Ready-to-eat (RTE) foods have been considered to be reservoirs of antibiotic resistance bacteria, which constitute direct threat to human health, but the potential microbiological risks of RTE foods remain largely unexplored. In this study, the metagenomic approach was employed to characterize the comprehensive profiles of bacterial community and antibiotic resistance gene (ARG) in 18 RTE food samples (8 RTE meat, 7 RTE vegetables and 3 RTE fruit) in southern China. In total, the most abundant phyla in RTE foods were Proteobacteria, Firmicutes, Cyanobacteria, Bacteroidetes and Actinobacteria. 204 ARG subtypes belonging to 18 ARG types were detected with an abundance range between 2.81 × 10⁻⁵ and 7.7 × 10⁻¹ copy of ARG per copy of 16S rRNA gene. Multidrug-resistant genes were the most predominant ARG type in the RTE foods. Chloramphenicol, macrolide-lincosamide-streptogramin, multidrug resistance, aminoglycoside, bacitracin, tetracycline and β-lactam resistance genes were dominant, which were also associated with antibiotics used extensively in human medicine or veterinary medicine/promoters. Variation partitioning analysis indicated that the join effect of bacterial community and mobile genetic elements (MGEs) played an important role in the resistome alteration. This study further deepens the comprehensive understanding of antibiotic resistome and the correlations among the antibiotic resistome, microbiota, and MGEs in the RTE foods.

Microbial multidrug resistance (MDR) poses a huge threat to human health. Bacterial acquisition of MDR relies primarily on class 1 integron-involved horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). To date, no strategies other than the use of antibiotics can efficiently cope with MDR. Here, we report that an engineered CRISPR interference (CRISPRi) system can markedly reduce MDR by blocking a class 1 integron in Escherichia coli. Using CRISPRi to block plasmid R388 class 1 integron, E. coli recombinants showed halted growth upon exposure to relevant antibiotics. A microplate alamarBlue assay showed that both subgenomic RNAs (sgRNAs) R3 and R6 led to 8- and 32-fold decreases in half-maximal inhibitory concentrations (IC₅₀) for trimethoprim and sulfamethoxazole, respectively. Reverse transcription and quantitative PCR (RT-qPCR) revealed that the strain employing sgRNA R6 exhibited 97% and 84% decreases in the transcriptional levels of the dfrB2 cassette and sul1, two typical ARGs, respectively. RT-qPCR analysis also demonstrated that the strain recruiting sgRNA R3 showed a 96% decrease in the transcriptional level of intI1, and a conjugation assay revealed a 1,000-fold decrease in HGT rates of ARGs. Overall, the sgRNA R3 targeting the 31 bp downstream of the Pc promoter on the intI1 nontemplate strand outperformed other sgRNAs in reducing integron activity. Furthermore, this CRISPRi system is reversible, genetically stable, and titratable by varying the concentration of the inducer. To our knowledge, this is the first report on exploiting a CRISPRi system to reduce the class 1 integron in E. coli. This study provides valuable insights for future development of CRISPRi-based antimicrobial agents and cellular therapy to suppress MDR.

Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic-resistant genes (ARGs) among human pathogens. The spread of ARGs can be halted or diminished by interfering with the conjugation process. In this study, we explored the possibility of using an rbsB gene as a single target to inhibit plasmid-mediated horizontal gene transfer in Escherichia coli by CRISPR interference (CRISPRi) system. Three single-guide RNAs (sgRNAs) were designed to target the rbsB gene. The transcriptional levels of the rbsB gene, the conjugation-related genes, and the conjugation efficiency in the CRISPRi strain were tested. We further explored the effect of the repressed expression of the rbsB gene on the quorum sensing (QS) system and biofilm formation. The results showed that the constructed CRISPRi system was effective in repressing the transcriptional level of the rbsB gene at a rate of 66.4%. The repressed expression of the rbsB gene resulted in the reduced conjugation rate of RP4 plasmid by 88.7%, which significantly inhibited the expression of the conjugation-related genes (trbBp, trfAp, traF and traJ) and increased the global regulator genes (korA, korB and trbA). The repressed rbsB gene expression reduced the depletion of autoinducer 2 signals (AI-2) by 12.8% and biofilm formation by a rate of 68.2%. The results of this study indicated the rbsB gene could be used as a universal target for the inhibition of conjugation. The constructed conjugative CRISPRi system has the potential to be used in ARG high-risk areas.

In-depth knowledge of the microbes responsible for biogenic amine (BA) production during soy sauce fermentation remains limited. Herein, the variations in the BA profiles, microbial communities, and microbes involved in BA production during the fermentation of soy sauce through Japanese-type (JP) and Cantonese-type (CP) processes were compared. BA analysis revealed that the three most abundant BA species were putrescine, tyramine, and histamine in the later three stages (1187.68, 785.16, and 193.20 mg/kg on average, respectively). The BA profiles differed significantly, with CP samples containing higher contents of putrescine, tyramine, and histamine (P < 0.05) at the end of fermentation. Metagenomic analysis indicated that BA-producing genes exhibited different abundance profiles, with most genes, including speA, speB, arg, speE, and tyrDC, having higher abundances in microbial communities during the CP process. In total, 15 high-quality metagenome-assembled genomes (MAGs) were retrieved, of which 10 encoded at least one BA production-related gene. Enterococcus faecium (MAG10) and Weissella paramesenteroides (MAG5) might be the major tyramine producers. The metabolic pathways for BA production were predicted. The high putrescine content in CP might be associated with the high abundance of Staphylococcus gallinarum (MAG8). This study provides a comprehensive understanding of the diversity and abundance of genes involved in BA synthesis, especially at the species level, during food fermentation.