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The emergence of the plasmid-borne colistin resistance gene mcr-1 threats public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmids conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer and 18 genes were identified, including five with annotations unrelated to conjugation. The Cappable-seq and RNA-seq analysis confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. Plasmid invasion and co-culture competition assays revealed that pixR is essential for the spread and persistence of mcr-1-bearing IncX4 plasmids in bacterial populations, and effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence.IMPORTANCEThe spread of clinical important antibiotic resistance genes is frequently related to some epidemic plasmids. However, the underlying molecular mechanisms contributing to the successful spread of these epidemic plasmids remains unclear. The significant of our research indicated that efficient conjugation could promote the invasion and persistence of plasmids within a bacterial population, resulting in the successful dissemination of epidemic plasmids in nature. Our data also highlight the importance of developing plasmid conjugation inhibitors to solve the antibiotic resistance crisis.

The New Delhi Metallo-β-lactamase (NDM) producing Enterobacteriaceae is spreading worldwide. Although the blaNDM gene has been identified in animal associated Enterobacteriaceae isolates in many countries, little is known about its occurrence in animal products in Pakistan. In this study, 13 Escherichia coli isolates were collected from chicken meat samples in Pakistan. Two isolates, 15978 and C4109, exhibited reduced susceptibility (MIC ≥1 μg/mL) to imipenem, and carried blaNDM-5 and blaNDM-7 gene, respectively. Whole-genome sequencing and Oxford Nanopore MinION sequencing revealed that 15978 and C4109 belonged to ST156 and ST167, respectively. blaNDM-7 was carried by an IncX3 plasmid that has disseminated worldwide, whereas blaNDM-5 was located on an F36: A-: B32 plasmid, which shared high identity with two plasmids carried by E. coli isolates from other countries (one from a patient in Canada). To the best of our knowledge, this is the first report characterizing blaNDM-carrying plasmids from chicken meat samples in Pakistan. The dissemination of almost identical blaNDM-5-bearing F36:A-:B32 and blaNDM-7-bearing IncX3 plasmids in different countries highlights the importance of international trade and travel in the spread of antimicrobial resistance strains and plasmids worldwide.

The plasmid-encoded colistin resistance gene mcr-1 challenges the use of polymyxins and poses a threat to public health. Although IncI2-type plasmids are the most common vector for spreading the mcr-1 gene, the mechanisms by which these plasmids adapt to host bacteria and maintain resistance genes remain unclear. Herein, we investigated the regulatory mechanism for controlling the fitness cost of an IncI2 plasmid carrying mcr-1. A putative ProQ/FinO family protein encoded by the IncI2 plasmid, designated as PcnR (plasmid copy number repressor), balances the mcr-1 expression and bacteria fitness by repressing the plasmid copy number. It binds to the first stem-loop structure of the repR mRNA to repress RepA expression, which differs from any other previously reported plasmid replication control mechanism. Plasmid invasion experiments revealed that pcnR is essential for the persistence of the mcr-1-bearing IncI2 plasmid in the bacterial populations. Additionally, single-copy mcr-1 gene still exerted a fitness cost to host bacteria, and negatively affected the persistence of the IncI2 plasmid in competitive co-cultures. These findings demonstrate that maintaining mcr-1 plasmid at a single copy is essential for its persistence, and explain the significantly reduced prevalence of mcr-1 following the ban of colistin as a growth promoter in China.

Summary Background Until now, polymyxin resistance has involved chromosomal mutations but has never been reported via horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae. Methods The mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model. Findings Polymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10 −1 to 10 −3 cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa . In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011–14, and 16 (1%) of 1322 samples from inpatients with infection. Interpretation The emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria. Funding Ministry of Science and Technology of China, National Natural Science Foundation of China.

The colonization of dairy herds and subsequent contamination of raw milk by Staphylococcus aureus (S. aureus), especially those expressing a multi-drug resistance (MDR), biofilm and toxins producing ability, remains an important issue for both the dairy producer and public health. In this study, we investigated the prevalence, antimicrobial resistance, virulence, and genetic diversity of S. aureus in raw milk taken from 2 dairy farms in Beijing, China. Ninety (46.2%, 90/195) samples were positive for S. aureus. Resistant to penicillin (PEN) (31.3%), ciprofloxacin (18.8%) and enrofloxacin (15.6%) were the most often observed. Isolates cultured from farm B showed significantly higher resistance to penicillin (73.9%), ciprofloxacin (34.8%), enrofloxacin (34.8%), tilmicosin (17.4%), and erythromycin (17.4%) than those from farm A (p < 0.05). Totally, 94.8% S. aureus harbored at least one virulence gene and the pvl (93.8%), sec (65.6%), and sea (60.4%) genes were the most frequently detected. The pvl and sec genes were more often detected in isolates from farm A (97.3% and 84.9% respectively) than those from farm B (p < 0.05). Of all 77 staphylococcus enterotoxin (SE)-positive isolates, more than 90% could produce enterotoxins and 70.1% could produce two types. Biofilm related genes (icaA/D, clf/B, can, and fnbA) were detected in all96 isolates. All 96 isolates could produce biofilm with 8.3, 70.8, and 18.8% of the isolates demonstrating weak, moderate and strong biofilm formation, respectively. A total of 5 STs, 7 spa types (1 novel spa type t17182), 3agr types (no agrII), and 14 SmaI-pulso-types were found in this study. PFGE cluster II-CC1-ST1-t127-agr III was the most prevalent clone (56.3%). Isolates of agr III (PFGE Cluster I/II-CC1-ST1-t127/2279) had higher detection of virulence genes than those of agr I and agr IV. TheMSSA-ST398-t1456-agr I clone expressed the greatest MDRbut with no virulence genes and weakly biofilm formation. Our finding indicated a relatively high prevalence of S. aureus with less antimicrobial resistance but often positive for enterotoxigenicity and biofilm formation. This study could help identify predominant clones and provide surveillance measures to eliminate and decrease the contamination of S. aureus in raw milk of dairy cows with mastitis.

The objective of this study was to assess the impact of diverse plasmids bearing colistin resistance gene mcr-1 on host fitness. Forty-seven commensal E. coli isolates recovered from the pig farm where mcr-1 was first identified were screened for mcr-1. mcr-1-bearing plasmids were characterized by sequencing. The fitness impact of mcr-1-bearing plasmids was evaluated by in vitro competition assays. Twenty-seven (57.5%) E. coli isolates were positive for mcr-1. The mcr-1 genes were mainly located on plasmids belonging to IncI2 (n = 5), IncX4 (n = 11), IncHI2/ST3 (n = 8), IncFII (n = 2), and IncY (n = 2). InHI2 plasmids also carried other resistance genes (floR, blaCTX-M, and fosA3) and were only detected in isolates from nursery pigs. Sequences of the representative mcr-1-bearing plasmids were almost identical to those of the corresponding plasmid types reported previously. An increase in the fitness of IncI2- and IncX4-carrying strains was observed, while the presence of IncHI2, IncFII and IncY plasmids showed a fitness cost although an insignificant fitness increase was initially observed in IncFII or IncY plasmids-containing strains. Acquisition of IncI2-type plasmid was more beneficial for host E. coli DH5α than either IncHI2 or IncX4 plasmid, while transformants with IncHI2-type plasmid presented a competitive disadvantage against IncI2 or IncX4 plasmid containing strains. In conclusion, IncI2, IncX4, and IncHI2 were the major plasmid types driving the dissemination of mcr-1 in this farm. Increased fitness or co-selection by other antimicrobials might contribute to the further dissemination of the three epidemic mcr-1-positive plasmids (IncI2, IncX4, and IncHI2) in this farm and worldwide.

mcr-1, the first plasmid-mediated colistin-resistance gene, can mediate polymyxin resistance and be transferred horizontally via plasmids. Many studies have confirmed its distribution via epidemic plasmids (IncI2, IncX4, IncHI2, etc.), as well as mobile genetic elements, among Enterobacteriaceae isolated from animals, humans, and the environment in 35 countries. These studies provide the basis of understanding the complicated mechanism of colistin resistance mediated by MCR-1 and its global dissemination and epidemic properties, and also enrich antimicrobial resistance mechanisms. Here, we review the latest advances in the prevalence, resistance mechanism, transfer mechanism, and genetic environments of mcr-1 in isolates recovered from various samples worldwide. Finally, we discuss the clinical risk and the corresponding solutions, aiming to provide a basis for researchers and clinical scientists to face the serious challenge of antimicrobial resistance together.