Purpose: The aim of this study was to determine the prevalence and transmission mechanism of mcr-3 in Aeromonas spp. isolated from chicken cloaca. Materials and Methods: A. veronii w55 was isolated from chicken in 2008. PCR assay was used to detect mcr genes and putative circular intermediate. Susceptibility testing was identified by the microdilution method. WGS was performed to obtain the whole sequence. S1-PFGE and DNA southern hybridization were used to study the location of mcr-3.6. Results: PCR-based analysis indicated that 1 out of 55 Aeromonas spp. isolates was mcr-3-positive. Whole-genome sequencing revealed that the strain A. veronii w55 belonged to novel sequence type ST514 and had two adjacent chromosomally located mcr variants, mcr-3.6 and mcr-3-like. The mcr-3.6 and mcr-3-like genes showed 93.67% and 82.84% nucleotide sequence identity, respectively, to original mcr-3 from E. coli. A. veronii w55 also exhibited resistance to extended-spectrum β-lactams and was positive for bla_PER-3, and this is the first time to report bla_PER-3 in A. veronii. Genetic environment analysis revealed that the segment of mcr-3.6-mcr-3-like-dgkA was flanked by five insertion sequence elements originated from Aeromonas species, and the structure of ISAs2-ISAhy2-ISAs20-mcr-3.6-mcr-3-like-dgkA-ISAs2 was designated as a novel transposon Tn 6518, in which an 8405-bp circular intermediate carrying two mcr-3 variants can be looped out. Conclusion: This result suggested the mcr-3 variant genes could be disseminated between various Aeromonas species via transposon-mediated transmission.

The optimum conditions of a direct competitive enzyme-linked immunosorbent assay (dcELISA) in regard to different monoclonal antibodies (MAbs), coating conditions, blocking conditions and physicochemical factors of the assay buffer were investigated to develop a broad-specific and sensitive immunoassay for detection of antibacterial synergists (ASGs) in milk, eggs, chicken and pork. Under optimized conditions, a highly broad-specific and sensitive dcELISA for ASGs was obtained based on 5C4 and hapten-C-HRP, demonstrating a 50% specific binding (IC₅₀) for five ASGs of 0.208–9.24 ng mL⁻¹ with cross-reactivity (CR) of 138.2–7.5%. The optimized dcELISA was used to quantify the five ASGs in milk, egg, chicken muscle and beef with the average recoveries of 79.5–105.4% and coefficients of variation (CV) less than 13.4%. The established method with broad specificity and uniform affinity, offered a simple, sensitive, and high-throughput screening tool for the detection of multi- ASGs in animal-derived food.

In a previous study, we developed a dihydropteroate synthase (DHPS)-based biosensor for multi-sulfonamides (SAs), which could detect 29 SAs with IC₅₀ values below 100 ng mL⁻¹. The purpose of the present study was to apply the DHPS-based biosensor to different food matrices and evaluate the influence of matrix effects on the performance of the biosensor by using sulfamethazine (SMZ) as a reference analyte. The results show that different sample pretreatment procedures were required for each food sample to ensure the accuracy and precision of the biosensor. The limit of detection (LOD) for SMZ in chicken, pork, egg and honey was 17.82, 20.55, 23.22, and 5.57 μg kg⁻¹, respectively. The recovery values ranged from 47.6% to 135.1%, and the coefficients of variation (CV) were less than 25.6%. Taken together, our findings show that the DHPS-DHPPP biosensor is a suitable screening method for the screening of multi-SAs in different food matrices.

Considering that the strip method is simple and convenient for users, a Europium nanosphere-based time-resolved fluorescent immunochromatographic assay (TRFICA) for the rapid screening of 4,4′-dinitrocarbanilide (DNC) was developed to improve the performance of strip assays. After optimization, TRFICA showed IC₅₀, the limit of detection, and cut-off values of 0.4, 0.07, and 5.0 ng mL⁻¹, respectively. No significant cross-reactivity (CR < 0.1%) with 15 DNC analogs was observed in the developed method. TRFICA was validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 77.3% to 92.7%, with coefficients of variation of <14.9%. Moreover, the time needed for the detection procedure, including the sample pre-treatment, was less than 30 min for TRFICA, which had never been achieved before in other immunoassays. The newly developed strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique for DNC analysis in chicken muscle.