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Objectives The recently discovered tigecycline-inactivating enzyme Tet(X4) can confer high-level tigecycline resistance on its hosts, which makes it a public health concern. This study focused on isolation and screening of Tet(X4)-positive Enterobacteriaceae from the gut microbiota of a cohort of healthy individuals in Singapore. Methods MinION and Illumina sequencing was performed to obtain the complete genome sequences of Escherichia coli 2EC1-1 and 94EC. Subsequently, 109 human faecal samples were screened retrospectively for eravacycline-resistant Enterobacteriaceae strains, which were further tested for tet(X4) by PCR. The taxonomy of the isolated strains was determined by 16S rRNA gene PCR and Sanger sequencing. Results Comparative genomic analysis of E. coli 2EC1-1 and 94EC revealed that both carry tet(X4), which is encoded by IncI1-type plasmids p2EC1-1 and p94EC-2, respectively. Retrospective screening of faecal samples collected from 109 healthy individuals showed that the faecal carriage rate of Tet(X4)-producing Enterobacteriaceae is 10.1% (95% CI = 5.1%–17.3%), suggesting that tet(X4) is widely distributed in the gut microbiota of healthy individuals in Singapore. Conclusions To the best of our knowledge, this is the first report on the prevalence of tet(X4) in the gut microbiota of a healthy human cohort, as well as the first description of this resistance mechanism outside of China. Our findings suggest that surveillance of tet(X4) in community settings is vital to monitor the spread of this resistance mechanism.

Multidrug-resistant (MDR) Escherichia coli strains that carry extended-spectrum β-lactamases (ESBLs) or colistin resistance gene mcr-1 have been identified in the human gut at an increasing incidence worldwide. In this study, we isolated and characterized MDR Enterobacteriaceae from the gut microbiota of healthy Singaporeans and show that the detection rates for ESBL-producing and mcr-positive Enterobacteriaceae are 25.7% (28/109) and 7.3% (8/109), respectively. Whole-genome sequencing analysis of the 37 E. coli isolates assigned them into 25 sequence types and 6 different phylogroups, suggesting that the MDR E. coli gut colonizers are highly diverse. We then analyzed the genetic context of the resistance genes and found that composite transposons played important roles in the cotransfer of blaCTX-M-15/55 and qnrS1, as well as the acquisition of mcr-1. Furthermore, comparative genomic analysis showed that 12 of the 37 MDR E. coli isolates showed high similarity to ESBL-producing E. coli isolates from raw meat products in local markets. By analyzing the core genome single nucleotide polymorphisms (SNPs) shared by these isolates, we identified possible clonal transmission of an MDR E. coli clone between human and raw meat, as well as a group of highly similar IncI2 (Delta) plasmids that might be responsible for the dissemination of mcr-1 in a much wider geographic region. Together, these results suggest that antibiotic resistance may be transmitted between different environmental settings by the expansion of MDR E. coli clones, as well as by the dissemination of resistance plasmids. IMPORTANCE The human gut can harbor both antibiotic-resistant and virulent Escherichia coli which may subsequently cause infections. In this study, we found that multidrug-resistant (MDR) E. coli isolates from the gut of healthy Singaporeans carry a diverse range of antibiotic resistance mechanisms and virulence factor genes and are highly diverse. By comparing their genomes with the extended-spectrum β-lactamase (ESBL)-producing E. coli isolates from raw meat products that were sampled at a similar time from local markets, we detected an MDR E. coli clone that was possibly transmitted between humans and raw meat products. Furthermore, we also found that a group of resistance plasmids might be responsible for the dissemination of colistin resistance gene mcr-1 in Singapore, Malaysia, and Europe. Our findings call for better countermeasures to block the transmission of antibiotic resistance.

Multidrug resistant (MDR) Escherichia coli strains that carry extended-spectrum β-lactamases (ESBLs) or colistin resistance gene mcr-1 have been identified in the human gut at an increasing incidence worldwide. In this study, we sampled and characterized MDR Enterobacteriaceae from the gut microbiota of healthy Singaporeans and show that the prevalence of ESBL-producing and mcr-positive Enterobacteriaceae is 26.6% and 7.3%, respectively. Whole-genome sequencing of 37 E. coli isolates identified 25 sequence types and assigned them into six different phylogroups, suggesting that the human intestinal MDR E. coli strains are highly diverse. In addition, we found that E. coli isolates belonging to phylogroup D, B2 and F carry a higher number of virulence genes, whereas isolates of phylogroup A, B1 and E carry fewer virulence factor genes but are frequent carriers of florfenicol resistance gene floR and colistin resistance gene mcr-1. Comparison of the seven mcr-1-positive E. coli isolates revealed that mcr-1 is carried by conjugative plasmids or embedded in composite transposons, which could potentially mobilize mcr-1 to other pathogenic Enterobacteriaceae strains or MDR plasmids. Finally, we found that 12 out of the 37 MDR E. coli isolates in this study show high similarity to ESBL-producing E. coli isolates from raw meats from local markets, suggesting a potential transmission of MDR E. coli from meat products to the human gut microbiota. Our findings show diverse antibiotic resistance and virulence profiles of intestinal E. coli and call for better countermeasures to block the transmission of MDR E. coli via the food chain.ImportanceThe human gut can harbor both antibiotic resistant and virulent E. coli which may subsequently cause infections. In this study, the antibiotic resistance and virulence traits of antibiotic-resistant E. coli isolates from human gut microbiota of healthy subjects were investigated. The isolated E. coli strains carry a diverse range of antibiotic resistance mechanisms and virulence factor genes, are highly diverse to each other, and are likely to originate from raw meat products from the local markets. Of particular concern are seven E. coli isolates which carry colistin resistance gene mcr-1. This gene can be mobilized into other pathogens and MDR plasmids, thereby spreading resistance to the last-resort antibiotic colistin. Our findings also suggest that raw meat could serve as important source to transmit MDR bacteria into the human gut microbiota.