We determined the longitudinal persistence of ceftiofur-resistant Escherichia coli from a chicken breeding farm in China. A total of 150 samples were collected from 5 breeding periods in a flock of layer hens, and the prevalence of ceftiofur-resistant E. coli fluctuated across the 5 chicken breeding stages: eggs, 3.33%; growing period, 100%; early laying period, 36.7%; peak laying period, 66.7% and late laying period, 80%. The most prevalent ceftiofur resistance genes were bla_CTX-M-55, bla_CMY and bla_NDM, and ST101 was the most prevalent and persistent sequence type across the breeding periods. Our results indicated that this breeder flock was heavily contaminated by ST101 ceftiofur-resistant E. coli and that its presence should be intensively monitored on chicken farms.

Anthropogenic environments take an active part in shaping the human microbiome. Herein, we studied skin and nasal microbiota dynamics in response to the exposure in confined and controlled swine farms to decipher the impact of occupational exposure on microbiome formation. The microbiota of volunteers was longitudinally profiled in a 9-months survey, in which the volunteers underwent occupational exposure during 3-month internships in swine farms. By high-throughput sequencing, we showed that occupational exposure compositionally and functionally reshaped the volunteers’ skin and nasal microbiota. The exposure in farm A reduced the microbial diversity of skin and nasal microbiota, whereas the microbiota of skin and nose increased after exposure in farm B. The exposure in different farms resulted in compositionally different microbial patterns, as the abundance of Actinobacteria sharply increased at expense of Firmicutes after exposure in farm A, yet Proteobacteria became the most predominant in the volunteers in farm B. The remodeled microbiota composition due to exposure in farm A appeared to stall and persist, whereas the microbiota of volunteers in farm B showed better resilience to revert to the pre-exposure state within 9 months after the exposure. Several metabolic pathways, for example, the styrene, aminobenzoate, and N-glycan biosynthesis, were significantly altered through our PICRUSt analysis, and notably, the function of beta-lactam resistance was predicted to enrich after exposure in farm A yet decrease in farm B. We proposed that the differently modified microbiota patterns might be coordinated by microbial and non-microbial factors in different swine farms, which were always environment-specific. This study highlights the active role of occupational exposure in defining the skin and nasal microbiota and sheds light on the dynamics of microbial patterns in response to environmental conversion.

Heteroresistance has seriously affected the evaluation of antibiotic efficacy against pathogenic bacteria, causing misjudgment of antibiotics’ sensitivity in clinical therapy, leading to treatment failure, and posing a serious threat to current medical health. However, the mechanism of Staphylococcus aureus heteroresistance to ciprofloxacin remains unclear. In this study, heteroresistance to ciprofloxacin in S. aureus strain 529 was confirmed by antimicrobial susceptibility testing and population analysis profiling (PAP), with the resistance of subclonal 529_HR based on MIC being 8-fold that of the original bacteria. A 7-day serial MIC evaluation and growth curves demonstrate that their phenotype was stable, with 529_HR growing more slowly than 529, but reaching a plateau in a similar proportion. WGS analysis showed that there were 11 nonsynonymous mutations and one deletion gene between the two bacteria, but none of these SNPs were directly associated with ciprofloxacin resistance. Transcriptome data analysis showed that the expression of membrane potential related genes (qoxA, qoxB, qoxC, qoxD, mprF) was downregulated, and the expression of multidrug resistance efflux pump gene mepA was upregulated. The combination of ciprofloxacin and limonene restored the 529_HR MIC from 1 mg/L to 0.125 mg/L. Measurement of the membrane potential found that 529_HR had a lower potential, which may enable it to withstand the ciprofloxacin-induced decrease in membrane potential. In summary, we demonstrated that upregulation of mepA gene expression and a reduction in membrane potential are the main heteroresistance mechanisms of S. aureus to ciprofloxacin. Additionally, limonene may be a potentially effective agent to inhibit ciprofloxacin heteroresistance phenotypes.

Salmonella enterica Serovar Typhimurium (S. Typhimurium) is a major global foodborne pathogen. In our previous study, the S. Typhimurium ATCC 14028 strain harboring plasmid pHXY0908 survived at fourfold its ciprofloxacin MIC (MIC0908 = 0.125 µg/mL), but the mechanism remained unclear. Heteroresistance, where minority subpopulations show elevated resistance, is among the key contributors to chronic, recurrent, and high-fatality infections. This study aimed to elucidate the molecular mechanisms by which plasmid pHXY0908 promotes ciprofloxacin heteroresistance in S. Typhimurium ATCC 14028. Ciprofloxacin heteroresistance in ATCC 14,028-pHXY0908 was confirmed by population analysis profiling, antimicrobial susceptibility testing, and stability assessment. Whole-genome sequencing (WGS) and transcriptome (RNA-seq) analyses were performed to identify genomic variations and global transcriptional changes linked to the development of heteroresistance. WGS revealed minimal DNA mutations in subclones with increased MIC, with no single nucleotide variants detected in known ciprofloxacin resistance genes. Notably, plasmid pHXY0908 acquisition altered chromosomal interaction domains (CIDs), potentially affecting gene regulation. Weighted gene co-expression network analysis (WGCNA) of transcriptomic profiles identified a “brown” module comprising 168 eigengenes that were significantly correlated with MIC values. Within this module, the efflux pump genes acrA, acrB, and tolC exhibited high gene significance and module membership scores. Additional candidate genes potentially contributing to ciprofloxacin heteroresistance were identified, including sodB, yhiH, yfaV, STM14_2687, and pHXY0908-99. Notably, STM14_2687 (a LysR-type transcriptional regulator), negatively regulated acrAB-tolC; its overexpression suppressed acrA and enhancing susceptibility to ciprofloxacin. Furthermore, functional validation demonstrated that plasmid pHXY0908 could confer heteroresistance to ciprofloxacin in other Salmonella enterica strains, Escherichia coli, and Klebsiella pneumoniae, suggesting its broad-host-range potential in mediating heteroresistance. Our study reveals that transcriptional regulation drives ciprofloxacin heteroresistance in S. Typhimurium ATCC 14028 carrying plasmid pHXY0908. We show that regulation of efflux pumps (e.g., acrAB-tolC), superoxide dismutase (sodB), DNA protection systems, and regulatory networks contributes to heteroresistance development. Plasmid pHXY0908 acquisition was found to confer ciprofloxacin heteroresistance across species, suggesting its potential for horizontal transfer and increased transmission risk. Notably, STM14_2687 was identified and validated as a transcription factor that represses acrAB-tolC expression and may serve as a potential therapeutic target.

The process of urbanization has brought with it several novel lifestyles, but it remains to be seen whether such lifestyles are the potential driver behind the spread of antimicrobial resistance (AMR) in modern society. Hence, this study employs the pet café as a proof of concept to observe how one pathway of AMR transmission occurs within a megacity. A total of 111 samples were collected from consumers, workers, animals and the surrounding environment from three pet cafés in Guangzhou, and 163 bacterial strains were isolated, with Escherichia coli (n=60) being the most dominant species. The sequence type and genomic diversity of E. coli were observed in all three cafés. Notably, 19 highly related ST328 strains were isolated in a single pet café from both workers (skin and faeces) and animals (faeces), suggesting transmission between distinct hosts. The number of SNPs between ST328 E. coli isolated in this study and strains from other provinces in China was minimal, with the possibility of clonal transmission. In terms of AMR, 90% of the isolates exhibited resistance to at least three distinct classes of antimicrobials (multidrug resistance). Multiple antimicrobial resistance genes (ARGs) such as tet(X4) were detected in this study, and plasmid, especially hybrid plasmid, is the main transmission vector of these ARGs. Our findings highlight that the pet café is a neglected site for the transfer of ARGs among Enterobacteriaceae, with a propensity for continuous contamination through either clonal or horizontal transmission of ARGs.