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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.)

Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. In this study, we characterized 76C. difficile isolates, obtained from three Mediterranean mussel (Mytilus galloprovincialis) farms in Slovenia from November 2014 to October 2015 (sampling period 1) and from January to December 2021 (sampling period 2). The overall isolation rate of C. difficile from all the examined mussels was 59.8 %. A statistically significant trend of seasonal variation was observed, with a higher isolation rate in the colder months of the year (87.9 %; sea temperature ≤ 15 °C) compared with the warmer months (31.8 %; sea temperature > 15 °C). Whole-genome sequencing (WGS) revealed that the isolates belonged to 31 different sequence types (STs), which were associated with three clades (1, 4, and 5) and two cryptic clades (C-II and C-III). Five isolates, which belonged to ST11 (clade 5), harbored all the main toxin genes (A + B + CDT + ) and chromosomal mutations conferring fluoroquinolone resistance. Core genome multilocus sequence typing (cgMLST) revealed four clusters of 2-3 isolates, three of which included isolates from different farms, suggesting that clonal C. difficile strains are circulating among the Slovenian mussel farms. The results highlight the presence of hypervirulent strains in mussels; therefore, at-risk population groups should be alerted to the risks associated with consuming shellfish., 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.)

Vibrio parahaemolyticus is a critical foodborne pathogen causing gastroenteritis worldwide. The occurrence of transmission and outbreaks attributed to V. parahaemolyticus has exhibited a notable upward trend during the past two decades. However, comprehensive information on this pathogen in inland cities in China remains scarce. This study shed light on the molecular characteristics, genetic associations, and significant transmission risks through foodborne and fecal-oral routes of 115 V. parahaemolyticus strains obtained from nine inland cities in Jilin Province. Strains were divided into 90 sequence types (STs), with 41 STs that were novel. The predominant sequence type was ST3 (14.78 %, 17/115). The strains exhibited the highest resistance rates to cefazolin and ampicillin. A total of ninety-four antibiotic resistance genes (ARGs) categorized groups based on antibiotic class were identified. The tet(34) (112/115, 97.39 %) and bla CARB (114/115, 99.13 %) genes responsible for tetracycline and β-lactams resistance were present in most isolates. Interestingly, V. parahaemolyticus with the tet(34) gene may not be resistant to tetracycline. Strains with strong biofilm formation ability carry more resistance genes. Atypical virulence genes and virulence genome islands (VPaI) were also identified in the isolated strains. All strains encoded type III secretion system 1 (T3SS1), while 114 isolates encoded type VI secretion system 2 (T6SS2). Notably, T6SS1 was present in 59.55 % of food strains, and pathogenicity genomic islands VPaI-1 to VPaI-5 were found in food-associated isolates. Based on the results of the phylogenetic analysis, clinical strains were closely related within a single cluster, which refers to strains that are more similar to each other than to those outside the group based on specific genetic profiles, while the food isolates were highly diverse. Furthermore, some isolates included in our study indicated potential transmission possibly through sharing of some SNPs between food and clinical-positive V. parahaemolyticus strains from different countries. The study elucidates the genetic characteristics, diversity, and virulence potential of V. parahaemolyticus isolates, thereby enhancing the understanding of the potential risks associated with the cross-border transmission of this pathogen., 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.)

Salmonella enterica subsp. enterica serovar Typhimurium variant 4,[5],12:i:- (referred to as S. 4,[5],12:i:-) has emerged rapidly as the predominant Salmonella serovar in pigs, often associated with the acquisition of antibiotic resistance (ABR) and heavy metal resistance (HMR) genes. Our study analysed 78 strains of S. 4,[5],12:i:- (n = 57) and S. Typhimurium (n = 21), collected from 1999 to 2021, to investigate the evolution of mobile genetic elements (MGEs) containing HMR and ABR genes. Five MGEs harbouring HMR genes were identified: pUO-STVR2, pSTM45, pUO-STmRV1, SGI-4 and MREL. Among the strains, 91.23 % (52/57) of S. 4,[5],12:i:- carried at least one of these elements, compared to only 14.29 % (3/21) of S. Typhimurium. Since 2008, S. 4,[5],12:i:- have shifted from predominantly carrying pUO-STmRV1 to the emergence of SGI-4 and MREL, reducing ABR genes, reflecting the European Union ban on the use of antibiotics as feed additives. Increased resistance to copper and silver in S. 4,[5],12:i:-, conferred by SGI-4 and MREL, reflected that their acquisition was linked to the ongoing use of heavy metals in food-animal production. However, strains carrying SGI-4 and MREL still exhibit multidrug resistance, emphasising the need for targeted interventions to mitigate multidrug-resistant Salmonella spread in veterinary and public health settings., 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.)

This study investigated the antibacterial effect of ultrasound (US) combined with Litsea cubeba essential oil nanoemulsion (LEON) on Salmonella Typhimurium in kiwifruit juice and effect on the quality and sensory properties of kiwifruit juice. In this study, LEON prepared by ultrasonic emulsification method had a good particle size distribution and high stability. The US+LEON treatment significantly (P < 0.05) improved antibacterial efficacy, compared to the control, and would not destroy the nutritional components containing ascorbic acid, flavonoids, total phenol and total soluble solids. Meanwhile, US+LEON treatment enhanced 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS) radical scavenging capacity and ferric ion reducing antioxidant power (FRAP). In terms of sensory properties, US and LEON had a significant (P < 0.05) effect on the odor and overall morphology of kiwifruit juice. The enhance of antibacterial efficacy and the retention of nutrients by combined treatments shows that US+LEON is a promising antibacterial method that will provide new ideas for the processing and safety of fruit juices, and the US parameters and LEON concentration should be adjusted to reduce the effect on food sensory properties in future studies., 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.)

Bacillus cereus is a ubiquitous foodborne pathogen commonly found in various foods. Its ability to form spores, biofilms and diarrhoeal and/or emetic toxins further exacerbates the risk of food poisoning. Violacein is a tryptophan derivative with excellent antibacterial activity. However, the knowledge on the antibacterial action of violacein against B. cereus was lacking, and thus this study aimed to investigate the antibacterial activity and mechanism. The antibacterial results demonstrated that minimum inhibitory concentration and minimum bactericidal concentration of violacein were 3.125 mg/L and 12.50 mg/L, respectively. Violacein could effectively inhibit planktonic growth, spore germination and biofilm formation of B. cereus (P < 0.001). Meanwhile, violacein significantly downregulated the expression of toxin genes, including nheA (P < 0.05), nheB (P < 0.001), bceT (P < 0.01), cytK (P < 0.001), hblC (P < 0.001) and hblD (P < 0.001). Results of extracellular alkaline phosphatase, nucleotide and protein leakage assays and scanning and transmission electron microscopy observation tests showed violacein destroyed cell walls and membranes of B. cereus. In addition, 6.25 mg/kg of violacein could significantly inhibit B. cereus in grass carp fillets (P < 0.05). These results demonstrate that violacein has great potential as an effective natural antimicrobial preservative to control food contamination and poisoning events caused by B. cereus., 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.)