Persistence and Transmission Dynamics of mcr-1 and mcr-9 in Enterobacteriaceae from Pig Farms in Fujian, China after the Colistin Ban

Persistence and Transmission Dynamics of mcr-1 and mcr-9 in Enterobacteriaceae from Pig Farms in Fujian, China after the Colistin Ban | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Persistence and Transmission Dynamics of mcr-1 and mcr-9 in Enterobacteriaceae from Pig Farms in Fujian, China after the Colistin Ban Lingxian Yi, Huamin Lai, Jianshuo Liu, Donghong Huang, Jiaming Huang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6934660/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Polymyxins are last-resort antibiotics for multidrug-resistant Gram-negative bacteria infections. However, their efficacy is threatened by the emergence of plasmid-mediated colistin resistance genes ( mcr-1 to mcr-10 ). Antibiotic use in agriculture has been recognized as a major driver of antimicrobial resistance, prompting China to ban colistin as a feed additive in 2017. This study investigated the persistence and transmission mechanisms of mcr genes in Enterobacteriaceae isolated from pigs, farm workers, and the surrounding environment on four pig farms in Fujian Province, China. Totally, 930 samples were collected, including pig rectal swabs, farm worker fecal samples, as well as environmental samples from both inside and outside the farms. From these, a total of 263 Klebsiella pneumoniae and 521 Escherichia coli isolates wererecovered, with isolation rate of 28.3% and 56.0%, respectively. PCR screening revealed that mcr-1 and mcr-9 were the only detected variants. Among K. pneumoniae isolates , 39 (14.8%) carried mcr-1 and 21(8.0%) carried mcr-9 . In E. coli , mcr-1 and mcr-9 were detected in 8 (1.5%) and 6 (1.1%) isolates, respectively. In addition, three Raoultella ornithinolytica and one Enterobacter roggenkampii isolates carried mcr-9 genes were identified . Besides animal sources, mcr -positive strains were also identified in environmental samples, particularly from inside the farms, and in farm workers, indicating potential zoonotic and environmental transmission.Antimicrobial susceptibility testing revealed that all mcr -positive isolates exhibited multi-antibiotic resistance, with mcr-1 -positive strains displaying broader resistance profiles than mcr-9 -positive strains. The minimum inhibitory concentrations (MICs)of colistin ranged from 2-32 μg/mL for mcr-1 -positive isolates and 1-8 μg/mL for mcr-9 -positive isolates. Whole-genome sequencing and conjugation experiments showed that mcr-1 was primarily located on IncHI2 (n=5), IncX4 (n=14), and IncI2 (n=15) plasmids, while mcr-9 was predominantly carried by IncHI2 plasmids (n=4) and IncF(n=2) plasmids. Notably, mcr-9 -positive plasmids showed higher conjugation efficiency, lower fitness cost, and greater persistence within bacterial hosts compared to mcr-1 -positive IncHI2 plasmids. These finding suggest that the increasing prevalence of mcr-9 in Enterobacteria may be driven by its enhanced transferability and stability, even in the absence of antibiotic selection pressure. This highlights the urgent need for continued environmental surveillance and targeted interventions to solve the dissemination of plasmid-mediated antibiotic resistance in livestock production. mcr colistin resistance Enterobacteriaceae One Health plasmid Environmental reservoirs Figures Figure 1 Figure 2 Figure 3 Introduction The end of the 'golden age of antibiotics' is approaching as global antibiotic resistance continues to rise, with carbapenems-producing Enterobacteriaceae (CPE) emerging as a critical public health threat[1]. Due to the increasing prevalence of multidrug-resistant Gram-negative bacteria and limited development of effective new antibiotics, colistin has become a crucial last-resort treatment for CPE infections[2]. Following the initial discovery of the mcr-1 gene in 2015, multiple variants ( mcr-1 through mcr-10 ) have been identified globally in diverse bacterial species[3-8]. Escherichia coli remains the most common carrier, with mcr-1 -positive strains showing the highest prevalence, followed by mcr-9 and mcr-3 variants[9]. In addition, geographical variations in prevalence have been noted: mcr-9 -positive E. coli strains predominate in Europe, North America, and Africa, while mcr-3 -positive strains are more prevalent in Asia[10, 11]. However, the molecular mechanisms underlying the global dissemination of these mcr variants remain unclear. In response to the escalating detection rates of mcr genes, several countries, including China, Japan, and Thailand, have implemented strict restrictions on the use of colistin as a growth promoter in food-producing animals[12-14]. These regulatory interventions have resulted in a marked reduction in mcr -positive Escherichia coli in animal populations, particularly mcr-1 -positive strains[8]. Also, the detection rates of mcr gene in pig feces, especially in colon, have reported decreased in China after the ban on colistin as a growth promoter[15]. However, previous reports have mainly focused on the prevalence of mcr gene in the pig feces, while their persistence and distribution in the broader farming environment remain largely unclear. This study aims to investigate the distribution of mcr genes in pig faeces, farm environments and farm workers across four agricultural sites in Fujian province during 2022-2023. Additionally, the study explores the molecular mechanisms contributions the recent increase in mcr-9 -positive Enterobacteriaceae , highlighting an emerging trend towards mcr-9 gene predominance within Enterobacteriaceae bacteria. Methods Sampling and isolation During 2022-2023, we collected non-duplicate environmental and fecal samples from four swine farms in Fujian province (Ningde, Yongtai, Longyan and Fuqing). Samples included swine faeces, drinking water, drinking devices, feed, feed troughs, worker faeces, worker boots, soil, and surfaces (ground and walls) from both interior and exterior farm environments. All samples were individually collected in sterile sampling bags and transported to the laboratory in cold storage (4°C) within 6 hours of collection. For bacterial isolation, each sample were enriched in 2 mL Luria-Bertani (LB broth, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) broth and incubated overnight at 37°C with 200 rpm shaking. The enriched cultures were subsequently plated on MacConkey agar (Mac agar, Beijing Solarbio Science & Technology Co., Ltd) with 2 μg/mL of colistin. One to two morphologically distinct colonies from each MacConkey plate were sub-cultured onto Levine Eosin-Methylene Blue agar (EMB agar, Qingdao Hi-Tech Industrial Park Hope Bio-Technology Co., Ltd, Qingdao, China). Morphologically distinct colonies were subjected to species identification through 16S rRNA gene sequencing. Additionally, 13 colistin resistant extended-spectrum 𝛽-lactamase strains, including 12 K. pneumoniae and 1 Escherichia coli, were obtained from a Grade A tertiary hospital in Fujian province, China. These isolates were isolated from clinical samples, including urine, blood and sputum. Detection of mcr genes and antimicrobial susceptibility testing (AST) All Enterobacteriaceae isolates were screened for colistin resistance genes ( mcr-1 to mcr-10 ) using primers listed in Table S1. Positive amplicons were confirmed through Sanger sequencing and subsequent analysis using the Basic Local Alignment Search Tool (BLAST) against the National Center for Biotechnology Information (NCBI) nr database. Minimum inhibitory concentrations (MICs) for 11 antimicrobial agents against mcr -positive isolates were determined using broth microdilution methods according to Clinical and Laboratory Standards Institute guidelines (CLSI M100)[16]. Interpretative criteria followed CLSI breakpoints, except for cefquinome according to CLSI supplement VET08, and tigecycline interpreted using European Committee on Antimicrobial Susceptibility Testing (EUCAST v14.0) clinical breakpoints[17, 18]. Escherichia coli ATCC 25922 served as the quality control strain. Conjugation Assays and Plasmid Characterization Conjugation experiments were performed using broth mating techniques according to previous reports[19]. Streptomycin-resistant Escherichia coli C600 or rifampicin-resistant Klebsiella pneumoniae strain 13883 served as the recipient strain, while mcr -positive isolates served as donors. Both donor and recipient strains were cultured in LB broth until reaching an optical density (OD 600 ) of 0.8. Equal volumes of donor and recipient cultures were mixed and incubated without agitation at 37°C for 18 hours. The resulting mixed culture was centrifuged and washed once with LB medium. Cell pellets were resuspended in 100 μL LB broth and plated on MacConkey agar supplemented with either streptomycin (3,000 μg/mL) or rifampin (512 μg/mL) alone or in combination with colistin (2 μg/mL). Conjugation frequencies were calculated as the ratio of transconjugant to recipient colony-forming units (CFUs). Transconjugants were confirmed through antibiotic selection and PCR verification with primers for mcr-1 or mcr-9 listed in Table S1. Eric-PCR were performed using the primers listed in Table S1 to confirm that the genomic fingerprint of transconjugants matched that of the recipient strain[20]. Whole-genome sequencing (WGS) and bioinformatic analysis Genomic DNA from nine mcr -positive isolates was extracted using the E.Z.N.A. Bacterial DNA Kit (Omega Bio-tek, Shanghai, China). The gDNA was separated into two parts. One was randomly fragmented to construct a library with an average insertion of 300 bp. The library was sequenced on illumina NovaSeq 6000 platform performing using a pair-end 150bp sequencing strategy. Clean reads were obtained by quality filtering and subjected to denovo assembly using SPAdes (v3.13.1). For the strains FJZZ12 and FJNS5, the second parts of gDNA were straightly processed through end-repair, 3’ adenylated, adapters and Motor protein ligations. The product was purified using Agencourt AMPure XP Beads (Beckman, A63881). Finally, fragments larger than 1KB were screened for Single-molecule nanopore DNA sequencing on MinION Flow Cell (ONT, R9.4.1). The raw reads were filtered and assembled using Canu with default parameters. The genomic sequences were proofread using nextpolish (v1.4.1) and Pilon (v1.18). Gene prediction and annotation were generated using the Bakta (v1.8.2) and the National Center for Biotechnology Information (NCBI) nr database. Sequence analyses were performed using the Center for Genomic Epidemiology (CGE) pipeline: multilocus sequence typing (MLST) for strain typing, PlasmidFinder for plasmid replicon identification, ResFinder for antimicrobial resistance gene detection and KmerFinder (v3.0.2) for bacterial species prediction. Comparative plasmid analyses were conducted using EasyFig and SnapGene software. Growth Kinetics of mcr -Positive Strains and Stability Test of mcr -Positive Plasmids To investigate the impact of mcr -positive plasmids on the fitness of the bacterial host strain BW25113, the plasmid pFJZZ12- mcr-9 , pFJNS5- mcr-9 and pFJY27- mcr-1 were chosen as the representative for IncFIB and IncHI2 plasmid type following previous methods[21]. Briefly, overnight cultures of BW25113 and transformants BW25113/pFJZZ12- mcr-9 , BW25113/pFJNS5- mcr-9 and BW25113/pFJY27- mcr-1 were measured for OD 600 . The initial OD 600 of each strain was adjusted to 0.01 in 50 mL of LB medium without antibiotics. Every hour, 200 µL of culture from each group was sampled in triplicate to measure OD 600 in a 96-well plate, with LB broth serving as a blank control. Plasmid stability was evaluated following established protocols. Briefly, overnight cultures of BW25113/pFJZZ12- mcr-9 , BW25113/pFJNS5- mcr-9 and BW25113/pFJY27- mcr-1 were diluted 1:1000 every 12 hours into 10 mL LB medium without antibiotics for continuous passaging over 7 days. On day 1, 3, 5 and 7, 100 µL of appropriately diluted culture was plated on LB plates and incubated overnight at 37 °C. the following day, 50 colonies were randomly selected and streak onto LB plates containing 2 µg/mL colistin. PCR was performed using primers listed in Table S1 to confirm the presence of mcr-1 or mcr-9 genes, assessing plasmid retention. Plasmid fitness assays of mcr -positive strains The fitness impacts of mcr -positive plasmids were evaluated through pairwise competition experiments. BW25113/pFJY27- mcr-1 was competed against BW25113/pFJZZ12- mcr-9 , and BW25113/pFJY27- mcr-1 was competed against BW25113/pFJNS5- mcr-9 . All assays were performed in biological triplicates, following previously methods with minor modifications[19]. Briefly, three single colonies from each strain were individually cultured overnight at 37 °C in LB broth supplemented with 2 µg/mL colistin. Cultures were mixed at a 1:1 ratio and diluted 1:1,000 into fresh antibiotic-free LB broth every 24 hours, for five days. At the beginning of the competition and at 24-hour intervals (days 1–5), aliquots of the cultures were appropriately diluted and plated onto LB agar plates containing 2 µg/mL colistin. All colonies grown were streaked onto LB plates containing 128 µg/mL florfenicol to select for pFJNS5- mcr-9 or 4 µg/mL cefotaxime to select for pFJZZ12- mcr-9 . Fifty colonies from these selective plates were randomly picked for PCR confirmation of the mcr-9 gene. Based on previous sequencing results, the plasmid pFJNS5- mcr-9 carried the florfenicol resistance gene floR , and the plasmid pFJZZ12- mcr-9 carried the cefotaxime resistance gene bla CTX-M-9 . The mcr-1 -positive plasmid pFJY27- mcr-1 lacked these resistance genes and thus did not grow on these selective plates, allowing clear differentiation of strains. Plasmid invasion and competition assays of mcr -positive strains Plasmid invasion assays were performed following previously established methods with slight modifications[19]. Klebsiella pneumoniae strain 13883 was used as the recipient strain, with plasmid-carrying 13883 transformants as donors. Three strain combinations were evaluated: (1) plasmid-free 13883, 13883/pFJY27- mcr-1 and 13883/pFJZZ12- mcr-9 ; and (2) plasmid-free 13883, 13883/pFJY27- mcr-1 and 13883/pFJNS5- mcr-9 . Each strain was cultured overnight in LB medium supplemented with 2 µg/mL colistin at 37 °C. The recipient strain 13883 were diluted to approximately 10⁻⁷ CFU/mL, and donor strains were diluted to approximately 10⁻⁵ CFU/mL co-cultured in 5 mL fresh LB medium at 37 °C with 80 rpm rotation. Cultures were diluted 1:100 into fresh LB broth every 24 hours for a total of 96 hours. At 24, 48, 72, and 96 hours, aliquots were serially diluted and plated onto selective LB agar plates containing 256 µg/mL rifampicin alone, 256 µg/mL rifampicin plus 2 µg/mL colistin, and 256 µg/mL rifampicin plus 128 µg/mL florfenicol or 256 µg/mL rifampicin plus 4 µg/mL cefotaxime. Colony counts from these plates were used to quantify population as follows: CFU of 13883/pFJY27- mcr-1 = (colonies on colistin and rifampicin plates × dilution factor) – (colonies on florfenicol and rifampicin plates or cefotaxime and rifampicin plates × dilution factor). CFU of 13883 (recipient) = colonies on rifampicin plates × dilution factor – colonies on colistin and rifampicin plates × dilution factor. CFU of 13883/pFJNS5 -mcr-9 or 13883/pFJZZ12 -mcr-9 = (colonies on florfenicol and rifampicin plates or cefotaxime and rifampicin plates) × dilution factor. Results Prevalence of mcr positive- bacteria in pig farms A total of 930 non-duplicate swab samples of were collected from both swine and the environment of four pig farms, including 537 samples from pig feces, 232 environmental samples within the pig farms and 124 water, soil samples outside the pig farms and 37 farmer feces. The mcr gene could be detected in pig feces, environmental samples both inside and outside the pig farm and in farmer feces. The isolation rates were listed in Table S2. Among the isolates, we found that 39/263 (14.8%) and 21/263 (8.0%) K. pneumoniae strains were positive for mcr-1 and mcr-9 ， respectively. Additionally, 7/521（1.3%）and 7/521(1.3%) E. coli strains were found to carry mcr-1 and mcr-9 ，respectively. In addition, we identified 3 Raoultella ornithinolytica strains T9，YSC9 and YSC3 collected from drinking water device inside the farm A and from soil outside the farm A, as well as one Enterobacter roggenkampii NS5carried mcr-9 gene isolated from drinking water inside the farm B . No other mcr genes were detected except for mcr-1 and mcr-9 . For 60 mcr positive Klebsiella pneumoniae strains, 30 strains were collected from pig feces，19 isolates were collected from environmental samples inside the pig farm and 7 were collected from environmental samples outside the pig farm and 4 were collected from farmer feces. The mcr-1 positive rate of these four farms were 12.12%,15.38%, 14.04% and 17.33%, while the mcr-9 positive rate within these three farms were 11%, 6.15%, 15.79% and 1.33% (Figure 1). For 14 mcr positive E. coli strains, 10 isolates were collected from pig feaces, 1 isolate were collected from environmental samples inside the pig farms, 1 isolated from environmental samples outside the pig farms and 2 were isolated from farmer feaces. The mcr-1 positive rate of these four farms were 0.90%, 5.17%, 0 and 0 while the mcr-9 positive rate within these four farms were 1.80%, 3.45%, 0 and 0.6%. (Figure 1) Besides, seven colistin resistance strains were obtained from hospital in Fujian Province and four tested positive for mcr-1 gene, Y46, Y19, Y51 and Y27. The information and MIC values of these four strains were listed in Table S2. Antimicrobial susceptibility profile of mcr positive strains The dilution method and micro-broth dilution method were employed to assess the susceptibility of 74 mcr positive strains towards 11 antimicrobial agents of 5 distinct antibiotic classes. The results revealed that the minimum inhibitory concentration (MIC) values of colistin for these 39 mcr-1 Klebsiella pneumoniae strains were mostly in the range of 2–32 μg/mL. Among them, the resistance rate of 39 mcr-1 -positive Klebsiella pneumoniae strains to colistin, cefazolin, doxycycline, gentamicin, cefotaxime, tetracycline, streptomycin, ciprofloxacin, cefepime and cefquinome were of 89.7%, 89.7%, 87.2%, 92.3%, 79.5%,100%, 84.6%, 74.4%, 35.9%, and 76.9%, respectively. All strains were susceptible to tigecycline. The colistin MIC of these 21 mcr-9 -positive K. pneumoniae strains primarily ranged from 1 to 2 μg/mL, with most strains exhibiting a susceptible phenotype with colistin MIC≦2. The resistance rates to colistin, cefazolin, doxycycline, gentamicin, cefotaxime, tetracycline, streptomycin, ciprofloxacin and cefquinome were 42.9%, 85.7%, 100%, 57.1%, 42.9%, 100%, 85.7%, 28.6% and 33.3%, respectively. In contrast, all strains were susceptible to cefepime and tigecycline. All mcr-1 and mcr-9 positive strains exhibited multidrug resistance phenotypes. Notably, 13/39(33.33%) mcr-1 positive K. pneumoniae strains were resistance to over 9 antibiotics, whereas 11/21(52.38%) mcr-9 positive K. pneumoniae strains showed resistance to less than 4 antibiotics. For 14 mcr positive E. coli , the resistance rate of 7 mcr-1 E. coli strains to cefazolin, doxycycline, gentamicin, cefotaxime, streptomycin, ciprofloxacin and cefquinome were 42.9%, 71.4%, 42.9%, 57.1%, 57.1%, 14.3% and 42.9%. all were resistance to colistin and tetracycline and were susceptible to cefepime and tigecycline. The resistance rate of 7 mcr-9 E. coli strains to cefazolin, tetracycline, doxycycline, gentamicin, cefotaxime, streptomycin and ciprofloxacin were 14.3%, 100%, 100%, 42.9%, 14.3%, 57.1% and 14.3%, all were susceptible to colistin, tigecycline, cefepime, cefquinome. 4/7（57.1%） mcr-1 positive E.coli strains were resistance to over 4 antibiotics and only one (14.3%) mcr-9 positive E.coli strains showed resistance to over 4 antibiotics. Besides, 3 R. ornithinolytica T9, YSC9 and YSC3 showed diverse antibiotic profile, they show resistant to 9, 7 and 3 antibiotics, respectively. One Enterobacter roggenkampii NS5 was resistant to colistin, doxycycline,cefotaxime and tetracycline. Of note, YSC9 exhibited a high MIC of colistin=256 μg/mL. (Table 1) Genome feature of plasmids harboring mcr gene The mcr -positive strains were all subject to conjugation assays and 40 conjugations were obtained. Eric-PCR were performed to confirm the profile were matched to recipient C600 strains and PCR were used to confirm the presence of mcr genes. Whole-genome sequencing and plasmid replicon PCR revealed that mcr-1 was primarily located on IncHI2 (n=5), IncX4 (n=14), and IncI2 (n=15) plasmids, while mcr-9 was predominantly carried by IncHI2 plasmids (n=4) and IncF(n=2) plasmids. Among them, the Enterobacter roggenkampii strain NS5 comprised a 4,796,092 bp chromosome and three plasmids pFJNS5- mcr-9 , pFJNS5-2 and pFJNS5-3 with the size of 187,381bp, 4783bp, and 3406bp, respectively. ResFinder analysis showed that NS5 contained bla MIR-1 located on the chromosone and bla TEM-116 gene located on plasmid pFJNS5-3. While the IncFIB plasmid pFJNS5- mcr-9 contained colistin resistant gene mcr-9 , florfenicol resistant gene floR , sulfonamide resistant gene sul2 and sul3 , quinolone resistant gene qnrS1 , tetracycline resistant gene tet(A) , trimethoprim resistant gene dfrA12 , aminoglycoside resistant gene aadA2 , aadA1 and aph(‘3’)-Ia , chloramphenicol resistant gene cmlA1 and bla LAP-2 gene encoding class A 𝜷-lactamase. The type of the other two plasmids is undetected. Comparing the plasmids sequence with that in the NCBI database, we found that pFJNS5- mcr-9 shared >99% identity with plasmids pEc61A (CP053104), pYUSHP2-2(CP073773), pM1(CP090910), pCUVET18-121(CP114980), plasmid unnamed2 (CP138221), plasmid unnamed(CP149842) and pW139_2(CP163059), all identified in Enterobacter hormaechei strains, plasmid unnamed1(CP129956) identified in Enterobacter asburiae strain and a plasmid pNK_H8_077(CP152556) identified in Klebsiella pneumoniae with 66%, 68%, 54%, 52%,66%,68% ,64%,60% and 61% coverage, respectively. They shared conserved region including region related to transfer, stable heritage and replication. While antibiotic resistant gene and inserted sequences were diverse in variable region. Sequencing analysis revealed that the genome of R. ornithinolytica T9 contained three plasmids, which belongs to Col, IncHI2A and IncQ1 plasmids. The plasmid pFJT9 carried sul1 , qacE △ , aadA2 and mcr-9 gene, belonging to IncHI2/HI2A group. Sequence comparing showed that pFJT9 exhibited high similarity with mcr-9 plasmids p14269_1( Enterobacter hormaechei ,CP083850), p12379_1 ( Enterobacter kobei , CP083858), unknown ( Salmonella enterica , OX442404), pZHH-1( Enterobacter hormaechei , CP059712), pK672-MCR9 ( Enterobacter hormaechei ,CP183850), p131 ( Salmonella enterica , CP099761) and pMRVIM0813 ( Enterobacter cloacae , KP975077), p12795_1 ( Enterobacter roggenkampii ,CP083854) deposited in NCBI database, pFJZZ12- mcr- 9 and pFJY27- mcr-1 isolated from pig feces and clinical samples in this study with >50% coverage . Linear comparing sequence of pFJZZ12- mcr-9 to that of pFJY27- mcr- 1 ， we found that the sequence length of pFJY27- mcr-1 with mcr-1 gene is much shorter than that of pFJZZ12- mcr-9 with mcr-9 gene. pFJY27- mcr-1 only contained colistin resistance gene mcr -1 , while plasmids pFJZZ12- mcr-9 contained aminoglycosides resistance genes aadA2 , aadA3 and ant(2”)-Ia , cephalosporins resistance gene bla CTX-M-9 , the trimethoprim resistance gene dfrA16 , the fluoroquinolones resistance gene qnrA1 and 3 copies of the sulfonamide resistance gene sul1 . IncHI2 type mcr-9 positive plasmids confer a growth advantage over IncHI2 type plasmids harboring mcr-1. Plasmids pFJY27 -mcr-1 , pFJZZ12- mcr-9 and pFJNS5- mcr-9 were chosen as representative plasmids for the IncHI2 plasmid carrying mcr-1 , the IncHI2 plasmid carrying mcr-9 and the IncFIB plasmid carrying mcr-9, respectively.A 12-hour continuous culture experiment was conducted to observe the growth kinetics of transformants BW25113/pFJY27 -mcr-1 (IncHI2) , BW25113/pFJZZ12- mcr-9 (IncHI2) and BW25113/pFJNS5- mcr-9 (IncFIB), and the plasmid-free recipient strain BW25113. As illustrated in Figure 3A, strain BW25113/pFJY27 -mcr-1 exhibited slower growth compared to the plasmid-free host strain BW25113, suggesting a fitness cost associated with carrying this plasmid. In contrast, no significant growth difference was observed between BW25113 and strains BW25113/pFJZZ12- mcr-9 and BW25113/pFJNS5- mcr-9 , indicating that the acquisition of these mcr-9 -carrying plasmids did not impose a noticeable growth burden on the host E. coli strain without the antibiotic selection pressure. Plasmids carrying mcr-9 confer a competitive advantage over plasmids carrying mcr-1 As shown in Figure S1, after seven days of serial passage in the absence of antibiotics, Escherichia coli BW25113/pFJY27- mcr-1 , BW25113/pFJZZ12- mcr-9 , and BW25113/pFJNS5- mcr-9 were all stably maintained within the host cells. A competitive fitness assay was conducted in vitro by co-culturing Escherichia coli BW25113 strains carrying the IncHI2-type plasmids with BW25113/pFJY27- mcr-1 or BW25113/pFJZZ12- mcr-9 to explore the adaptability of mcr-1 and mcr-9 plasmids. As shown in Figure 3B, the strain carrying the IncHI2 plasmid BW25113/pFJY27- mcr-1 was clearly outcompeted by the strain carrying the IncHI2 plasmid BW25113/pFJZZ12- mcr-9 , with RF values below 1 starting from day 2. The relative fitness of the mcr-1 -carrying strain declined progressively, from 0.9588 to 0.4566 over the course of serial passages. A similar competitive fitness assay was performed with strains BW25113/pFJY27- mcr-1 and BW25113/pFJNS5- mcr-9 , carrying IncHI2-type plasmid and IncFIB plasmid, respectively. As shown in Figure 3B, the strain carrying the BW25113/pFJY27- mcr-1 consistently exhibited RF values below 1, indicating lower competitiveness compared to the strain carrying the plasmid BW25113/pFJNS5- mcr-9 . Over serial passages, the relative fitness of the mcr-1-carrying strain decreased from 0.7663 to 0.5551. IncHI2 type mcr-9 positive plasmids harbor higher plasmid spread ability and plasmid persistence in Klebsiella pneumoniae stains population Conjugation transfer abilities of mcr-1 and mcr-9 positive plasmids were evaluated by using Escherichia coli BW25113/pFJY27- mcr-1, BW25113/pFJZZ12- mcr-9 , and BW25113/pFJNS5- mcr-9 as donor strains and Klebsiella pneumoniae 13883 as the recipient strain. As shown in Figure 3C, the conjugation frequency of pFJY27- mcr-1 was 2.5×10⁻⁵, while that of pFJZZ12- mcr-9 was 9.56×10⁻⁵, and pFJNS5- mcr-9 exhibited a conjugation frequency of 5.42×10⁻⁴. A co-culture experiment was conducted with Escherichia coli BW25113 strains carrying plasmid pFJY27- mcr-1 and mcr-9 plasmids pFJZZ12- mcr-9 or pFJNS5- mcr-9 , and a plasmid-free Klebsiella pneumoniae 13883 engineered strain. As shown in Figure 3D, plasmid invasion was observed as early as day 1 of serial passage. The strains 13883/pFJNS5- mcr-9 or 13883/pFJZZ12- mcr-9 was found to significantly outnumber the strain 13883/pFJY27- mcr-1 , indicating that the mcr-9 positive plasmids pFJZZ12- mcr-9 andpFJNS5- mcr-9 exhibits a higher invasion capacity and faster invasion speed compared to plasmid pFJY27- mcr-1. Discussion The emergence and spread of plasmid-mediated mcr genes have been documented worldwide, posing a threat to public health. This study aimed to investigate the persistence of mcr genes in Enterobacteria isolated from the pig farms of Fujian province in China after the ban on antibiotic use as feed additive in livestock, which was implemented by the Chinese government since 2017[12]. Also, the factors that contribute to the prevalence of mcr-9 in these four farms. A total of 930 samples were examined for the presence of mcr genes, with only mcr-1 and mcr-9 being detected. The prevalence of mcr-9 was 8.0%, slightly lower than the 14.8% detection for mcr-1 gene in K. pneumoniae . In E. coli isolates, mcr-1 and mcr-9 were detected in 8 (1.5%) and 6 (1.1%) strains, respectively. These observations are consistent with previously study, where mcr-9 consistently detected as the one of the most prevalent mcr subtype following mcr-1 [9]. Among 1000 sequenced Klebsiella pneumoniae strains in prior research, mcr-9 was reported as both the earliest and second-most prevalent subtype after mcr-1 [22]. In a global survey of MCR gene family distribution across 3250 isolates, mcr-1 and mcr-9 were found to be the most widely disseminated, with isolates identified in 61 and 40 countries across six continents, respectively[9]. The widespread geographic distribution underscores the significant global dissemination potential of the mcr-9 gene, while the mechanism of its widespread is unclear. Beside K. pneumoniae and E. coli , the mcr-9 isolates were also identified in Enterobacter roggenkampii isolated from drinking water and in Raoultella ornithinolytica isolated from both drinking water devices and soil in the study , indicating that zoonotic transmission of mcr positive plasmids between animals, the environment and humans is also occurring. The presence of mcr gene identified in the environmental samples suggest that the environmental can be regarded as a reservoir for the spread of antimicrobial resistance without antibiotic selection pressure. The identification of mcr-9 -positive isolates in drinking water devices and soil further reinforces the idea that environmental surveillance must be a key component of AMR control strategies. The antimicrobial susceptibility results revealed that 42 mcr-1 -positive strains (35 K. pneumoniae and 7 E. coli ) exhibited resistance to colistin, with MIC values predominantly between 2–32 μg/mL, while only 9 mcr-9 -positive strains had MIC values ≧ 4 μg/mL. Previous surveillance of the mcr genes has primarily focused on monitoring mcr genes in colistin-resistant bacteria. The observation indicated an underestimation of the true prevalence of mcr-9 due to its frequent association with low colistin MIC value. Most of the mcr-1 and mcr-9 -positive bacteria demonstrated multidrug resistance, while the mcr-1 positive strains seemed to be associated with a more extensive resistance phenotype than mcr-9 positive strains. The broader resistance profiles of mcr-1 -positive strains influence their relative fitness and persistence under antibiotic selection pressure, helping to explain the higher prevalence of mcr-1 compared to mcr-9 . Comparative genomic analyses of the pFJNS5- mcr-9 plasmid shared >99% identity with plasmids isolated in Enterobacter hormaechei , Enterobacter asburiae , and Klebsiella pneumoniae strains in the NCBI database, with coverage ranging from 52% to 68%. These plasmids shared conserved plasmid backbone but the antibiotic resistance gene and insertion sequences, indicating the frequent recombination and horizontal gene transfer event of antibiotic resistance genes. Similarity, the plasmid pFJT9- mcr-9 exhibited high sequence similarity to mcr-9 plasmids available in the NCBI database identified in diverse Enterobacteria , indicating the extensive mobilization potential and broad host range of the mcr-9 gene. Plasmid persistence within a bacterial community is typically linked to efficient acquisition and associated fitness advantage[19]. Hence, we investigated the dynamics of mcr-1 and mcr-9 positive plasmids through a series of conjugation frequency assays, plasmid stability tests, fitness trials, and plasmid competition-invasion assays to elucidate the factors underlying the higher detection rate of the mcr-9 gene in pig farms in Fujian Province, China. Plasmids pFJY27- mcr-1 and pFJZZ12- mcr-9 were selected as the representative of IncHI2 plasmid with mcr-1 and mcr-9, respectively, due to similar plasmid backbones, while plasmid pFJNS5- mcr-9 served as a representative IncFIB plasmid harboring the mcr-9 gene. The conjugation frequency for both mcr-1 and mcr-9 positive IncHI2 plasmids was 10⁻⁴~10⁻⁵, indicating their high transfer capability. In addition, the mcr-9 positive plasmids appeared to exhibit a slightly higher conjugation rate compared to the mcr-1 positive plasmids. However, the sample size is limited, further studies involving larger sample sizes and diverse plasmid types are needed to confirm the observation. Both mcr-1 and mcr-9 positive plasmids remained stably maintained within the host E. coli BW25113 strains over seven days of serial passaging without antibiotic pressure. Growth kinetics analysis demonstrated that mcr-1 positive IncHI2 plasmids negatively affected host bacterial growth, while mcr-9 positive IncHI2 and IncFIB plasmids had no significant impact on host growth. This indicated that carriage of the mcr-9 gene dose not impose a notable fitness burden on bacterial host. The lower fitness cost associated with mcr-9 positive plasmids suggests that these plasmids may persist longer within bacterial populations comparing to mcr-1 positive plasmids in the absence of colistin selection pressure. In vitro competition assays between E. coli BW25113 strains carrying mcr-1 or mcr-9- positive plasmids demonstrated that, in the absence of antibiotic selection, mcr-1 -positive plasmids conferred a higher fitness cost to their hosts than mcr-9 plasmids, placing mcr-1- positive strains at a competitive disadvantage over successive generations without antibiotic selection. These results further underlined the survival advantage of mcr-9- positive bacteria in nutrient-competitive environments without colistin selection pressure, suggesting an emerging trend of increasing prevalence of mcr-9 -postive plasmids within pig farm ecosystem in China. Additionally, the invasion capability of these plasmids was evaluated, we found that the mcr-9 positive IncHI2 or IncFIB plasmids invades recipient K. pneumoniae strains at significantly higher rates compared to mcr-1 positive IncHI2 plasmids. After four days, the concentration of bacteria harboring mcr-9 -positive plasmids (pFJNS5 or pFJZZ12) significantly outnumber those of bacteria carrying the mcr-1- positive plasmid (pFJY27), although plasmid-free bacterial populations remained dominant. The enhanced invasion potential observed for mcr-9 -positive plasmids could be attributed to their higher conjugation frequencies and lower associated fitness costs. Conclusion This study investigates the persistence and dissemination dynamics of plasmid-mediated mcr genes—particularly mcr-1 and mcr-9 —within Enterobacteriaceae isolated from four pig farms in Fujian Province, China, following the ban on colistin as a feed additive. While both mcr-1 and mcr-9 were detected, mcr-1 remained more prevalent. In the study, mcr-9 -positive plasmids were identified not only in K. pneumoniae and E. coli , but also in Enterobacter roggenkampii and Raoultella ornithinolytica , with environmental sources such as drinking water, soil, and water devices serving as reservoirs—highlighting the role of the environment in facilitating plasmid-mediated resistance transmission. The lower fitness cost, stable maintenance, and higher conjugation and invasion efficiencies of mcr-9 -positive IncHI2 and IncFIB plasmids suggest that these plasmids are more likely to persist and propagate in bacterial populations in the absence of antibiotic selection pressure. This biological advantage may contribute to a gradual increase in the prevalence of mcr-9- positive bacteria within livestock environments. Overall, these findings underscore the urgent need for enhanced surveillance of mcr-9 , especially in environmental reservoirs, and a deeper understanding of its ecological and evolutionary advantages. Although mcr-9 gene is frequently associated with colistin-susceptible phenotypes, its expression can be induced under colistin exposure. Moreover, mcr-9 harboring plasmids are often linked to multiantibiotic resistance, limiting treatment options. Targeted strategies should be developed to monitor, prevent, and mitigate the spread of mcr- positive plasmids, integrating a One Health approach that addresses animal, environmental, and human health dimensions of antimicrobial resistance. Declarations Acknowledgements Not applicable. Ethical approval and Consent to Participate This study did not involve any experiments on live animals. All fecal and environmental samples were collected with permission from commercial pig farm owners and veterinarians. The clinical sample collection was approved by the Animal Experimentation Committee of the Second Affiliated Hospital of Fujian Medical University (approval number:2022408). Consent for publication Not applicable. Authors’ contributions Lingxian Yi: Conceptualization, Methodology, Data analysis, Writing-original draft, review, editing and Funding acquisition. Huamin Lai, Jianshuo Liu and Donghong Huang: Investigation and Methodology. Daojin Yu and Jiaming Huang: Conceptualization, supervision, project administration. Clinical trial number Not applicable. Funding Declaration This research was supported by the grants from the Natural Science Foundation of Fujian Province (No. 2024J08038), the Education Research Fund for Young Academic of Fujian Province (JAT220060) and the Natural Science Foundation of Fujian Province (2023J01728). Data Availability The complete nucleotide sequence of pFJNS5- mcr-9 and pFJZZ12- mcr-9 has been updated at GenBank (PV817785 and PV817786). Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work the author(s) used ChatGPT -4o in order to check for spelling and clarity of language. After using this tool, the author(s) reviewed and edited the content as needed and take full responsibility for the content of the published article. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Reference Wilson H, Torok ME: Extended-spectrum beta-lactamase-producing and carbapenemase-producing Enterobacteriaceae . Microb Genom 2018, 4 (7). Moffatt JH, Harper M, Boyce JD: Mechanisms of Polymyxin Resistance . Adv Exp Med Biol 2019, 1145 :55-71. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X et al : Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study . Lancet Infect Dis 2016, 16 (2):161-168. Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, Malhotra-Kumar S: Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016 . Euro Surveill 2016, 21 (27). Roer L, Hansen F, Stegger M, Sonksen UW, Hasman H, Hammerum AM: Novel mcr-3 variant, encoding mobile colistin resistance, in an ST131 Escherichia coli isolate from bloodstream infection, Denmark, 2014 . Euro Surveill 2017, 22 (31). Yang YQ, Li YX, Lei CW, Zhang AY, Wang HN: Novel plasmid-mediated colistin resistance gene mcr-7.1 in Klebsiella pneumoniae . J Antimicrob Chemother 2018, 73 (7):1791-1795. Rebelo AR, Bortolaia V, Kjeldgaard JS, Pedersen SK, Leekitcharoenphon P, Hansen IM, Guerra B, Malorny B, Borowiak M, Hammerl JA et al : Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes . Euro Surveill 2018, 23 (6). Wang Y, Xu C, Zhang R, Chen Y, Shen Y, Hu F, Liu D, Lu J, Guo Y, Xia X et al : Changes in colistin resistance and mcr-1 abundance in Escherichia coli of animal and human origins following the ban of colistin-positive additives in China: an epidemiological comparative study . Lancet Infect Dis 2020, 20 (10):1161-1171. Ling Z, Yin W, Shen Z, Wang Y, Shen J, Walsh TR: Epidemiology of mobile colistin resistance genes mcr-1 to mcr-9 . J Antimicrob Chemother 2020, 75 (11):3087-3095. Li Y, Dai X, Zeng J, Gao Y, Zhang Z, Zhang L: Characterization of the global distribution and diversified plasmid reservoirs of the colistin resistance gene mcr-9 . Sci Rep 2020, 10 (1):8113. Mmatli M, Mbelle NM, Osei Sekyere J: Global epidemiology, genetic environment, risk factors and therapeutic prospects of mcr genes: A current and emerging update . Front Cell Infect Microbiol 2022, 12 :941358. Walsh TR, Wu Y: China bans colistin as a feed additive for animals . Lancet Infect Dis 2016, 16 (10):1102-1103. Olaitan AO, Dandachi I, Baron SA, Daoud Z, Morand S, Rolain JM: Banning colistin in feed additives: a small step in the right direction . Lancet Infect Dis 2021, 21 (1):29-30. Hayashi W, Tanaka H, Taniguchi Y, Iimura M, Soga E, Kubo R, Matsuo N, Kawamura K, Arakawa Y, Nagano Y et al : Acquisition of mcr-1 and Cocarriage of Virulence Genes in Avian Pathogenic Escherichia coli Isolates from Municipal Wastewater Influents in Japan . Appl Environ Microbiol 2019, 85 (22). Shen C, Zhong LL, Yang Y, Doi Y, Paterson DL, Stoesser N, Ma F, El-Sayed Ahmed MAE, Feng S, Huang S et al : Dynamics of mcr-1 prevalence and mcr-1-positive Escherichia coli after the cessation of colistin use as a feed additive for animals in China: a prospective cross-sectional and whole genome sequencing-based molecular epidemiological study . Lancet Microbe 2020, 1 (1):e34-e43. CLSI: CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 32nd ed.CLSI supplement M100. 2022. Institute. CaLS: Performance Standards for Antimicrobial Disk and Dilution Susceptibility Testing for Bacteria Isolated From Animals (5th ed.)CLSI supplement VET08 . Wayne, PA: CLSI 2023. (EUCAST). ECoAST: Breakpoint tables for interpretation of MICs and zone diameters, version 14.0. 2024. Yi L, Durand R, Grenier F, Yang J, Yu K, Burrus V, Liu JH: PixR, a Novel Activator of Conjugative Transfer of IncX4 Resistance Plasmids, Mitigates the Fitness Cost of mcr-1 Carriage in Escherichia coli . mBio 2022, 13 (1):e0320921. Khosravi AD, Hoveizavi H, Mohammadian A, Farahani A, Jenabi A: Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR . Acta Cir Bras 2016, 31 (3):206-211. Wu R, Yi LX, Yu LF, Wang J, Liu Y, Chen X, Lv L, Yang J, Liu JH: Fitness Advantage of mcr-1-Bearing IncI2 and IncX4 Plasmids in Vitro . Front Microbiol 2018, 9 :331. Liu M, Wu J, Zhao J, Xi Y, Jin Y, Yang H, Chen S, Long J, Duan G: Global epidemiology and genetic diversity of mcr-positive Klebsiella pneumoniae: A systematic review and genomic analysis . Environ Res 2024, 259 :119516. Table Table 1. The information of 3 mcr-9 -positive Raoultella ornithinolytica and an Enterobacter roggenkampii strains. Organism Species Origin Site Location Colistin MIC other rsistantce phenotype(s) Other resistance gene(s) T9 Raoultella ornithinolytica soil outside farm Farm A IncHI2 256 STR, TGC, DOX, CIP, GEN, CTX, TET, CQM aac(6')-Ib-cr , aadA2 , aph(3'')-Ib , aph(6)-Id bla DHA-1 , bla OXA-1 catB3 , dfrA12 , floR fosA , qnrB2 , qnrS1 , sul1 sul2, tet(A) , tet(D) YSC9 drinking water device Farm A - 2 STR, DOX, FEP, GEN, CTX, TET, CQM - YSC3 drinking water device Farm B - 4 DOX,TET - NS5 Enterobacter roggenkampii drinking water Farm B IncFIB 8 DOX，CTX, TET aadA2 , aph(3')-Ia, bla LAP-2 , bla MIR-1 , cmlA1 , dfrA12 , floR, qnrS1 , sul2 , sul3 , tet(A) Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6934660","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476190395,"identity":"e6350e6b-f034-4448-9c8d-be063ccf8dfa","order_by":0,"name":"Lingxian Yi","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Lingxian","middleName":"","lastName":"Yi","suffix":""},{"id":476190396,"identity":"b7da8440-c99c-4ae2-9a19-46c9e5d3ad79","order_by":1,"name":"Huamin Lai","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Huamin","middleName":"","lastName":"Lai","suffix":""},{"id":476190397,"identity":"47aa91f2-0aa8-4fdc-906d-1fbe87157c95","order_by":2,"name":"Jianshuo Liu","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Jianshuo","middleName":"","lastName":"Liu","suffix":""},{"id":476190398,"identity":"9ea56c27-cf62-4c0c-9cca-6f6d6ce80e4b","order_by":3,"name":"Donghong Huang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Donghong","middleName":"","lastName":"Huang","suffix":""},{"id":476190399,"identity":"d1312179-a1cc-4bc4-aaff-cef6320c120d","order_by":4,"name":"Jiaming Huang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiaming","middleName":"","lastName":"Huang","suffix":""},{"id":476190400,"identity":"c3b3bd4c-52c9-4204-9db1-e4b109411f44","order_by":5,"name":"Daojin Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACeyA2SGA4zMAP4j0AcghqMWyAapFsYGBsSCBGi8EBMJUGYhCr5fjhAwUPd9gkbj7eY/4gocLGmIH98NENeLWcSUswSDwjkbjtzBnDhoQzaWYMPGlpN/A7LMfAILENqOVGjmFDYtthGwYJHjP8Ws6/gWjZPINoLTegtmyQgGgxI6jFcMazBJAW4xlnjhXOAPrFmI2QX+z5k48Z/myTkO1vb97w4UOFjWE/++FjeLUAARtqVLARUA4CzA+IUDQKRsEoGAUjGQAAWw1QXX76j/MAAAAASUVORK5CYII=","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Daojin","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2025-06-20 02:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6934660/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6934660/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85460177,"identity":"cf5bc2e1-e434-4a5a-9be6-e532cf2eb4a1","added_by":"auto","created_at":"2025-06-26 07:23:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1761282,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of \u003cem\u003emcr-1\u003c/em\u003eand \u003cem\u003emcr-9\u003c/em\u003e-positive isolates from various sample sources collected at four pig farms.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6934660/v1/5a14856413c2de65c614b811.png"},{"id":85459456,"identity":"c63e8c08-30d7-4093-94ed-366f6d15f6fc","added_by":"auto","created_at":"2025-06-26 07:15:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4254496,"visible":true,"origin":"","legend":"\u003cp\u003eSequence comparison of Inc FIB: pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e and IncHI2 type plasmid: pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e from this study and GenBank. (A) Plasmid pFJNS5-mcr-9 and other similar plasmids deposit in Genbank. (B) Plasmid pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e and other similar plasmids deposit in Genbank. (C) Linear comparison of pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e and pFJY27-\u003cem\u003emcr-1\u003c/em\u003ein this study.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6934660/v1/1e7d892e41b2a61779bf6b31.png"},{"id":85458614,"identity":"b16ba489-ba9d-4b4c-aa15-60b4746b32bb","added_by":"auto","created_at":"2025-06-26 07:07:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1892767,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e-positive plasmids on bacterial host fitness and population dynamics. (A) Growth kinetics of \u003cem\u003eE. coli \u003c/em\u003eBW25113 strains carrying plasmids pFJNS-\u003cem\u003emcr-9, \u003c/em\u003eBW25113/pFJZZ12-\u003cem\u003emcr-9 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003eBW25113/pFJY27-\u003cem\u003emcr-1 \u003c/em\u003ecompared with the plasmid-free strain BW25113\u003cem\u003e.\u003c/em\u003e (B) Relative fitness comparisons between BW25113 strains carrying plasmids pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e and pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e versus the strains carrying pFJY27-\u003cem\u003emcr-1\u003c/em\u003e. All competitions assays were performed with three biological replicates. (C) Conjugation frequency of plasmids pFJZZ12-\u003cem\u003emcr-9, \u003c/em\u003epFJNS5-\u003cem\u003emcr-9 and \u003c/em\u003epFJY27-\u003cem\u003emcr-1. \u003c/em\u003e(D) Plasmid invasion dynamics assessed in co-cultures assays using strains 13883/pFJZZ12-\u003cem\u003emcr-9, \u003c/em\u003e13883/pFJNS5-\u003cem\u003emcr-9 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003e13883/pFJY27-\u003cem\u003emcr-1, \u003c/em\u003eeach\u003cem\u003e \u003c/em\u003emixed with a 1000-fold excess of plasmid-free 13883 at the beginning of the assay. All invasion assays were performed three biological replicates. Bars represent the standard deviation (SD) from these biological replicates.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6934660/v1/48349283a024e44f7e731752.png"},{"id":92971020,"identity":"3d631e45-f1cb-4752-b040-6f6c278943b1","added_by":"auto","created_at":"2025-10-07 16:48:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10108425,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6934660/v1/9d5711bf-22bc-4bc3-bdaa-dffbdfba6bdf.pdf"},{"id":85458617,"identity":"26a31deb-845d-4225-8f6e-d3bf1c8ad08e","added_by":"auto","created_at":"2025-06-26 07:07:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":53968,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6934660/v1/81cb4b8449e1f7a67e83dd84.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Persistence and Transmission Dynamics of mcr-1 and mcr-9 in Enterobacteriaceae from Pig Farms in Fujian, China after the Colistin Ban","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe end of the 'golden age of antibiotics' is approaching as global antibiotic resistance continues to rise, with carbapenems-producing \u003cem\u003eEnterobacteriaceae\u003c/em\u003e (CPE) emerging as a critical public health threat[1]. Due to the increasing prevalence of multidrug-resistant Gram-negative bacteria and limited development of effective new antibiotics, colistin has become a crucial last-resort treatment for CPE infections[2].\u003c/p\u003e\n\u003cp\u003eFollowing the initial discovery of the \u003cem\u003emcr-1\u003c/em\u003e gene in 2015, multiple variants (\u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003ethrough \u003cem\u003emcr-10\u003c/em\u003e) have been identified globally in diverse bacterial species[3-8]. \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eremains the most common carrier, with \u003cem\u003emcr-1\u003c/em\u003e-positive strains showing the highest prevalence, followed by \u003cem\u003emcr-9\u003c/em\u003e and \u003cem\u003emcr-3\u003c/em\u003e variants[9]. In addition, geographical variations in prevalence have been noted: \u003cem\u003emcr-9\u003c/em\u003e-positive \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003estrains predominate in Europe, North America, and Africa, while \u003cem\u003emcr-3\u003c/em\u003e-positive strains are more prevalent in Asia[10, 11]. However, the molecular mechanisms underlying the global dissemination of these \u003cem\u003emcr\u003c/em\u003e variants remain unclear.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn response to the escalating detection rates of \u003cem\u003emcr\u003c/em\u003e genes, several countries, including China, Japan, and Thailand, have implemented strict restrictions on the use of colistin as a growth promoter in food-producing animals[12-14]. These regulatory interventions have resulted in a marked reduction in \u003cem\u003emcr\u003c/em\u003e-positive \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003ein animal populations, particularly \u003cem\u003emcr-1\u003c/em\u003e-positive strains[8]. Also, the detection rates of \u003cem\u003emcr\u003c/em\u003e gene in pig feces, especially in colon, have reported decreased in China after the ban on colistin as a growth promoter[15]. However, previous reports have mainly focused on the prevalence of \u003cem\u003emcr\u003c/em\u003e gene in the pig feces, while their persistence and distribution in the broader farming environment remain largely unclear. This study aims to investigate the distribution of \u003cem\u003emcr\u003c/em\u003e genes in pig faeces, farm environments and farm workers across four agricultural sites\u0026nbsp;in Fujian province during 2022-2023. Additionally, the study\u0026nbsp;explores the molecular mechanisms contributions the recent increase in \u003cem\u003emcr-9\u003c/em\u003e-positive \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, highlighting an emerging trend towards \u003cem\u003emcr-9\u003c/em\u003e gene predominance within \u003cem\u003eEnterobacteriaceae\u003c/em\u003e bacteria.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSampling and isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring 2022-2023, we collected non-duplicate environmental and fecal samples from four swine farms in Fujian province (Ningde, Yongtai, Longyan and Fuqing). Samples included swine faeces, drinking water, drinking devices, feed, feed troughs, worker faeces, worker boots, soil, and surfaces (ground and walls) from both interior and exterior farm environments. All samples were individually collected in sterile sampling bags and transported to the laboratory in cold storage (4°C) within 6 hours of collection. For bacterial isolation, each sample were enriched in 2 mL Luria-Bertani (LB broth, Beijing Solarbio Science \u0026amp; Technology Co., Ltd., Beijing, China) broth and incubated overnight at 37°C with 200 rpm shaking. The enriched cultures were subsequently plated on MacConkey agar (Mac agar, Beijing Solarbio Science \u0026amp; Technology Co., Ltd) with 2 μg/mL of colistin. One to two morphologically distinct colonies from each MacConkey plate were sub-cultured onto Levine Eosin-Methylene Blue agar (EMB agar, Qingdao Hi-Tech Industrial Park Hope Bio-Technology Co., Ltd, Qingdao, China). Morphologically distinct colonies were subjected to species identification through 16S rRNA gene sequencing. Additionally, 13 colistin resistant extended-spectrum\u0026nbsp;𝛽-lactamase strains, including 12\u0026nbsp;\u003cem\u003eK. pneumoniae\u0026nbsp;\u003c/em\u003eand 1\u003cem\u003e\u0026nbsp;Escherichia coli,\u0026nbsp;\u003c/em\u003ewere\u0026nbsp;obtained from a Grade A tertiary hospital in Fujian province, China. These isolates were isolated from clinical samples, including urine, blood and sputum.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of \u003cem\u003emcr\u003c/em\u003e genes and antimicrobial susceptibility testing (AST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll\u0026nbsp;\u003cem\u003eEnterobacteriaceae\u003c/em\u003e isolates were screened for colistin resistance genes (\u003cem\u003emcr-1\u003c/em\u003e to \u003cem\u003emcr-10\u003c/em\u003e) using primers listed in Table S1. Positive amplicons were confirmed through Sanger sequencing and subsequent analysis using the Basic Local Alignment Search Tool (BLAST) against the National Center for Biotechnology Information (NCBI) nr database.\u003c/p\u003e\n\u003cp\u003eMinimum inhibitory concentrations (MICs) for 11 antimicrobial agents against\u003cem\u003e\u0026nbsp;mcr\u003c/em\u003e-positive isolates were determined using broth microdilution methods according to Clinical and Laboratory Standards Institute guidelines (CLSI M100)[16]. Interpretative criteria followed CLSI breakpoints, except for cefquinome according to CLSI supplement VET08, and tigecycline interpreted using European Committee on Antimicrobial Susceptibility Testing (EUCAST v14.0) clinical breakpoints[17, 18]. \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 served as the quality control strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConjugation Assays and Plasmid Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConjugation experiments were performed using broth mating techniques according to previous reports[19]. Streptomycin-resistant \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eC600 or rifampicin-resistant\u0026nbsp;\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strain 13883\u0026nbsp;served as the recipient strain, while \u003cem\u003emcr\u003c/em\u003e-positive isolates served as donors. Both donor and recipient strains were cultured in LB broth until reaching an optical density (OD\u003csub\u003e600\u003c/sub\u003e) of 0.8. Equal volumes of donor and recipient cultures were mixed and incubated without agitation at 37°C for 18 hours.\u003c/p\u003e\n\u003cp\u003eThe resulting mixed culture was centrifuged and washed once with LB medium. Cell pellets were resuspended in 100 μL LB broth and plated on MacConkey agar supplemented with either streptomycin (3,000 μg/mL) or rifampin (512 μg/mL) alone or in combination with colistin (2 μg/mL). Conjugation frequencies were calculated as the ratio of transconjugant to recipient colony-forming units (CFUs). Transconjugants were confirmed through antibiotic selection and PCR verification with primers for \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eor \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003elisted in Table S1. Eric-PCR were performed using the primers listed in Table S1 to confirm that the genomic fingerprint of transconjugants matched that of the recipient strain[20].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-genome sequencing\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(WGS) and bioinformatic analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA from nine \u003cem\u003emcr\u003c/em\u003e-positive isolates was extracted using the E.Z.N.A. Bacterial DNA Kit (Omega Bio-tek, Shanghai, China). The gDNA was separated into two parts. One was randomly fragmented to construct a library with an average insertion of 300 bp. The library was sequenced on illumina NovaSeq 6000 platform performing using a pair-end 150bp sequencing strategy. Clean reads were obtained by quality filtering and subjected to denovo assembly using SPAdes (v3.13.1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the strains FJZZ12 and FJNS5, the second parts of gDNA were straightly processed through end-repair, 3’ adenylated, adapters and Motor protein ligations. The product was purified using Agencourt AMPure XP Beads (Beckman, A63881). Finally, fragments larger than 1KB were screened for Single-molecule nanopore DNA sequencing on MinION Flow Cell (ONT, R9.4.1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe raw reads were filtered and assembled using Canu with default parameters. The genomic sequences were proofread using nextpolish (v1.4.1) and Pilon (v1.18). Gene prediction and annotation were generated using the Bakta (v1.8.2) and the National Center for Biotechnology Information (NCBI) nr database. Sequence analyses were performed using the Center for Genomic Epidemiology (CGE) pipeline: multilocus sequence typing (MLST) for strain typing, PlasmidFinder for plasmid replicon identification, ResFinder for antimicrobial resistance gene detection and KmerFinder (v3.0.2) for bacterial species prediction. Comparative plasmid analyses were conducted using EasyFig and SnapGene software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth Kinetics of \u003cem\u003emcr\u003c/em\u003e-Positive Strains and Stability Test of \u003cem\u003emcr\u003c/em\u003e-Positive Plasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the impact of \u003cem\u003emcr\u003c/em\u003e-positive plasmids on the fitness of the bacterial host strain BW25113, the plasmid pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e, pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e and pFJY27-\u003cem\u003emcr-1\u003c/em\u003e were chosen as the representative for IncFIB and IncHI2 plasmid type following previous methods[21].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBriefly, overnight cultures of BW25113 and transformants BW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e, BW25113/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e and BW25113/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e were measured for OD\u003csub\u003e600\u003c/sub\u003e. The initial OD\u003csub\u003e600\u003c/sub\u003e of each strain was adjusted to 0.01 in 50 mL of LB medium without antibiotics.\u0026nbsp;Every hour, 200 µL of culture from each group was sampled in triplicate to measure OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003ein a 96-well plate, with LB broth serving as a blank control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasmid stability was evaluated following established protocols. Briefly, overnight cultures of BW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e, BW25113/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e and BW25113/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e were diluted 1:1000 every 12 hours into 10 mL LB medium without antibiotics for continuous passaging over 7 days. On day 1, 3, 5 and 7, 100 µL of appropriately diluted culture was plated on LB plates and incubated overnight at 37 °C. the following day, 50 colonies were randomly selected and streak onto LB plates containing 2 µg/mL colistin. PCR was performed using primers listed in Table S1 to confirm the presence of \u003cem\u003emcr-1\u003c/em\u003e or \u003cem\u003emcr-9\u003c/em\u003e genes, assessing plasmid retention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid fitness assays of \u003cem\u003emcr\u003c/em\u003e-positive strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fitness impacts of \u003cem\u003emcr\u003c/em\u003e-positive plasmids were evaluated through pairwise competition experiments. BW25113/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e was competed against BW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e, and BW25113/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e was competed against BW25113/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e. All assays were performed in biological triplicates, following previously methods with minor modifications[19]. Briefly, three single colonies from each strain were individually cultured overnight at 37 °C in LB broth supplemented with 2 µg/mL colistin. Cultures were mixed at a 1:1 ratio and diluted 1:1,000 into fresh antibiotic-free LB broth every 24 hours, for five days.\u003c/p\u003e\n\u003cp\u003eAt the beginning of the competition and at 24-hour intervals (days 1–5), aliquots of the cultures were appropriately diluted and plated onto LB agar plates containing 2 µg/mL colistin. All colonies grown were streaked onto LB plates containing 128 µg/mL florfenicol to select for pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e or 4 µg/mL cefotaxime to select for pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e. Fifty colonies from these selective plates were randomly picked for PCR confirmation of the \u003cem\u003emcr-9\u003c/em\u003e gene. Based on previous sequencing results, the plasmid pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e carried the florfenicol resistance gene \u003cem\u003efloR\u003c/em\u003e, and the plasmid pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e carried the cefotaxime resistance gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e. The \u003cem\u003emcr-1\u003c/em\u003e-positive plasmid pFJY27-\u003cem\u003emcr-1\u003c/em\u003e lacked these resistance genes and thus did not grow on these selective plates, allowing clear differentiation of strains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid invasion and competition assays of \u003cem\u003emcr\u003c/em\u003e-positive strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmid invasion assays were performed following previously established methods with slight modifications[19]. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strain 13883 was used as the recipient strain, with plasmid-carrying 13883 transformants as donors. Three strain combinations were evaluated: (1) plasmid-free 13883, 13883/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e and 13883/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e; and (2) plasmid-free 13883, 13883/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e and 13883/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eEach strain was cultured overnight in LB medium supplemented with 2 µg/mL colistin at 37 °C. The recipient strain 13883 were diluted to approximately 10⁻⁷ CFU/mL, and donor strains were diluted to approximately 10⁻⁵ CFU/mL co-cultured in 5 mL fresh LB medium at 37 °C with 80 rpm rotation. Cultures were diluted 1:100 into fresh LB broth every 24 hours for a total of 96 hours. At 24, 48, 72, and 96 hours, aliquots were serially diluted and plated onto selective LB agar plates containing 256 µg/mL rifampicin alone, 256 µg/mL rifampicin plus 2 µg/mL colistin, and 256 µg/mL rifampicin plus 128 µg/mL florfenicol or 256 µg/mL rifampicin plus 4 µg/mL cefotaxime. Colony counts from these plates were used to quantify population as follows: CFU of 13883/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e = (colonies on colistin and rifampicin plates × dilution factor) – (colonies on florfenicol and rifampicin plates or cefotaxime and rifampicin plates × dilution factor). CFU of 13883 (recipient) = colonies on rifampicin plates × dilution factor – colonies on colistin and rifampicin plates × dilution factor. CFU of 13883/pFJNS5\u003cem\u003e-mcr-9\u003c/em\u003e or 13883/pFJZZ12\u003cem\u003e-mcr-9\u003c/em\u003e = (colonies on florfenicol and rifampicin plates or cefotaxime and rifampicin plates) × dilution factor.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePrevalence of \u003cem\u003emcr\u003c/em\u003e positive- bacteria in pig farms\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 930 non-duplicate swab samples of were collected from both swine and the environment of four pig farms, including 537 samples from pig feces, 232 environmental samples within the pig farms and 124 water, soil samples outside the pig farms and 37 farmer feces. The \u003cem\u003emcr\u003c/em\u003e gene could be detected in pig feces, environmental samples both inside and outside the pig farm and in farmer feces. \u0026nbsp; \u0026nbsp;The isolation rates were listed in Table S2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the isolates, we found that 39/263 (14.8%) and 21/263 (8.0%) \u003cem\u003eK. pneumoniae\u003c/em\u003e strains were positive for \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e，\u0026nbsp;respectively. Additionally, 7/521（1.3%）and 7/521(1.3%) \u003cem\u003eE. coli\u003c/em\u003e strains were found to carry \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e，respectively. In addition, we identified 3 \u003cem\u003eRaoultella ornithinolytica\u003c/em\u003e strains T9，YSC9 and YSC3 collected from drinking water device inside the farm A and from soil outside the farm A, \u0026nbsp;as well as one \u003cem\u003eEnterobacter roggenkampii\u0026nbsp;\u003c/em\u003eNS5carried \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003egene isolated from drinking water inside the farm B\u003cem\u003e.\u003c/em\u003e \u0026nbsp;No other \u003cem\u003emcr\u0026nbsp;\u003c/em\u003egenes were detected except for \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFor 60 \u003cem\u003emcr\u0026nbsp;\u003c/em\u003epositive\u0026nbsp;\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains, 30 strains were collected from pig feces，19 isolates were collected from environmental samples inside the pig farm and 7 were collected from environmental samples outside the pig farm and 4 were collected from farmer feces. The \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003epositive rate of these four farms were 12.12%,15.38%, 14.04% and 17.33%, while the \u003cem\u003emcr-9\u003c/em\u003e positive rate within these three farms were 11%, 6.15%, 15.79% and 1.33% (Figure 1).\u003c/p\u003e\n\u003cp\u003eFor 14\u003cem\u003e\u0026nbsp;mcr\u003c/em\u003e positive \u003cem\u003eE. coli\u003c/em\u003e strains, 10 isolates were collected from pig feaces, 1 isolate were collected from environmental samples inside the pig farms, 1 isolated from environmental samples outside the pig farms and 2 were isolated from farmer feaces. The \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003epositive rate of these four farms were 0.90%, 5.17%, 0 and 0 while the \u003cem\u003emcr-9\u003c/em\u003e positive rate within these four farms were 1.80%, 3.45%, 0 and 0.6%. (Figure 1)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides, seven colistin resistance strains were obtained from hospital in Fujian Province and four tested positive for \u003cem\u003emcr-1\u003c/em\u003e gene, Y46, Y19, Y51 and Y27. The information and MIC values of these four strains were listed in Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial susceptibility profile of \u003cem\u003emcr\u0026nbsp;\u003c/em\u003epositive strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dilution method and micro-broth dilution method were employed to assess the susceptibility of 74 \u003cem\u003emcr\u0026nbsp;\u003c/em\u003epositive strains towards 11 antimicrobial agents of 5 distinct antibiotic classes. The results revealed that the minimum inhibitory concentration (MIC) values of colistin for these 39\u0026nbsp;\u003cem\u003emcr-1\u003c/em\u003e\u003cem\u003e\u0026nbsp;Klebsiella pneumoniae\u003c/em\u003e strains were mostly in the range of 2\u0026ndash;32 \u0026mu;g/mL. Among them, the resistance rate of 39 \u003cem\u003emcr-1\u003c/em\u003e-positive \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains to colistin, cefazolin, doxycycline, gentamicin, cefotaxime, tetracycline, streptomycin, ciprofloxacin, cefepime and cefquinome were of 89.7%, 89.7%, 87.2%, 92.3%, 79.5%,100%, 84.6%, 74.4%, 35.9%, and 76.9%, respectively. All strains were susceptible to tigecycline. The colistin MIC of these 21 \u003cem\u003emcr-9\u003c/em\u003e-positive \u003cem\u003eK. pneumoniae\u003c/em\u003estrains primarily ranged from 1 to 2 \u0026mu;g/mL, with most strains exhibiting a susceptible phenotype with colistin MIC≦2. The resistance rates to colistin, cefazolin, doxycycline, gentamicin, cefotaxime, tetracycline, streptomycin, ciprofloxacin and cefquinome were 42.9%, 85.7%, 100%, 57.1%, 42.9%, 100%, 85.7%, 28.6%\u0026nbsp;and 33.3%,\u0026nbsp;respectively. In contrast, all strains were susceptible to cefepime and tigecycline. All \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e positive strains exhibited multidrug resistance phenotypes. Notably, 13/39(33.33%) \u003cem\u003emcr-1\u003c/em\u003e positive \u003cem\u003eK. pneumoniae\u003c/em\u003e strains were resistance to over 9 antibiotics, whereas 11/21(52.38%) \u003cem\u003emcr-9\u003c/em\u003e positive \u003cem\u003eK. pneumoniae\u003c/em\u003e strains showed resistance to less than 4 antibiotics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor 14 \u003cem\u003emcr\u003c/em\u003e positive \u003cem\u003eE. coli\u003c/em\u003e, the resistance rate of 7 \u003cem\u003emcr-1\u003c/em\u003e \u003cem\u003eE. coli\u003c/em\u003e strains to cefazolin, doxycycline, gentamicin, cefotaxime, streptomycin, ciprofloxacin and cefquinome were 42.9%, 71.4%, 42.9%, 57.1%, 57.1%, 14.3% and 42.9%. all were resistance to colistin and tetracycline and were susceptible to cefepime and tigecycline. The resistance rate of 7 \u003cem\u003emcr-9 E. coli\u0026nbsp;\u003c/em\u003estrains to cefazolin, tetracycline, doxycycline, gentamicin, cefotaxime, streptomycin and ciprofloxacin were 14.3%, 100%, 100%, 42.9%, 14.3%, 57.1% and 14.3%, all were susceptible to colistin, tigecycline, cefepime, cefquinome. 4/7（57.1%）\u0026nbsp;\u003cem\u003emcr-1\u003c/em\u003e positive\u003cem\u003eE.coli\u003c/em\u003e strains were resistance to over 4 antibiotics and only one (14.3%) \u003cem\u003emcr-9\u003c/em\u003e positive E.coli strains showed resistance to over 4 antibiotics.\u003c/p\u003e\n\u003cp\u003eBesides, 3 \u003cem\u003eR. ornithinolytica\u003c/em\u003e T9, YSC9 and YSC3 showed diverse antibiotic profile, they show resistant to 9, 7 and 3 antibiotics, respectively. One \u003cem\u003eEnterobacter roggenkampii\u0026nbsp;\u003c/em\u003eNS5 was resistant to colistin, doxycycline,cefotaxime and tetracycline. Of note, YSC9 exhibited a high MIC of colistin=256 \u0026mu;g/mL. (Table 1)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome feature of plasmids harboring \u003cem\u003emcr\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003emcr\u003c/em\u003e-positive strains were all subject to conjugation assays and 40 conjugations were obtained. Eric-PCR were performed to confirm the profile were matched to recipient C600 strains and PCR were used to confirm the presence of \u003cem\u003emcr\u0026nbsp;\u003c/em\u003egenes. \u0026nbsp;Whole-genome sequencing and plasmid replicon PCR revealed that \u003cem\u003emcr-1\u003c/em\u003e was primarily located on IncHI2 (n=5), IncX4 (n=14), and IncI2 (n=15) plasmids, while \u003cem\u003emcr-9\u003c/em\u003e was predominantly carried by IncHI2 plasmids (n=4) and IncF(n=2) plasmids.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong them, the\u003cem\u003eEnterobacter roggenkampii\u0026nbsp;\u003c/em\u003estrain NS5 comprised a 4,796,092 bp chromosome and three plasmids pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e, pFJNS5-2 and pFJNS5-3 with the size of 187,381bp, 4783bp, and 3406bp, respectively. ResFinder analysis showed that NS5 contained \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR-1\u0026nbsp;\u003c/sub\u003elocated on the chromosone and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM-116\u003c/sub\u003e gene located on plasmid pFJNS5-3. While the IncFIB plasmid pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e contained colistin resistant gene \u003cem\u003emcr-9\u003c/em\u003e, florfenicol resistant gene \u003cem\u003efloR\u003c/em\u003e, sulfonamide resistant gene \u003cem\u003esul2\u003c/em\u003e and \u003cem\u003esul3\u003c/em\u003e, quinolone resistant gene\u003cem\u003e\u0026nbsp;qnrS1\u003c/em\u003e, tetracycline resistant gene \u003cem\u003etet(A)\u003c/em\u003e, trimethoprim resistant gene \u003cem\u003edfrA12\u003c/em\u003e, aminoglycoside resistant gene \u003cem\u003eaadA2\u003c/em\u003e, \u003cem\u003eaadA1\u003c/em\u003e and \u003cem\u003eaph(\u0026lsquo;3\u0026rsquo;)-Ia\u003c/em\u003e, chloramphenicol resistant gene \u003cem\u003ecmlA1\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eLAP-2\u003c/sub\u003e gene encoding class A\u0026nbsp;𝜷-lactamase. The type of the other two plasmids is undetected. Comparing the plasmids sequence with that in the NCBI database, we found that pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e shared \u0026gt;99% identity with plasmids pEc61A (CP053104), pYUSHP2-2(CP073773), pM1(CP090910), pCUVET18-121(CP114980), plasmid unnamed2 (CP138221), plasmid unnamed(CP149842) and pW139_2(CP163059), all identified in \u003cem\u003eEnterobacter hormaechei\u0026nbsp;\u003c/em\u003estrains, plasmid unnamed1(CP129956) identified in \u003cem\u003eEnterobacter asburiae\u003c/em\u003e strain and a plasmid pNK_H8_077(CP152556) identified in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e with 66%, 68%, 54%, 52%,66%,68% ,64%,60% and 61% coverage, respectively. They shared conserved region including region related to transfer, stable heritage and replication. While antibiotic resistant gene and inserted sequences were diverse in variable region.\u003c/p\u003e\n\u003cp skip=\"true\"\u003eSequencing analysis revealed that the genome of \u003cem\u003eR. ornithinolytica\u003c/em\u003e T9 contained three plasmids, which belongs to Col, IncHI2A and IncQ1 plasmids. The plasmid pFJT9 carried \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003eqacE\u003c/em\u003e\u003cem\u003e△\u003c/em\u003e, \u003cem\u003eaadA2\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e gene, belonging to IncHI2/HI2A group. Sequence comparing showed that pFJT9 exhibited high similarity with \u003cem\u003emcr-9\u003c/em\u003e plasmids p14269_1(\u003cem\u003eEnterobacter hormaechei\u003c/em\u003e,CP083850), p12379_1 (\u003cem\u003eEnterobacter kobei\u003c/em\u003e, CP083858), unknown (\u003cem\u003eSalmonella enterica\u003c/em\u003e, OX442404), pZHH-1(\u003cem\u003eEnterobacter hormaechei\u003c/em\u003e, CP059712), pK672-MCR9 (\u003cem\u003eEnterobacter hormaechei\u003c/em\u003e,CP183850), p131 (\u003cem\u003eSalmonella enterica\u003c/em\u003e, CP099761) and pMRVIM0813 (\u003cem\u003eEnterobacter cloacae\u003c/em\u003e, KP975077), p12795_1 (\u003cem\u003eEnterobacter roggenkampii\u003c/em\u003e,CP083854) deposited in NCBI database, pFJZZ12-\u003cem\u003emcr-\u003c/em\u003e\u003cem\u003e9\u003c/em\u003eand pFJY27-\u003cem\u003emcr-1\u003c/em\u003e isolated from pig feces and clinical samples in this study with \u0026gt;50% coverage . \u003c/p\u003e\n\u003cp skip=\"true\"\u003eLinear\u0026nbsp;\u0026nbsp;comparing sequence of pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e to that of pFJY27-\u003cem\u003emcr-\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e， we found that the sequence length of pFJY27-\u003cem\u003emcr-1\u003c/em\u003e with \u003cem\u003emcr-1\u003c/em\u003e gene is much shorter than that of pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e with \u003cem\u003emcr-9\u003c/em\u003e gene. pFJY27-\u003cem\u003emcr-1\u003c/em\u003e only contained colistin resistance gene \u003cem\u003emcr\u003c/em\u003e\u003cem\u003e-1\u003c/em\u003e, while plasmids pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e contained aminoglycosides resistance genes \u003cem\u003eaadA2\u003c/em\u003e, \u003cem\u003eaadA3\u003c/em\u003e and \u003cem\u003eant(2\u0026rdquo;)-Ia\u003c/em\u003e, cephalosporins resistance gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M-9\u003c/sub\u003e, the trimethoprim resistance gene \u003cem\u003edfrA16\u003c/em\u003e, the fluoroquinolones resistance gene \u003cem\u003eqnrA1\u003c/em\u003e and 3 copies of the sulfonamide resistance gene \u003cem\u003esul1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncHI2 type mcr-9 positive plasmids\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econfer a growth advantage over IncHI2 type plasmids harboring mcr-1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids pFJY27\u003cem\u003e-mcr-1\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e and pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e were chosen as representative plasmids for the IncHI2 plasmid carrying \u003cem\u003emcr-1\u003c/em\u003e, the IncHI2 plasmid carrying \u003cem\u003emcr-9\u003c/em\u003e and the IncFIB plasmid carrying \u003cem\u003emcr-9,\u0026nbsp;\u003c/em\u003erespectively.A 12-hour continuous culture experiment was conducted to observe the growth kinetics of transformants BW25113/pFJY27\u003cem\u003e-mcr-1\u003c/em\u003e(IncHI2)\u003cem\u003e,\u0026nbsp;\u003c/em\u003eBW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e(IncHI2) and BW25113/pFJNS5-\u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003e(IncFIB), and the plasmid-free recipient strain BW25113. As illustrated in Figure 3A, strain BW25113/pFJY27\u003cem\u003e-mcr-1\u003c/em\u003e exhibited slower growth compared to the plasmid-free host strain BW25113, suggesting a fitness cost associated with carrying this plasmid. In contrast, no significant growth difference was observed between BW25113 and strains BW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e and BW25113/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e, indicating that the acquisition of these\u003cem\u003e\u0026nbsp;mcr-9\u003c/em\u003e-carrying plasmids did not impose a noticeable growth burden on the host \u003cem\u003eE. coli\u003c/em\u003e strain without the antibiotic selection pressure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmids carrying \u003cem\u003emcr-9\u003c/em\u003e confer a competitive advantage over plasmids carrying \u003cem\u003emcr-1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure S1, after seven days of serial passage in the absence of antibiotics, \u003cem\u003eEscherichia coli\u003c/em\u003e BW25113/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e, BW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e, and BW25113/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e were all stably maintained within the host cells.\u003c/p\u003e\n\u003cp\u003eA competitive fitness assay was conducted in vitro by co-culturing \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eBW25113 strains carrying the IncHI2-type plasmids with BW25113/pFJY27-\u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eor BW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e to explore the adaptability of \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e plasmids. As shown in Figure 3B, the strain carrying the IncHI2 plasmid BW25113/pFJY27-\u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003ewas clearly outcompeted by the strain carrying the IncHI2 plasmid BW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e, with RF values below 1 starting from day 2. The\u0026nbsp;relative fitness of the \u003cem\u003emcr-1\u003c/em\u003e-carrying strain declined progressively, from 0.9588 to 0.4566 over the course of serial passages.\u003c/p\u003e\n\u003cp\u003eA similar competitive fitness assay was performed with strains BW25113/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e and BW25113/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e, carrying IncHI2-type plasmid and IncFIB plasmid, respectively. As shown in Figure 3B, the strain carrying the BW25113/pFJY27-\u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003econsistently exhibited RF values below 1, indicating lower competitiveness compared to the strain carrying the plasmid BW25113/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e. Over serial passages, the relative fitness of the mcr-1-carrying strain decreased from 0.7663 to 0.5551.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncHI2 type mcr-9 positive plasmids harbor higher plasmid spread ability and plasmid persistence in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;stains population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConjugation transfer abilities of \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e positive plasmids were evaluated by using \u003cem\u003eEscherichia coli\u003c/em\u003e BW25113/pFJY27-\u003cem\u003emcr-1,\u003c/em\u003e BW25113/pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e, and BW25113/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e as donor strains and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e 13883 as the recipient strain. As shown in Figure 3C, the conjugation frequency of pFJY27-\u003cem\u003emcr-1\u003c/em\u003e was 2.5\u0026times;10⁻⁵, while that of pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e was 9.56\u0026times;10⁻⁵, and pFJNS5-\u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003eexhibited a conjugation frequency of 5.42\u0026times;10⁻⁴.\u003c/p\u003e\n\u003cp\u003eA co-culture experiment was conducted with \u003cem\u003eEscherichia coli\u003c/em\u003e BW25113 strains carrying plasmid pFJY27-\u003cem\u003emcr-1 and mcr-9\u0026nbsp;\u003c/em\u003eplasmids pFJZZ12-\u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003eor pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e, and a plasmid-free \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e 13883 engineered strain. As shown in Figure 3D, plasmid invasion was observed as early as day 1 of serial passage. The strains 13883/pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e or 13883/pFJZZ12-\u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003ewas found to significantly outnumber the strain 13883/pFJY27-\u003cem\u003emcr-1\u003c/em\u003e, indicating that the mcr-9 positive plasmids pFJZZ12-\u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003eandpFJNS5-\u003cem\u003emcr-9\u003c/em\u003e exhibits a higher invasion capacity and faster invasion speed compared to plasmid pFJY27-\u003cem\u003emcr-1.\u003c/em\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe emergence and spread of plasmid-mediated \u003cem\u003emcr\u0026nbsp;\u003c/em\u003egenes have been documented worldwide, posing a threat to public health. This study aimed to investigate the persistence of \u003cem\u003emcr\u003c/em\u003e genes in \u003cem\u003eEnterobacteria\u003c/em\u003e isolated from the pig farms of Fujian province in China after the ban on antibiotic use as feed additive in livestock, which was implemented by the Chinese government since 2017[12]. Also, the factors that contribute to the prevalence of \u003cem\u003emcr-9\u003c/em\u003e in these four farms.\u003c/p\u003e\n\u003cp\u003eA total of 930 samples were examined for the presence of \u003cem\u003emcr\u0026nbsp;\u003c/em\u003egenes, with only \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eand \u003cem\u003emcr-9\u003c/em\u003e being detected. The prevalence of \u003cem\u003emcr-9\u003c/em\u003e was 8.0%, slightly lower than the 14.8% detection for \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003egene in \u003cem\u003eK. pneumoniae\u003c/em\u003e. In \u003cem\u003eE. coli\u003c/em\u003e isolates, \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eand \u003cem\u003emcr-9\u003c/em\u003e were detected in 8 (1.5%) and 6 (1.1%) strains, respectively. These observations are consistent with previously study, where \u003cem\u003emcr-9\u003c/em\u003e consistently detected as the one of the most prevalent \u003cem\u003emcr\u0026nbsp;\u003c/em\u003esubtype following \u003cem\u003emcr-1\u003c/em\u003e[9]. Among 1000 sequenced\u0026nbsp;\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains in prior research, \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003ewas reported as both the earliest and second-most prevalent subtype after \u003cem\u003emcr-1\u003c/em\u003e[22]. In a global survey of MCR gene family distribution across 3250 isolates, \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eand \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003ewere found to be the most widely disseminated, with isolates identified in 61 and 40 countries across six continents, respectively[9].\u0026nbsp;The widespread geographic distribution underscores the significant global dissemination potential of the \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003egene, while the mechanism of its widespread is unclear.\u003c/p\u003e\n\u003cp\u003eBeside\u0026nbsp;\u003cem\u003eK. pneumoniae\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE. coli\u003c/em\u003e, the \u003cem\u003emcr-9\u003c/em\u003e isolates were also identified in\u0026nbsp;\u003cem\u003eEnterobacter roggenkampii\u0026nbsp;\u003c/em\u003eisolated from drinking water and in\u003cem\u003e\u0026nbsp;Raoultella ornithinolytica\u0026nbsp;\u003c/em\u003eisolated from both drinking water devices and soil in the study\u003cem\u003e,\u0026nbsp;\u003c/em\u003eindicating that zoonotic transmission of \u003cem\u003emcr\u0026nbsp;\u003c/em\u003epositive plasmids between animals, the environment and humans is also occurring. The presence of\u003cem\u003e\u0026nbsp;mcr\u003c/em\u003e gene identified in the environmental samples suggest that the environmental can be regarded as a reservoir for the spread of antimicrobial resistance without antibiotic selection pressure. The identification of \u003cem\u003emcr-9\u003c/em\u003e-positive isolates in drinking water devices and soil further reinforces the idea that environmental surveillance must be a key component of AMR control strategies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe antimicrobial susceptibility results revealed that 42 \u003cem\u003emcr-1\u003c/em\u003e-positive strains (35 \u003cem\u003eK. pneumoniae\u003c/em\u003e and 7 \u003cem\u003eE. coli\u003c/em\u003e) exhibited resistance to colistin, with MIC values predominantly between 2–32 μg/mL, while only 9 \u003cem\u003emcr-9\u003c/em\u003e-positive strains had MIC values\u0026nbsp;≧\u0026nbsp;4 μg/mL. \u0026nbsp; Previous surveillance of the \u003cem\u003emcr\u0026nbsp;\u003c/em\u003egenes has primarily focused on monitoring \u003cem\u003emcr\u003c/em\u003e genes in colistin-resistant bacteria. The observation indicated an underestimation of the true prevalence of \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003edue to its frequent association with low colistin MIC value. Most of the \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eand \u003cem\u003emcr-9\u003c/em\u003e-positive bacteria demonstrated multidrug resistance, while the \u003cem\u003emcr-1\u003c/em\u003e positive strains seemed to be associated with a more extensive resistance phenotype than \u003cem\u003emcr-9\u003c/em\u003e positive strains. The broader resistance profiles of \u003cem\u003emcr-1\u003c/em\u003e-positive strains influence their relative fitness and persistence under antibiotic selection pressure, helping to explain the higher prevalence of \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003ecompared to \u003cem\u003emcr-9\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eComparative genomic analyses of the pFJNS5-\u003cem\u003emcr-9\u003c/em\u003e plasmid shared \u0026gt;99% identity with plasmids isolated in \u003cem\u003eEnterobacter hormaechei\u003c/em\u003e, \u003cem\u003eEnterobacter asburiae\u003c/em\u003e, and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains in the NCBI database, with coverage ranging from 52% to 68%. These plasmids shared conserved plasmid backbone but the antibiotic resistance gene and insertion sequences, indicating the frequent recombination and horizontal gene transfer event of antibiotic resistance genes. Similarity, the plasmid pFJT9-\u003cem\u003emcr-9\u003c/em\u003e exhibited high sequence similarity to \u003cem\u003emcr-9\u003c/em\u003e plasmids available in the NCBI database identified in diverse \u003cem\u003eEnterobacteria\u003c/em\u003e, indicating the extensive mobilization potential and broad host range of the \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003egene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasmid persistence within a bacterial community is typically linked to efficient acquisition and associated fitness advantage[19]. Hence, we investigated the dynamics of \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e positive plasmids through a series of conjugation frequency assays, plasmid stability tests, fitness trials, and plasmid competition-invasion assays to elucidate the factors underlying the higher detection rate of the \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003egene in pig farms in Fujian Province, China. Plasmids pFJY27-\u003cem\u003emcr-1\u003c/em\u003e and pFJZZ12-\u003cem\u003emcr-9\u003c/em\u003e were selected as the representative of IncHI2 plasmid with \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9,\u003c/em\u003e respectively, due to similar plasmid backbones, while plasmid pFJNS5-\u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003eserved as a representative IncFIB plasmid harboring the \u003cem\u003emcr-9\u003c/em\u003e gene. The conjugation frequency for both \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003epositive IncHI2 plasmids was 10⁻⁴~10⁻⁵, indicating their high transfer capability. In addition, the \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003epositive plasmids appeared to exhibit a slightly higher conjugation rate compared to the \u003cem\u003emcr-1\u003c/em\u003e positive plasmids. However, the sample size is limited, further studies involving larger sample sizes and diverse plasmid types are needed to confirm the observation.\u003c/p\u003e\n\u003cp\u003eBoth \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003epositive plasmids remained stably maintained within the host \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eBW25113 strains over seven days of serial passaging without antibiotic pressure. Growth kinetics analysis demonstrated that \u003cem\u003emcr-1\u003c/em\u003e positive IncHI2 plasmids negatively affected host bacterial growth, while \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003epositive IncHI2 and IncFIB plasmids had no significant impact on host growth. This indicated that carriage of the \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003egene dose not impose a notable fitness burden on bacterial host. The lower fitness cost associated with \u003cem\u003emcr-9\u003c/em\u003e positive plasmids suggests that these plasmids may persist longer within bacterial populations comparing to\u003cem\u003e\u0026nbsp;mcr-1\u0026nbsp;\u003c/em\u003epositive plasmids in the absence of colistin selection pressure.\u003c/p\u003e\n\u003cp\u003eIn vitro competition assays between \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eBW25113 strains carrying \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eor \u003cem\u003emcr-9-\u0026nbsp;\u003c/em\u003epositive plasmids demonstrated that, in the absence of antibiotic selection, \u003cem\u003emcr-1\u003c/em\u003e-positive plasmids conferred a higher fitness cost to their hosts than\u003cem\u003e\u0026nbsp;mcr-9\u003c/em\u003e plasmids, placing \u003cem\u003emcr-1-\u003c/em\u003epositive strains at a competitive disadvantage over successive generations without antibiotic selection. These results further underlined the survival advantage of \u003cem\u003emcr-9-\u003c/em\u003epositive bacteria in nutrient-competitive environments without colistin selection pressure, suggesting an emerging trend of increasing prevalence of \u003cem\u003emcr-9\u003c/em\u003e-postive plasmids within pig farm ecosystem in China.\u003c/p\u003e\n\u003cp\u003eAdditionally, the invasion capability of these plasmids was evaluated, we found that the \u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003epositive IncHI2 or IncFIB plasmids invades recipient \u003cem\u003eK. pneumoniae\u003c/em\u003e strains at significantly higher rates compared to \u003cem\u003emcr-1\u003c/em\u003e positive IncHI2 plasmids. After four days, the concentration of bacteria harboring \u003cem\u003emcr-9\u003c/em\u003e-positive plasmids (pFJNS5 or pFJZZ12) significantly outnumber those of bacteria carrying the \u003cem\u003emcr-1-\u003c/em\u003epositive plasmid (pFJY27), although plasmid-free bacterial populations remained dominant. The enhanced invasion potential observed for \u003cem\u003emcr-9\u003c/em\u003e-positive plasmids could be attributed to their higher conjugation frequencies and lower associated fitness costs.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study investigates the persistence and dissemination dynamics of plasmid-mediated \u003cem\u003emcr\u003c/em\u003e genes—particularly\u003cem\u003e\u0026nbsp;mcr-1\u0026nbsp;\u003c/em\u003eand \u003cem\u003emcr-9\u003c/em\u003e—within Enterobacteriaceae isolated from four pig farms in Fujian Province, China, following the ban on colistin as a feed additive. While both \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e were detected,\u003cem\u003e\u0026nbsp;mcr-1\u0026nbsp;\u003c/em\u003eremained more prevalent. In the study, \u003cem\u003emcr-9\u003c/em\u003e-positive plasmids were identified not only in\u003cem\u003e\u0026nbsp;K. pneumoniae\u003c/em\u003e and\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e, but also in \u003cem\u003eEnterobacter roggenkampii\u0026nbsp;\u003c/em\u003eand \u003cem\u003eRaoultella ornithinolytica\u003c/em\u003e, with environmental sources such as drinking water, soil, and water devices serving as reservoirs—highlighting the role of the environment in facilitating plasmid-mediated resistance transmission. The lower fitness cost, stable maintenance, and higher conjugation and invasion efficiencies of \u003cem\u003emcr-9\u003c/em\u003e-positive IncHI2 and IncFIB plasmids suggest that these plasmids are more likely to persist and propagate in bacterial populations in the absence of antibiotic selection pressure. This biological advantage may contribute to a gradual increase in the prevalence of \u003cem\u003emcr-9-\u003c/em\u003epositive bacteria within livestock environments. Overall, these findings underscore the urgent need for enhanced surveillance of \u003cem\u003emcr-9\u003c/em\u003e, especially in environmental reservoirs, and a deeper understanding of its ecological and evolutionary advantages. Although \u003cem\u003emcr-9\u003c/em\u003e gene is frequently associated with colistin-susceptible phenotypes, its expression can be induced under colistin exposure. Moreover, \u003cem\u003emcr-9\u003c/em\u003e harboring plasmids are often linked to multiantibiotic resistance, limiting treatment options. Targeted strategies should be developed to monitor, prevent, and mitigate the spread of \u003cem\u003emcr-\u003c/em\u003epositive plasmids, integrating a One Health approach that addresses animal, environmental, and human health\u0026nbsp;\u003cem\u003edimensions of antimicrobial resistance.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve any experiments on live animals. All fecal and environmental samples were collected with permission from commercial pig farm owners and veterinarians.\u0026nbsp;The clinical sample collection was approved by the Animal Experimentation Committee of the Second Affiliated Hospital of Fujian Medical University (approval number:2022408).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLingxian Yi:\u003c/strong\u003e Conceptualization, Methodology, Data analysis, Writing-original draft, review, editing and Funding acquisition. \u003cstrong\u003eHuamin Lai, Jianshuo Liu and Donghong Huang:\u0026nbsp;\u003c/strong\u003eInvestigation and Methodology. \u003cstrong\u003eDaojin Yu and Jiaming Huang:\u003c/strong\u003e Conceptualization, supervision, project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the grants from the Natural Science Foundation of Fujian Province (No. 2024J08038), the Education Research Fund for Young Academic of Fujian Province (JAT220060) and the Natural Science Foundation of Fujian Province (2023J01728).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete nucleotide sequence of pFJNS5-\u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003eand pFJZZ12-\u003cem\u003emcr-9\u0026nbsp;\u003c/em\u003ehas been updated at GenBank (PV817785 and PV817786).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the author(s) used ChatGPT -4o in order to check for spelling and clarity of language. After using this tool, the author(s) reviewed and edited the content as needed and take full responsibility for the content of the published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"Reference","content":"\u003col\u003e\n \u003cli\u003eWilson H, Torok ME: \u003cstrong\u003eExtended-spectrum beta-lactamase-producing and carbapenemase-producing Enterobacteriaceae\u003c/strong\u003e. \u003cem\u003eMicrob Genom\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e4\u003c/strong\u003e(7).\u003c/li\u003e\n \u003cli\u003eMoffatt JH, Harper M, Boyce JD: \u003cstrong\u003eMechanisms of Polymyxin Resistance\u003c/strong\u003e. \u003cem\u003eAdv Exp Med Biol\u0026nbsp;\u003c/em\u003e2019, 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right direction\u003c/strong\u003e. \u003cem\u003eLancet Infect Dis\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e21\u003c/strong\u003e(1):29-30.\u003c/li\u003e\n \u003cli\u003eHayashi W, Tanaka H, Taniguchi Y, Iimura M, Soga E, Kubo R, Matsuo N, Kawamura K, Arakawa Y, Nagano Y\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eAcquisition of mcr-1 and Cocarriage of Virulence Genes in Avian Pathogenic Escherichia coli Isolates from Municipal Wastewater Influents in Japan\u003c/strong\u003e. \u003cem\u003eAppl Environ Microbiol\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e85\u003c/strong\u003e(22).\u003c/li\u003e\n \u003cli\u003eShen C, Zhong LL, Yang Y, Doi Y, Paterson DL, Stoesser N, Ma F, El-Sayed Ahmed MAE, Feng S, Huang S\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eDynamics of mcr-1 prevalence and mcr-1-positive Escherichia coli after the cessation of colistin use as a feed additive for animals in China: a prospective cross-sectional and whole genome sequencing-based molecular epidemiological study\u003c/strong\u003e. \u003cem\u003eLancet Microbe\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e1\u003c/strong\u003e(1):e34-e43.\u003c/li\u003e\n \u003cli\u003eCLSI: \u003cstrong\u003eCLSI. 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The information of 3\u003cem\u003e\u0026nbsp;mcr-9\u003c/em\u003e-positive \u003cem\u003eRaoultella ornithinolytica\u003c/em\u003e and\u003cem\u003e\u0026nbsp;\u003c/em\u003ean \u003cem\u003eEnterobacter roggenkampii\u0026nbsp;\u003c/em\u003estrains.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"987\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eOrganism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003eOrigin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eSite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eLocation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003eColistin MIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eother rsistantce phenotype(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003eOther resistance gene(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cem\u003eRaoultella ornithinolytica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003esoil outside farm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eFarm A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eIncHI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eSTR, TGC, DOX, CIP, GEN, CTX, TET, CQM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cem\u003eaac(6\u0026apos;)-Ib-cr\u003c/em\u003e, \u003cem\u003eaadA2\u003c/em\u003e, \u003cem\u003eaph(3\u0026apos;\u0026apos;)-Ib\u003c/em\u003e, \u003cem\u003eaph(6)-Id\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eDHA-1\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-1\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ecatB3\u003c/em\u003e, \u003cem\u003edfrA12\u003c/em\u003e,\u003cem\u003e\u0026nbsp;floR\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003efosA\u003c/em\u003e, \u003cem\u003eqnrB2\u003c/em\u003e, \u003cem\u003eqnrS1\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003esul2, \u003cem\u003etet(A)\u003c/em\u003e, \u003cem\u003etet(D)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eYSC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003edrinking water device\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eFarm A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eSTR, DOX, FEP, GEN, CTX, TET, CQM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eYSC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003edrinking water device\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eFarm B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eDOX,TET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003eNS5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter roggenkampii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003edrinking water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eFarm B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eIncFIB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eDOX，CTX, TET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cem\u003eaadA2\u003c/em\u003e, aph(3\u0026apos;)-Ia, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eLAP-2\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR-1\u003c/sub\u003e, \u003cem\u003ecmlA1\u003c/em\u003e, \u003cem\u003edfrA12\u003c/em\u003e, floR, \u003cem\u003eqnrS1\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;sul3\u003c/em\u003e, \u003cem\u003etet(A)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mcr, colistin resistance, Enterobacteriaceae, One Health, plasmid, Environmental reservoirs","lastPublishedDoi":"10.21203/rs.3.rs-6934660/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6934660/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolymyxins are last-resort antibiotics for multidrug-resistant Gram-negative bacteria infections. However, their efficacy is threatened by the emergence of plasmid-mediated colistin resistance genes (\u003cem\u003emcr-1\u003c/em\u003e to \u003cem\u003emcr-10\u003c/em\u003e). Antibiotic use in agriculture has been recognized as a major driver of antimicrobial resistance, prompting China to ban colistin as a feed additive in 2017. This study investigated the persistence and transmission mechanisms of \u003cem\u003emcr\u003c/em\u003e genes in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e isolated from pigs, farm workers, and the surrounding environment on four pig farms in Fujian Province, China.\u003c/p\u003e\n\u003cp\u003eTotally, 930 samples were collected, including pig rectal swabs, farm worker fecal samples, as well as environmental samples from both inside and outside the farms. From these, a total of 263 \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and 521 \u003cem\u003eEscherichia coli \u003c/em\u003eisolates wererecovered, with isolation rate of 28.3% and 56.0%, respectively. PCR screening revealed that \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003e were the only detected variants. Among \u003cem\u003eK. pneumoniae \u003c/em\u003eisolates\u003cem\u003e,\u003c/em\u003e 39 (14.8%) carried \u003cem\u003emcr-1\u003c/em\u003e and 21(8.0%) carried \u003cem\u003emcr-9\u003c/em\u003e. In \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003emcr-1\u003c/em\u003e and \u003cem\u003emcr-9\u003c/em\u003ewere detected in 8 (1.5%) and 6 (1.1%) isolates, respectively. In addition, three \u003cem\u003eRaoultella ornithinolytica\u003c/em\u003e and one \u003cem\u003eEnterobacter roggenkampii \u003c/em\u003eisolates\u003cem\u003e \u003c/em\u003ecarried \u003cem\u003emcr-9 \u003c/em\u003egenes were identified\u003cem\u003e.\u003c/em\u003e Besides animal sources, \u003cem\u003emcr\u003c/em\u003e-positive strains were also identified in environmental samples, particularly from inside the farms, and in farm workers, indicating potential zoonotic and environmental transmission.Antimicrobial susceptibility testing revealed that all \u003cem\u003emcr\u003c/em\u003e-positive isolates exhibited multi-antibiotic resistance, with \u003cem\u003emcr-1\u003c/em\u003e-positive strains displaying broader resistance profiles than \u003cem\u003emcr-9\u003c/em\u003e-positive strains. The minimum inhibitory concentrations (MICs)of colistin ranged from 2-32 μg/mL for \u003cem\u003emcr-1\u003c/em\u003e-positive isolates and 1-8 μg/mL for \u003cem\u003emcr-9\u003c/em\u003e-positive isolates. Whole-genome sequencing and conjugation experiments showed that \u003cem\u003emcr-1\u003c/em\u003ewas primarily located on IncHI2 (n=5), IncX4 (n=14), and IncI2 (n=15) plasmids, while \u003cem\u003emcr-9\u003c/em\u003e was predominantly carried by IncHI2 plasmids (n=4) and IncF(n=2) plasmids. Notably, \u003cem\u003emcr-9\u003c/em\u003e-positive plasmids showed higher conjugation efficiency, lower fitness cost, and greater persistence within bacterial hosts compared to \u003cem\u003emcr-1\u003c/em\u003e-positive IncHI2 plasmids. These finding suggest that the increasing prevalence of \u003cem\u003emcr-9\u003c/em\u003e in \u003cem\u003eEnterobacteria\u003c/em\u003emay be driven by its enhanced transferability and stability, even in the absence of antibiotic selection pressure. This highlights the urgent need for continued environmental surveillance and targeted interventions to solve the dissemination of plasmid-mediated antibiotic resistance in livestock production.\u003c/p\u003e","manuscriptTitle":"Persistence and Transmission Dynamics of mcr-1 and mcr-9 in Enterobacteriaceae from Pig Farms in Fujian, China after the Colistin Ban","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 07:07:29","doi":"10.21203/rs.3.rs-6934660/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd446275-78c3-487c-b510-88fc7d986400","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-07T16:40:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-26 07:07:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6934660","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6934660","identity":"rs-6934660","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}