The survival of Salmonella Enteritidis in the food chain is relevant to its biofilm formation capacity, which is influenced by suboptimal environmental conditions. Here, biofilm formation pattern of this bacterium was assessed in the presence of ethanol at sub-minimal inhibitory concentrations (sub-MICs) by microtiter plate assays, cell characteristic analyses, and gene expression tests. It was observed that ethanol at subinhibitory concentrations (1/4 MIC, 2.5%; 1/2 MIC, 5.0%) was able to stimulate biofilm formation in S. Enteritidis. The OD₅₉₅ value (optical density at 595 nm) used to quantify biofilm production was increased from 0.14 in control groups to 0.36 and 0.63 under 2.5% and 5.0% ethanol stresses, respectively. Ethanol was also shown to reduce bacterial swimming motility and enhance cell auto-aggregation ability. However, other cell characteristics such as swarming activity, initial attachment and cell surface hydrophobicity were not remarkedly impacted by ethanol. Reverse transcription quantitative real-time PCR (RT-qPCR) analysis further revealed that the luxS gene belonging to a quorum-sensing system was upregulated by 2.49- and 10.08-fold in the presence of 2.5% and 5.0% ethanol, respectively. The relative expression level of other biofilm-related genes (adrA, csgB, csgD, and sdiA) and sRNAs (ArcZ, CsrB, OxyS, and SroC) did not obviously change. Taken together, these findings suggest that decrease in swimming motility and increase in cell auto-aggregation and quorum sensing may result in the enhancement of biofilm formation by S. Enteritidis under sublethal ethanol stress.

Tigecycline resistance in bacteria has become a significant threat to food safety and public health, where the development of which is attributed to plasmid-mediated tet(X4) genes. In this study, the genomes of 613 tet(X4)-producing Escherichia coli (E. coli) isolates, available from public databases, are evaluated to determine their international prevalence and molecular characterization. These E. coli isolates have been disseminated in 12 countries across Asia and Europe. It was found that pigs and their products (n = 162) were the most common vehicle, followed by humans (n = 122), chickens (n = 60), and the environment (n = 49). Carbapenems-resistant genes bla_NDM-5 (1.3%) and bla_NDM-1 (0.2%) were identified, as well as colistin-resistant genes mcr-1.1 (12.6%) and mcr-3.1 (0.5%). It was noted that the tigecycline-resistant gene cluster tmexC-tmexD-toprJ1 was identified in seven (1.1%) isolates. Phylogenomic results indicated that tet(X4)-producing E. coli isolates fell into seven lineages (lineages I, II, III, IV, V, VI, and VII), and international spread mainly occurred in Asian countries, especially China, Pakistan, Singapore, and Malaysia. Four forms of tet(X4) transposon units were found, including the I-type (IS26-tet(X4)-ISCR2), II-type (ΔIS1R-tet(X4)-ISCR2), III-type (ISCR2-tet(X4)-ISCR2), and IV-type (ISCR2-tet(X4)-ΔISCR2). These findings underline further challenges for the spread of E. coli bearing tet(X4) gene.

Staphylococcus aureus, a major cause of foodborne illness globally, presents significant challenges due to its multidrug resistance and biofilm-forming capabilities. Pyruvate carboxylase (PycA), a metabolic master switch linking glycolysis and the tricarboxylic acid (TCA) cycle, is a potential target for controlling S. aureus. In this study, a pycA mutant was constructed and analyzed using phenotypic assays and proteomics to investigate its role in virulence and antimicrobial resistance. The results showed that deletion of pycA in the foodborne methicillin-resistant strain ATCC BAA1717 resulted in a 4- to 1024-fold reduction in resistance to β-lactams, aminoglycosides, and macrolides; a 23.24% impairment in biofilm formation; and a 22.32% decrease in staphyloxanthin production, a key antioxidant essential for survival in oxidative food environments. Proteomic analysis revealed downregulation of the TCA cycle, purine biosynthesis, surface adhesins (FnbA/B, SasG), and β-lactamase (BlaZ), linking PycA-mediated metabolism to phenotypes relevant to food safety. These findings underscore the importance of PycA as a metabolic regulator crucial for S. aureus resilience in food systems, suggesting novel strategies to combat foodborne staphylococcal infections through metabolic interference.

Salmonella is a major foodborne pathogen that poses an increasing threat due to the emergence of mcr-1-mediated colistin resistance. However, data on mcr-1-positive Salmonella in pork products are limited. In this study, 457 samples collected in 2023 from pig slaughterhouses in Guangdong province were investigated to determine the prevalence and genomic characteristics of mcr-1-positive Salmonella isolates. We found that 92 Salmonella isolates (20.1%, 92/457) were recovered, representing six serotypes, including Salmonella Typhimurium (n = 29) and Salmonella Rissen (n = 29). High resistance to tetracycline (90.2%, 83/92) and multidrug resistance (58.7%, 54/92) were observed. Critically, two colistin-resistant Salmonella Kentucky ST198 isolates (2.2%) harboring mcr-1 on transferable IncI2 plasmids were identified. Genomic analysis revealed a novel multidrug resistance region (MRR, ~57 kb) inserted into the bcfH locus (containing floR, qnrS1, bla_CTX-M-55, and aph (3’)-Ia) and a variant Salmonella Genomic Island 1 (SGI1-KI, containing tet (A), sul1, qacEΔ1 and aadA7) in these isolates. The MRR and SGI1-KI may enhance bacterial survival under antibiotic selection pressure. Phylogenetic analysis showed close relatedness to human clinical strains, suggesting food chain transmission. The findings highlight a high antimicrobial resistance burden, the emergence of transferable last-resort colistin resistance (mcr-1), and acquisition of complex resistance determinants (MRR, SGI1-KI), underscoring an urgent need for enhanced “One Health” surveillance.

Two-component systems (TCS) of Salmonella enterica serovar Enteritidis are composed of a histidine kinase and a response regulator (RR) and represent a critical mechanism by which bacteria develop resistance to environmental stress. Here, we characterized the functions of RRs in TCS in the formation of stress tolerance, motility and biofilm using twenty-six S. Enteritidis RR-encoding gene deletion mutants. The viability results unraveled their essential roles in resistance to elevated temperature (GlrR), pH alterations (GlrR, TctD, YedW, ArcA and YehT), high salt (PhoB, BaeR, CpxR, PhoP, UvrY and TctD), oxidative stress (PhoB, YedW, BaeR, ArcA, PhoP, UvrY, PgtA and QseB) and motility (ArcA, GlnG, PgtA, PhoB, UhpA, OmpR, UvrY and QseB) of S. Enteritidis. The results of the crystal violet staining, microscopy observation and Congo red binding assays demonstrated that the absence of ArcA, GlnG, PhoP, OmpR, ZraR or SsrB in S. Enteritidis led to a reduction in biofilms and an impairment in red/dry/rough macrocolony formation, whereas the absence of UvrY exhibited an increase in biofilms and formed a brown/smooth/sticky macrocolony. The results indicated the regulatory effects of these RRs on the production of biofilm matrix, curli fimbriae and cellulose. Our findings yielded insights into the role of TCSs, making them a promising target for combating S. Enteritidis.

The emergence of extensively drug-resistant (XDR) foodborne pathogens poses grave threats to food safety. This study characterizes the genome of an XDR Salmonella Kentucky isolate (Sal23C1) co-harboring cfr, mcr-1 and tet(A) from Shanghai chicken meat in 2022, which was the only isolate co-harboring these three key resistance genes among 502 screened Salmonella isolates. Genomic analysis revealed that the multidrug resistance gene cfr, which confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A, was identified within a Tn3-IS6-cfr-IS6 structure on the transferable plasmid p3Sal23C1 (32,387 bp), showing high similarity to the Citrobacter braakii plasmid pCE32-2 (99% coverage, 99.98% identity). Concurrently, the mcr-1 gene resided in a pap2-mcr-1 structure on the transferable IncI2 plasmid p2Sal23C1 (63,103 bp). Notably, both genes could be co-transferred to recipient bacteria via conjugative plasmids at frequencies of (1.15 ± 0.98) × 10⁻⁶. Furthermore, a novel ~79 kb multidrug resistance region (MRR) chromosomally inserted at the bcfH locus was identified, carrying fosA3, mph(A), rmtB, qnrS1 and bla_CTX-M-55. Additionally, a novel Salmonella Genomic Island 1 variant (SGI1-KI) harbored aadA7, qacEΔ1, sul1 and the tet(A) variant. The acquisition of these antibiotic resistance genes in this isolate enhanced bacterial resistance to 21 antimicrobials, including resistance to the critical last-resort antibiotics tigecycline and colistin, which left virtually no treatment options for potential infections. Taken together, this is the first comprehensive genomic report of an XDR poultry-derived Salmonella Kentucky isolate co-harboring cfr, mcr-1 and the tet(A) variant. The mobility of these resistance genes, facilitated by IS6 elements and conjugative plasmids, underscores significant public health risks associated with such isolates in the food chain.