding β-lactamase genes, suggesting they may not be transferable. This study provides a foundational basis for the safety evaluation of Akkermansia species as probiotics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The emergence of RND efflux pump gene cluster tmexCD-toprJ threats the clinical use of tigecycline as a last-resort antibiotic. Co-existence of extended spectrum β-lactamases and tmexCD-toprJ can accelerate the emergence of multidrug resistant or extensively drug-resistant Pseudomonas aeruginosa, leading to the production of high-risk clones. This study identified a novel gene cluster, tmexC6D6.2-toprJ1b, on the chromosome of a high-risk ST233 XDR P. aeruginosa from Chinese retail chicken samples. Genetic feature analysis revealed that a tnfxB6-tmexC6D6.2-toprJ1b-strBA-floR-tet(G)-IS6100-sul1-aadA2-int1-intA structure formed a putative transposition unit. tmexC6D6.2-toprJ1b shared high similarity at the nucleotide level with other tmexCD-toprJ gene clusters. tnfxB6 regulator was located downstream from the tmexC6D6.2-toprJ1b gene cluster and it did not affect bacterial resistance phenotype. The expression of tmexC6D6.2-toprJ1b could reduce the growth of E. coli and bring a moderate fitness cost. Further studies are needed to decipher the mechanism of the silencing of tnfxB6 in mediating the high-level resistance of tmexC6D6.2-torpJ1b and continuously monitor the coexistence of the novel tmexCD-toprJ gene cluster and the ESBLs-related resistance genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Animal food products are important sources of zoonotic agents, increasing the risk of exposure to antibiotic-resistant bacteria from farm to fork. Therefore, we aimed to detect and fully characterise Extended-Spectrum Beta-Lactamase (ESBL)-producing E. coli from the poultry sector in a One Health approach. From December 2021 to March 2022, 48 chicken meat samples were collected from 16 establishments in La Rioja (Northern Spain). Antibiotic susceptibility testing was assessed by the disk-diffusion method. Forty E. coli isolates were recovered from 33 of the 48 chicken meat samples tested (68.8%) when plated on MacConkey-agar. In addition, six ESBL-E. coli (6/48, 12.5%) were obtained on cefotaxime-supplemented MacConkey-agar, which were Whole-Genome Sequenced. A large diversity of clones and ESBL genes was observed, namely ST1140-E/bla CTX-M-32 (n = 1), ST752-A/bla TEM-52 (n = 1), ST117-B2/bla CTX-M-1 /bla SHV-12 (n = 2), ST10-A/bla SHV-12 (n = 1) and ST223-B1/bla SHV-12 (n = 1). Three IncI1-plasmids (pST3-CC3) were found carrying the bla SHV-12 /bla CTX-M-1 /bla CTX-M-32 genes in two genetic environments: i) IS26-smc-glpR-bla SHV-12 -IS26; and ii) wbuC-bla CTX-M-32 /bla CTX-M-1 -ISEcp1. The bla TEM-52 gene was carried on a P1-like phage-plasmid flanked by an IS4-mediated composite transposon. An IncHI2 plasmid harboured a bla SHV-12 gene flanked by an IS26-mediated composite transposon but also additional genes conferring resistance to aminoglycosides, chloramphenicol, and sulphonamides. To analyse the cross-sectoral relatedness of our ESBL-E. coli isolates, our six genomes were mapped with publicly available genomes (n = 2588) related to the STs detected, revealing that one of our genomes (X3078-ST117) displayed strong similarities (34-40 allelic differences) with few genomes belonging to ST117 from the poultry sector from Germany and USA. This study demonstrated that the proportion of ESBL-E. coli is still high in chicken meat in Spain. In addition, the ST117 clone and the IncI1-bla CTX-M-1-32 /bla SHV-12 plasmids might represent successful clones and plasmids adapted to the chicken host., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)