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Salmonella enterica resistant to colistin, third-generation cephalosporins (3GCs), and fluoroquinolones (FQs) has been deemed a high-priority pathogen by the World Health Organization (WHO). The objective of this study was to characterize 11 mcr-1-harboring Salmonella enterica serovar Typhimurium isolates from raw pork and ready-to-eat (RTE) pork products in Guangzhou, China. All isolates were multi-drug resistant and contained 6–24 antibiotic-resistant genes. The mcr-1 gene was localized in the most conserved structure (mcr-1-orf ) in eight isolates and in mobile structure (ISApl1-mcr-1-orf ) in three isolates. One raw pork isolate SH16SF0850, co-harbored mcr-1, blaCTX−M−14, and oqxAB genes. One isolate 17Sal008 carried mcr-1, blaCTX−M−14, qnrS2, and oqxAB genes located on a 298,622 bp IncHI2 plasmid pSal008, which was obtained from an RTE pork product for the first time. The pSal008 was closely related to a plasmid in an S. typhimurium isolate from a 1-year-old diarrheal outpatient in China and was found to be transferable to Escherichia coli J53 by conjugation. Genome sequence comparisons by core-genome Multi Locus Sequence Typing (cgMLST) based on all S. typhimurium isolates from China inferred highly probably epidemiological links between selected pork isolates and no possible epidemiologically links between RTE pork isolate 17Sal008 and other isolates. Our findings indicate that raw pork and pork products are potential reservoirs of mcr-1-harboring S. typhimurium and highlight the necessity for continuous monitoring of colistin, 3GCs, and FQs resistant S. typhimurium from different origins. [ABSTRACT FROM AUTHOR]

Considering the serious threat to food safety and public health posed by pathogens with colistin resistance, colistin was banned as a growth promoter in 2017 in China. In recent years, the resistance rate of Escherichia coli isolated from animal intestines or feces to colistin has decreased. However, the prevalence and characteristics of the mcr-1 gene in retail meat have not been well explored. Herein, 106 mcr-1-negative and 16 mcr-1-positive E. coli isolates were randomly recovered from 120 retail meat samples and screened using colistin. The 106 E. coli isolates showed maximum resistance to sulfafurazole (73.58%) and tetracycline (62.26%) but susceptibility to colistin (0.00%). All 16 mcr-1-positive E. coli isolates showed resistance to colistin, were extended spectrum beta-lactamase (ESBL)-positive and exhibited complex multidrug resistance (MDR). For these 16 isolates, 17 plasmid replicons and 42 antibiotic resistance genes were identified, and at least 7 antibiotic resistance genes were found in each isolate. Acquired disinfectant resistance genes were identified in 75.00% (12/16) of the isolates. Furthermore, comparative genomic and phylogenetic analysis results indicated that these 16 mcr-1-positive E. coli isolates and the most prevalent mcr-1-harboring IncI2 plasmid in this study were closely related to other previously reported mcr-1-positive E. coli isolates and the IncI2 plasmid, respectively, showing their wide distribution. Taken together, our findings showed that retail meat products were a crucial reservoir of mcr-1 during the colistin ban period and should be continuously monitored. [ABSTRACT FROM AUTHOR]

Background: The rising resistance to carbapenems in Gram-negative bacteria worldwide poses a major clinical and public health risk. This study aimed to characterise carbapenem- and colistin-resistance genes, blaNDM-1 and mcr-1 located on IncX4 plasmid in MDR Pseudomonas aeruginosa, isolated from paediatric patients undergoing chemotherapy as a result of leukaemia. Methods: In this study, six carbapenem-resistant strains of P. aeruginosa were isolated from two paediatric patients under chemotherapy treatment (1.8 years old female and 2.1 years male) from the Shenzhen Hospital, China, in the year 2019. Isolates were screened for conventional antibiotics such as tobramycin, cefepime, imipenem, and ciprofloxacin in additional colistin by using the broth dilution method. Furthermore, resistance determinants: mcr-1, blaNDM-1,blaKPC-1, and blaGES were screened using PCR and sequencing followed by multi-locus sequence typing. The horizontal gene transfer and location of mcr-1 and blaNDM-1 were determined by a liquid mating assay. In addition, Incompatibility type (Inc), PCR-based replicon type, and subgroup (MOB) of plasmid were studied. Results: The screening for conventional antibiotics isolates showed 100% resistance to all the tested antibiotics except tobramycin. All isolates harboured carbapenemase encoding blaNDM-1, of which three also had mcr-1 located on a single IncX4 transferable plasmid. MLST typing revealed that four strains had a novel (new) STs type, while two belonged to ST1966. Conclusion: This study identified for the first time colistin- and carbapenem-resistant MDR P. aeruginosa in paediatric patients with leukaemia in Shenzhen, China. It highlights the need for continuous surveillance in high-risk clones of MDR P. aeruginosa. Prudent use of antibiotics based on local antimicrobial susceptibility and clinical characteristics can help in reducing mortality in immunocompromised patients. [ABSTRACT FROM AUTHOR]

Colistin, a last-resort antibiotic, is used to treat infections caused by multi-drug-resistant Gram-negative bacteria. Colistin resistance can emerge by acquiring the mobile colistin gene, mcr-1, usually plasmid borne. Studies on mcr-1 and its transmissibility are limited in the Middle East and North Africa (MENA) region. Here, we investigated the occurrence of mcr-1 in 18 previously collected Escherichia coli isolates collected from chicken samples in Qatar; whole-genome sequencing was performed to determine the location (plasmid-borne and chromosomal) of mcr-1 in the isolates. Additionally, we assessed the transmissibility of plasmid-borne mcr-1 and its cost on fitness in E. coli biofilms. Our results showed that the E. coli isolates belonged to different sequence types, indicating that mcr-1 was occurring in strains with diverse genetic backgrounds. In silico analysis and transformation assays showed that all the isolates carried mcr-1 on plasmids that were mainly IncI2 types. All the mcr-1 plasmids were found to be transmissible by conjugation. In biofilms, a significant reduction in the number of CFU (≈0.055 logs CFU/mL) and colistin resistance (≈2.19 log CFU/mL) was observed; however, the reduction in resistance was significantly larger, indicating that the plasmids incur a high fitness cost. To our knowledge, this is the first study that investigates mcr-1 transmissibility and persistence in Qatar. Our findings highlight that mcr has the potential to spread colistin resistance to potentially disparate strains and niches in Qatar, posing a risk that requires intervention. [ABSTRACT FROM AUTHOR]

Ganfeng Liang,1,* Yuting Rao,2,3,* Shuang Wang,4 Xiaohui Chi,2 Hao Xu,2 Yang Shen1 1Department of Infectious Diseases, The First Hospital of Taizhou, Taizhou, People’s Republic of China; 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 3Department of Laboratory Medicine, Puyang Oil Field General Hospital, Puyang, People’s Republic of China; 4Institute of Bacterial Infection Disease Control, Shandong Centre for Disease Control and Prevention, Jinan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yang Shen; Hao Xu Email Shenyanghtt@163.com; xuhao0523@zju.edu.cnBackground: The emergence of the plasmid-borne colistin-resistant gene (mcr-1) poses a great threat to human health. What is worse, the recent observations of the coexistence of mcr-1 with carbapenemase encoding genes in some bacteria caused even more concern. Yet, there is a lack of observations of such strains in the human gut.Methods: The isolation of E. coli L889 was performed on selective medium plates. Antibiotic susceptibilities were determined by an agar dilution and a broth microdilution method. Multi-locus sequence typing (MLST) and acquired resistance genes were also characterized. Transferability of blaNDM-9/mcr-1-carrying plasmids was determined by conjugation, replicon typing and S1-Pulsed-field gel electrophoresis (S1-PFGE), and Southern blotting. The sequences of these plasmids were analyzed by using whole-genome sequencing with Illumina Novaseq and Nanopore platforms.Results: E. coli L889 was identified as ST1101 concomitantly carrying blaNDM-9 and mcr-1 from a stool sample. Antimicrobial susceptibility tests showed that it was resistant to various antimicrobial agents and only susceptible to tigecycline. Notably, blaNDM-9 was located on a ∼ 114-kb untypable plasmid, while mcr-1 was located on a ∼ 63-kb IncI2 plasmid.Conclusion: Our research, to our knowledge, first reported an ST1101 E. coli strain with an untypeable blaNDM-9-harbouring plasmid and an IncI2 mcr-1-carrying plasmid. The colonized E. coli strains potentially contribute to the dissemination and transfer of blaNDM-9 and mcr-1 to clinical isolates, which is a considerable threat to public health and should be closely monitored.Keywords: blaNDM-9, mcr-1, gut, Escherichia coli, ST1101

Chen Huang,1,2 Qingyi Shi,2 Shuntian Zhang,2 Hongcheng Wu,1 Yonghong Xiao2 1Department of Respiratory Medicine, Ningbo Medical Center Lihuili Hospital, Ningbo, People’s Republic of China; 2State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of ChinaCorrespondence: Hongcheng WuDepartment of Respiratory Medicine, Ningbo Medical Center Lihuili Hospital, Ningbo, 315000, People’s Republic of ChinaEmail doctorwu1967@126.comYonghong XiaoState Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, People’s Republic of ChinaTel +865 718 723 6421Email xiao-yonghong@163.comObjective: The spread of the plasmid-mediated colistin resistance gene mcr-1 poses a significant public health threat. Little information is available on the development of high-level colistin-resistant mutants (HLCRMs) in MCR-1-producing Escherichia coli (MCRPEC). The present study was designed to evaluate the impact of chromosomal modifications in pmrAB, phoPQ, and mgrB combined with mcr-1 on colistin resistance in E. coli.Methods: Five MCRPEC and three non-MCRPEC (E. coli ATCC25922 and two plasmid-curing) strains were used. The HLCRMs were selected through multi-stepwise colistin exposure. Moreover, two E. coli C600-pMCRs were constructed and used for selection of HLCRMs. Further analysis included mutation rates and DNA sequencing. Transcripts of pmrABC, phoP, mgrB, and mcr-1 were quantified by real-time quantitative PCR.Results: All tested HLCRMs were successfully isolated from their parental strains. Non-MCRPEC strains had higher minimum inhibitory concentrations (MICs) and mutation rates than MCRPEC strains. Nineteen amino acid substitutions were identified: seven in PmrA, six in PmrB, one in PhoP, three in PhoQ, and two in MgrB. Most were detected in non-MCRPEC strains. Sorting Intolerant From Tolerant predicted that four substitutions, PmrA Gly15Arg, Gly53Arg, PmrB Pro94Gln, and PhoP Asp86Gly, affected protein function. Two HLCRM isolates did not show amino acid substitutions in contrast to their parental MCRPEC isolates. No further mutations were detected in the second- and third-step mutants. Further transcriptional analysis showed that the up-regulation of pmrCAB expression was greater in the mutant of E. coli C600 than in E. coli C600-pMCR.Conclusion: Acquisition of the mcr-1 gene had a negative impact on the development of HLCRMs in E. coli, but was associated with low-level colistin resistance. Thus, colistin-based combination regimens may be effective against infections with MCR-1-producing isolates.Keywords: mutation, mcr-1, chromosomal resistance mechanisms, high-level colistin resistance, Escherichia coli

Xingwei Cao,1,* Qiaoshi Zhong,1,* Yinjuan Guo,2,3 Yaping Hang,1 Yanhui Chen,1 Xueyao Fang,1 Yanping Xiao,1 Hongying Zhu,1 Hong Luo,1 Fangyou Yu,2,3 Longhua Hu1 1Jiangxi Provincial Key Laboratory of Medicine, Clinical Laboratory of the Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200082, People’s Republic of China; 3Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200082, People’s Republic of China*These authors contributed equally to this workCorrespondence: Longhua Hu; Fangyou Yu Email longhuahu@163.com; wzjxyfy@163.comIntroduction: Polymyxin resistance caused by the plasmid-mediated mcr-1 gene in gram-negative bacilli poses a huge threat to our health. In recent years, many regions have reported that mcr-1 and β-lactamase genes can coexist in a single strain.Methods: In this study, 107 nonduplicate Klebsiella pneumoniae (K. pneumoniae) isolates were collected from a tertiary hospital in Jiangxi, China. Antimicrobial susceptibility testing of isolates was performed using gram-negative susceptibility cards on the VITEK system. The minimum inhibitory concentrations (MICs) of polymyxin B was detected using the microdilution broth method. The presence of resistance genes was assessed using polymerase chain reaction (PCR). We subjected isolates to genotyping using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and analyzed the transferability of plasmids with filter mating and electroporation. Subsequently, whole-genome sequencing was performed for plasmids.Results: Of the 107 K. pneumoniae isolates, 15 (14.0%) were resistant to polymyxin B. All polymyxin B-resistant isolates harbored at least one of the extended-spectrum β-lactamase genes tested. Only one isolate simultaneously harbored mcr-1, blaNDM− 5, blaCTX-M-55, and blaSHV-27 genes. MLST results showed that 15 carbapenem-resistant K. pneumoniae isolates belonged to five sequence types (STs). PFGE results displayed nine different PFGE clusters. Conjugation and transformation experiments and sequencing analysis showed that the strain had three plasmids, and mcr-1, blaNDM− 5, and blaCTX-M-55 were located on different plasmids.Conclusion: The present study demonstrated for the first time the coexistence of mcr-1, blaNDM− 5, and blaCTX-M-55 in a K. pneumoniae ST485 isolate. The three plasmids carrying the mcr-1, blaNDM− 5, and blaCTX-M-55 genes can be transmitted in Enterobacteriaceae strains, which may lead to more severe bacterial resistance.Keywords: Klebsiella pneumoniae, polymyxin B, mcr-1, blaNDM-5, blaCTX-M-55, plasmid

Zhenzhu Zheng, Ying Lei, Yinna Wang, Chunli Lin, Jiansheng Lin Department of Laboratory Medicine, Quanzhou Women’s and Children’s Hospital, Quanzhou, People’s Republic of ChinaCorrespondence: Jiansheng LinDepartment of Laboratory Medicine, Quanzhou Women’s and Children’s Hospital, 700 Fengze Street, Fengze District, Quanzhou, Fujian Province, 350122, People’s Republic of ChinaTel +86 0595 22196131Email 63678462@qq.comBackground: The purpose of this study was to evaluate the prevalence of mobile colistin resistance genes (mcr) in Gram-negative bacteria and to analyze the molecular characteristics of mcr-1 positive Salmonella typhimurium strain 75 and Escherichia coli strain 107 from the Quanzhou Women’s and Children’s Hospital in China.Methods: The genes mcr-1 through mcr-9 were screened via multiplex PCR. Antibiotic susceptibility was detected using a GN11 card with the VITEK-2 compact automated system. Whole genomes were sequenced using PacBio’s single molecule real-time (SMRT) technology.Results: In this study, mcr-1 was detected in only four strains, with a positivity rate of 0.65% (4/616). All the four strains were resistant to more than three different kinds of antibiotics. The mcr-1 positive S. typhimurium strain 75 harbored IncHI2 plasmid, which carried mcr-1 gene, while the mcr-1 positive E. coli strain 107 contained four plasmids including one mcr-1 harboring IncHI2 plasmid, one IncFII plasmid and two IncI1-I (Alpha) plasmids. Mobile elements carrying mcr-1 in the 75_plasmid and 107_plasmid-1 were located in the IS1086(ISApl1)-IS30A(ISApl1)-mcr-1-hp and IS1086(ISApl1)-mcr-1-hp regions, respectively. Tn6010 carrying drug efflux pump genes was found in 75_plasmid, while cn_31611_IS26 carrying multi-drug resistance (MDR) genes were found in 107_plasmid-1.Conclusion: This study found that mcr-1 was prevalent at a low frequency in the Quanzhou Women’s and Children’s Hospital. A similar genetic pattern of mcr-1 transmission was found in both E. coli and S. typhimurium.Keywords: mcr-1, Salmonella typhimurium, Escherichia coli, molecular characteristics

Abstract Background Antimicrobial-resistant bacteria and their antimicrobial resistance (AMR) genes can spread by hitchhiking in human guts. International travel can exacerbate this public health threat when travelers acquire AMR genes endemic to their destinations and bring them back to their home countries. Prior studies have demonstrated travel-related acquisition of specific opportunistic pathogens and AMR genes, but the extent and magnitude of travel’s effects on the gut resistome remain largely unknown. Methods Using whole metagenomic shotgun sequencing, functional metagenomics, and Dirichlet multinomial mixture models, we investigated the abundance, diversity, function, resistome architecture, and context of AMR genes in the fecal microbiomes of 190 Dutch individuals, before and after travel to diverse international locations. Results Travel markedly increased the abundance and α-diversity of AMR genes in the travelers’ gut resistome, and we determined that 56 unique AMR genes showed significant acquisition following international travel. These acquisition events were biased towards AMR genes with efflux, inactivation, and target replacement resistance mechanisms. Travel-induced shaping of the gut resistome had distinct correlations with geographical destination, so individuals returning to The Netherlands from the same destination country were more likely to have similar resistome features. Finally, we identified and detailed specific acquisition events of high-risk, mobile genetic element-associated AMR genes including qnr fluoroquinolone resistance genes, bla CTX-M family extended-spectrum β-lactamases, and the plasmid-borne mcr-1 colistin resistance gene. Conclusions Our results show that travel shapes the architecture of the human gut resistome and results in AMR gene acquisition against a variety of antimicrobial drug classes. These broad acquisitions highlight the putative risks that international travel poses to public health by gut resistome perturbation and the global spread of locally endemic AMR genes.

ABSTRACT: Objectives: The emergence and dissemination of colistin-resistant Enterobacterales has become a major global public-health threat. Here we investigated the genomic and phylogenetic characteristics of a multidrug-resistant Klebsiella pneumoniae strain (KP4823) carrying the mcr-1 gene recovered from a urinary tract infection in China. Methods: Antimicrobial susceptibility of K. pneumoniae KP4823 was determined by broth microdilution. Whole genomic DNA was extracted and sequenced using Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. Hybrid assembly with long and short reads was performed using Unicycler, and the genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The sequence type (ST), capsular type, and antimicrobial resistance and virulence genes were identified from the genome sequence. Core genome multilocus sequence typing (cgMLST) analysis was performed by BacWGSTdb 2.0 server. Results: Klebsiella pneumoniae KP4823 was resistant to colistin, ceftazidime, cefepime, cefotaxime, fosfomycin and aztreonam. The complete genome sequence of KP4823 consists of five contigs comprising 5 445 519 bp, including one chromosome and four plasmids. The isolate was assigned to ST101 with capsular serotype KL106. Several antimicrobial resistance genes were identified, including the colistin resistance gene mcr-1, which was located on a 34 685-bp IncX4 plasmid. The closest relative of K. pneumoniae KP4823 was another ST101 isolate (08EU827) recovered from Sweden in 2008, which differed by 191 cgMLST loci. Conclusion: Our study reports the genome sequence of a multidrug-resistant mcr-1-carrying K. pneumoniae in China. These data may help to understand the antimicrobial resistance mechanisms, genomic features and transmission dynamics of colistin resistance in clinical settings.

BackgroundMobile colistin resistance (mcr) genes modify Lipid A molecules of the lipopolysaccharide, changing the overall charge of the outer membrane.Results and discussionTen mcr genes have been described to date within eleven Enterobacteriaceae species, with Escherichia coli, Klebsiella pneumoniae, and Salmonella species being the most predominant. They are present worldwide in 72 countries, with animal specimens currently having the highest incidence, due to the use of colistin in poultry for promoting growth and treating intestinal infections. The wide dissemination of mcr from food animals to meat, manure, the environment, and wastewater samples has increased the risk of transmission to humans via foodborne and vector-borne routes. The stability and spread of mcr genes were mediated by mobile genetic elements such as the IncHI2 conjugative plasmid, which is associated with multiple mcr genes and other antibiotic resistance genes. The cost of acquiring mcr is reduced by compensatory adaptation mechanisms. MCR proteins are well conserved structurally and via enzymatic action. Thus, therapeutics found effective against MCR-1 should be tested against the remaining MCR proteins.ConclusionThe dissemination of mcr genes into the clinical setting, is threatening public health by limiting therapeutics options available. Combination therapies are a promising option for managing and treating colistin-resistant Enterobacteriaceae infections whilst reducing the toxic effects of colistin.

Salmonella enterica resistant to colistin, third-generation cephalosporins (3GCs), and fluoroquinolones (FQs) has been deemed a high-priority pathogen by the World Health Organization (WHO). The objective of this study was to characterize 11 mcr-1-harboring Salmonella enterica serovar Typhimurium isolates from raw pork and ready-to-eat (RTE) pork products in Guangzhou, China. All isolates were multi-drug resistant and contained 6–24 antibiotic-resistant genes. The mcr-1 gene was localized in the most conserved structure (mcr-1-orf ) in eight isolates and in mobile structure (ISApl1-mcr-1-orf ) in three isolates. One raw pork isolate SH16SF0850, co-harbored mcr-1, blaCTX−M−14, and oqxAB genes. One isolate 17Sal008 carried mcr-1, blaCTX−M−14, qnrS2, and oqxAB genes located on a 298,622 bp IncHI2 plasmid pSal008, which was obtained from an RTE pork product for the first time. The pSal008 was closely related to a plasmid in an S. typhimurium isolate from a 1-year-old diarrheal outpatient in China and was found to be transferable to Escherichia coli J53 by conjugation. Genome sequence comparisons by core-genome Multi Locus Sequence Typing (cgMLST) based on all S. typhimurium isolates from China inferred highly probably epidemiological links between selected pork isolates and no possible epidemiologically links between RTE pork isolate 17Sal008 and other isolates. Our findings indicate that raw pork and pork products are potential reservoirs of mcr-1-harboring S. typhimurium and highlight the necessity for continuous monitoring of colistin, 3GCs, and FQs resistant S. typhimurium from different origins.

ABSTRACT Colistin is one of the last-resort antibiotics for infections caused by multidrug-resistant Gram-negative bacteria. However, the wide spread of novel plasmid-carrying colistin resistance genes mcr-1 and its variants substantially compromise colistin's therapeutic effectiveness and pose a severe danger to public health. To detect colistin-resistant microorganisms induced by mcr genes, rapid and reliable antibiotic susceptibility testing (AST) is imminently needed. In this study, we identified an RNA-based AST (RBAST) to discriminate between colistin-susceptible and mcr-1-mediated colistin-resistant bacteria. After short-time colistin treatment, RBAST can detect differentially expressed RNA biomarkers in bacteria. Those candidate mRNA biomarkers were successfully verified within colistin exposure temporal shifts, concentration shifts, and other mcr-1 variants. Furthermore, a group of clinical strains were effectively distinguished by using the RBAST approach during the 3-h test duration with over 93% accuracy. Taken together, our findings imply that certain mRNA transcripts produced in response to colistin treatment might be useful indicators for the development of fast AST for mcr-positive bacteria. IMPORTANCE The emergence and prevalence of mcr-1 and its variants in humans, animals, and the environment pose a global public health threat. There is a pressing urgency to develop rapid and accurate methods to identify MCR-positive colistin-resistant bacteria in the clinical samples, providing a basis for subsequent effective antibiotic treatment. Using the specific mRNA signatures, we develop an RNA-based antibiotic susceptibility testing (RBAST) for effectively distinguishing colistin-susceptible and mcr-1-mediated colistin-resistant strains. Meanwhile, the detection efficiency of these RNA biomarkers was evidenced in other mcr variants-carrying strains. By comparing with the traditional AST method, the RBAST method was verified to successfully characterize a set of clinical isolates during 3 h assay time with over 93% accuracy. Our study provides a feasible method for the rapid detection of colistin-resistant strains in clinical practice.

Abstract Multidrug resistance is one of the worldwide public health concerns. Water represents the most suitable environment, for the exchange of antibiotic resistance genes among pathogenic to non-pathogenic bacteria. Therefore, we aimed to screen the presence of bla NDM-1, bla TEM, bla SHV, bla CTX-M and mcr1–5 genes among water samples from different locations of Lower Himachal Pradesh. We examined the genotypic incidences of bla NDM-1, bla TEM, bla SHV, bla CTXM and mcr1–5 by polymerase chain reaction. Survivability assay, fitness cost assay and biofilm assay were performed for phenotypic characterization. The presence of bla NDM-1 and its related variants were analysed and confirmed by sequencing-based approaches. A total of 73 bacterial strains were identified on M-lauryl sulphate agar medium. Out of 73 colistin-resistant isolates, 34 were E. coli and 39 were K. pneumoniae. Out of 34 samples, 2 (5.8%), 2 (5.8%), 5 (14.7%), 5 (14.7%) and 4 (11.76%) E. coli were bla TEM, bla SHV, bla CTXM-1, bla CTXM-2 and bla CTXM-15 positive, respectively. Among 39 K. pneumoniae, 15 (38.4%), 6 (15.3%), 10 (25.6%), 9 (23.07%) and 10 (25.6%) were bla TEM , bla SHV , bla CTXM-1 , bla CTXM-2 and bla CTXM-15 positive, respectively. Interestingly, we observed one E. coli (HG4) isolate with both bla NDM-1 and mcr-1 gene. Further analysis showed HG4 isolate has lesser survivability on the cotton swab, long lag phase and less biofilm production compared to colistin-sensitive isolates. Detection of E. coli with bla NDM-1 and mcr-1 in this geographical region is an alarming signal for tourists, community, health workers and policymakers. Hence, it is utmost important to take appropriate measures to control the dissemination of antibiotic resistance gene in such pristine locations.

Muhammad Fazal Hameed,1 Yanan Chen,1 Ying Wang,2 Muhammad Shafiq,3 Hazrat Bilal,1 Linqing Liu,4 Jinming Ma,1 Pengying Gu,4 Honghua Ge1 1Institutes of Physical Science and Information Technology, Anhui University, Hefei, Anhui, 230601, People’s Republic of China; 2Department of Laboratory Medicine, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui, 230036, People’s Republic of China; 3College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China; 4The Department of Geriatrics, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230036, People’s Republic of ChinaCorrespondence: Honghua GeInstitutes of Physical Science and Information Technology, Anhui University, Hefei, Anhui, 230601, People’s Republic of ChinaTel +86-551-63861773Email hhge@ahu.edu.cnPengying GuThe Department of Geriatrics, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230036, People’s Republic of ChinaEmail hellengpy@163.comPurpose: Antimicrobial resistance, especially carbapenem resistance Enterobacteriaceae and plasmid mediated mobile colistin resistance, is a serious issue worldwide. This study was designed to determine the epidemiological characteristics of plasmid mediated colistin resistance and carbapenem resistant Enterobacteriaceae from tertiary A hospital located in Hefei, China.Methods: Totally, 158 carbapenems resistant Enterobacteriaceae (CRE) were screened for antibiotic susceptibility, mcr-1, extended spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), and fosfomycin resistance genes using PCR and sequencing. The sequence types were identified by multilocus sequence typing (MLST). Plasmid profiles were determined by PCR based replicon typing (PBRT), and the plasmid sizes were confirmed by southern blotting.Results: The isolates showed high MIC50 and MIC90 for all antimicrobials, except tigecycline, meropenem, and colistin. The main Carbapenemase genes were blaKPC-2 (90.5%), blaNDM-1(3.7%), blaOXA-48(5.6%) and fosA3 (14.5%). The blaCTXM-15 found 36.7%, mcr-1 (3.7%) recorded in six isolates. PBRT revealed blaKPC-2 in K. pneumoniae on IncR, IncFII, and IncA/C. blaNDM-1 in E. coli on IncFII, whereas in E. cloacae noticed on IncHI2 plasmid. mcr-1 was recorded among IncFIIK, IncFII, and IncF in E. coli, K. pneumoniae, and E. cloacae. Resistance genes (mcr-1, blaNDM-1, blaKPC-2) harboring plasmids are successfully trans-conjugant to EC-600. A high incidence of ST11 was observed in K. pneumoniae carbapenem resistant isolates. While in E. coli, multiple STs were identified. However, mcr-1 in ST23 was identified for the first time in Anhui Province. Among Enterobacter cloacae, ST270 detected carrying blaNDM-1. Southern-hybridization confirmed the plasmid sizes 35– 150kb.Conclusion: This study indicates the co-carrying of mcr-1, blaKPC-2, and blaNDM-1 among clinical isolates, the prevalence of different Enterobacteriaceae STs is alarming, especially in E. coli. Holding such a resistance profile is a threat for humans and animals, which may be transferred between the strains through plasmid transfusion. Persistent control actions are immediately necessary to combat this hazard.Keywords: ESBL, MBL, KPC2, mcr-1, NDM-1, CTXM-15

Abstract Objectives The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is one of the major public health concerns as colistin is the last-resort antibiotic for treating infections caused by multidrug-resistant Gram-negative bacteria. We aimed to determine the prevalence of the prototype widespread colistin resistance genes (mcr-1 and mcr-2) among commensal and pathogenic Escherichia coli strains isolated from food-producing and companion animals in Iran. Results A total of 607 E. coli isolates which were previously collected from different animal sources between 2008 and 2016 used to uncover the possible presence of plasmid-mediated colistin resistance genes (mcr-1 and mcr-2) by PCR. Overall, our results could not confirm the presence of any mcr-1 or mcr-2 positive E. coli among the studied isolates. It is concluded that despite the important role of food-producing animals in transferring the antibiotic resistance, they were not the main source for carriage of mcr-1 and mcr-2 in Iran until 2016. This study suggests that the other mcr variants (mcr-3 to mcr-9) might be responsible for conferring colistin resistance in animal isolates in Iran. The possible linkage between pig farming industry and high level of mcr carriage in some countries needs to be clarified in future prospective studies.

Objectives: The resistance ofmcr-1-carrying Enterobacteriaceae to polymyxins, which are last-resort antibiotics, has raised great concern worldwide. In this study, four mcr-1-carrying plasmids isolated from Klebsiella pneumoniae clinical isolates were completely sequenced and the genome composition of the mcr-1-carrying plasmids was analysed. Methods: Antimicrobial resistance genes and antimicrobial susceptibility of fourmcr-1-carrying K. pneumoniae isolates were characterised. Comparison of mcr-1-carrying plasmids with closely related plasmids and analysis of the mcr-1 gene cassette were performed. Results: The genome composition of the fourmcr-1-carrying plasmids revealed the Tn6330 and Tn6390 cassettes embedded in two IncHI1-type plasmids, ΔTn6330 in an IncHI2-type plasmid, and mcr-1–pap2 in an IncX4-type plasmid. We also predicted the intermediate structures of the Tn6330 and Tn6390 cassettes. Conclusion: Dissemination of the colistin resistant genemcr-1 in Taiwan could have been driven by various plasmids and mobile gene cassettes. Evolution of the genetic environment has led to diversity in the mcr-1 gene among plasmids. This work sheds light on the urgent need for continued surveillance of the worldwide distribution of mcr-1 and evaluates the public-health risk of colistin resistance.

Avian pathogenic Escherichia coli (APEC) is responsible for significant economic losses in the poultry industry. This study aimed to molecularly detect carbapenem-resistant co-harboring mcr-1 avian pathogenic E. coli in broiler chickens infected with colibacillosis. A total of 750 samples were collected from colibacillosis-infected broilers, and conventional microbiological techniques were used to isolate and identify APEC. MALDI-TOF and virulence-associated genes (VAGs) were used for further identification. Phenotypic carbapenem resistance profiling was followed by molecular detection of carbapenem resistance genes (CRGs) and other resistance genes through PCR using specific primers. Isolates were also subjected to PCR for O typing, followed by allele-specific PCR to detect sequence type (ST) 95. Results showed that 154 (37%) isolates were confirmed as APEC, with 13 (8.4%) isolates found to be carbapenem-resistant (CR)-APEC. Among CR-APEC isolates, 5 (38%) were observed to co-harbor mcr-1. All CR-APEC showed the presence of five markers (ompT, hylF, iutA, iroN, and iss) APEC VAGs, and 89% of CR-APEC isolates displayed O78 type. Furthermore, 7 (54%) CR-APEC isolates were observed with ST95, all displaying O78 type. These results suggest that the improper use of antibiotics in poultry production systems is contributing to the emergence of pathogens such as CR-APEC co-harboring the mcr-1 gene.

We report the presence of the mcr-1 gene among 880 Escherichia coli clinical isolates collected in 13 hospitals from 12 Colombian cities between 2016 and 2019. Seven (0.8%) isolates were colistin resistant (MIC ≥ 4 µg/mL). These colistin-resistant isolates were screened for the presence of the mcr-1 gene; five carried the gene. These five isolates were subjected to whole genome sequencing (WGS) to identify additional resistomes and their ST. In addition, antimicrobial susceptibility testing revealed that all E. coli isolates carrying mcr-1 were susceptible to third generation-cephalosporin and carbapenems, except one, which carried an extended-spectrum β-lactamase (CTX-M-55), along with the fosfomycin resistance encoding gene, fosA. WGS indicated that these isolates belonged to four distinct sequence types (ST58, ST46, ST393, and a newly described ST14315) and to phylogroups B1, A, and D. In this geographic region, the spread of mcr-1 in E. coli is low and has not been inserted into high-risk clones such as ST131, which has been present in the country longer.

Simple Summary: This is a study related to NDM-5-producing E. coli in companion animals in China. Notably, an E. coli isolate possessing both blaNDM-5-bearing plasmid and mcr-1-bearing plasmid was identified in a dog from the same veterinary clinic, where we previously found the mobile IncX3–X4 hybrid plasmid encoding both blaNDM-5 and mcr-1 in a cat. This observation highlights the diversity of blaNDM-5- and mcr-1-harboring plasmids and the transferability of such resistant pathogens from companion animals to humans. Given that colistin is the last-resort antibiotic for treating human infections caused by carbapenem-resistant Enterobacteriaceae, the co-transfer of resistance to both antibiotics may seriously compromise the effectiveness of clinical therapy. Carbapenem and colistin are important antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. Here, we isolated the blaNDM-5-harboring Escherichia coli in companion animals in healthy or diseased companion animals from veterinary clinics in six cities in China from July to November 2016. A total of 129 rectal swabs of healthy or diseased dogs and cats were collected from veterinary clinics in six different cities in China, and the isolates were subjected to carbapenem and colistin susceptibility testing. Resistance genes were confirmed using PCR. Conjugation experiments were conducted to determine the transferability of antibiotic resistance genes (ARGs) in the strains. The isolated rate of blaNDM-5-harboring E. coli strains was 3.88% (five strains). These five strains were multidrug resistant to at least three antibiotics and corresponded to four sequence types including ST101. The blaNDM-5 gene was located on 46 kb IncX3 plasmids in these five strains, and the genetic contexts were shared and were nearly identical to the K. pneumoniae plasmid pNDM5-IncX3 from China. In addition, one strain (CQ6-1) co-harbored blaNDM-5-encoding-IncX3 plasmid along with a mcr-1-encoding-IncX4 plasmid, and their corresponding genetic environments were identical to the blaNDM-5-IncX3 and mcr-1-IncX4 hybrid plasmid reported previously from the same area and from the same clinic. The results indicated that the similar genetic contexts were shared between these isolates from companion animals, and the IncX3-type plasmids played a key role in the spread of blaNDM-5 among these bacteria. [ABSTRACT FROM AUTHOR]

Plasmid-borne colistin resistance mediated by mcr-1 is a growing problem, which poses a serious challenge to the clinical application of colistin for Gram-negative bacterial infections. Drug combination is one of the effective strategies to treat colistin-resistant bacteria. Here, we found a guanidine compound, namely, isopropoxy benzene guanidine (IBG), which boosted the efficacy of colistin against mcr-1 -positive Salmonella. This study aimed to develop a pharmacokinetics/pharmacodynamics (PK/PD) model by combining colistin with IBG against mcr-1 -positive Salmonella in an intestinal infection model. Antibiotic susceptibility testing, checkerboard assays and time-kill curves were used to investigate the antibacterial activity of the synergistic activity of the combination. PK studies of colistin in the intestine were determined through oral gavage of single dose of 2, 4, 8, and 16 mg/kg of body weight in broilers with intestinal infection. On the contrary, PD studies were conducted over 24 h based on a single dose ranging from 2 to 16 mg/kg. The inhibitory effect I max model was used for PK/PD modeling. The combination of colistin and IBG showed significant synergistic activity. The AUC0−24 h /MIC index was used to evaluate the relationship between PK and PD, and the correlation was >0.9085. The AUC0−24 h /MIC targets in combination required to achieve the bacteriostatic action, 3-log10 kill, and 4-log10 kill of bacterial counts were 47.55, 865.87, and 1894.39, respectively. These results can facilitate the evaluation of the use of IBG as a potential colistin adjuvant in the treatment of intestinal diseases in broilers caused by colistin-resistant Salmonella. [ABSTRACT FROM AUTHOR]

Research on resistance against polymyxins induced by the mcr-1 gene is gaining interest. In this study, using agar dilution method, polymerase chain reaction, and comparative genomic analysis, we investigated the colistin resistance mechanism of clinical E. coli isolates. The minimum inhibitory concentration (MIC) analysis results revealed that of the 515 isolates tested, bacteria with significantly increased MIC levels against colistin were isolated in 2019. Approximately one-fifth (17.14% to 19.65%) of the isolates showed MIC values ≥1 mg/L against colistin in 2015, 2016, and 2017. However, in 2019, up to three-quarters (74.11%, 146/197) of the isolates showed MIC values ≥1 mg/L against colistin indicating an increase in colistin resistance. Six isolates (EC7518, EC4968, EC3769, EC16, EC117, EC195, 1.13%, 6/515) were found to carry the mcr-1 gene and a novel mcr-1 variant with Met2Ile mutation was identified in EC3769. All six strains showed higher MIC levels (MIC=4 mg/L) than any mcr-1- negative strains (MIC ≤ 2 mg/L). Whole-genome sequencing of the six mcr-1 -positive isolates revealed that EC195 carried the highest number of resistance genes (n = 28), nearly a half more than those of the following EC117 (n = 19). Thus, EC195 showed a wider resistance spectrum and higher MIC levels against the antimicrobials tested than the other five isolates. Multi-locus sequence typing demonstrated that these mcr-1 -positive strains belonged to six different sequence types. The six mcr-1 genes were located in three different incompatibility group plasmids (IncI2, IncHI2 and IncX4). The genetic context of mcr-1 was related to a sequence derived from Tn 6330 (IS Apl1 - mcr-1 - pap2 -IS Apl1). Investigations into the colistin resistance mechanism and characterization of the molecular background of the mcr genes may help trace the development and spread of colistin resistance in clinical settings. [ABSTRACT FROM AUTHOR]

The emergence of the plasmid-mediated colistin resistance gene mcr-1 has resulted in the loss of available treatments for certain severe infections. Here we identified a potential inhibitor of MCR-1 for the treatment of infections caused by MCR-1-positive drug-resistant bacteria, especially MCR-1-positive carbapenem-resistant Enterobacteriaceae (CRE). A checkerboard minimum inhibitory concentration (MIC) test, a killing curve test, a growth curve test, bacterial live/dead assays, scanning electron microscope (SEM) analysis, cytotoxicity tests, molecular dynamics simulation analysis, and animal studies were used to confirm the in vivo/in vitro synergistic effects of pogostone and colistin. The results showed that pogostone could restore the bactericidal activity of colistin against all tested MCR-1-positive bacterial strains or MCR-1 mutant–positive bacterial strains (FIC < 0.5). Pogostone does not inhibit the expression of MCR-1. Rather, it inhibits the binding of MCR-1 to substrates by binding to amino acids in the active region of MCR-1, thus inhibiting the biological activity of MCR-1 and its mutants (such as MCR-3). An in vivo mouse systemic infection model, pogostone in combination with colistin resulted in 80.0% (the survival rates after monotherapy with colistin or pogostone alone were 33.3% and 40.0%) survival at 72 h after infection of MCR-1-positve Escherichia coli (E. coli) ZJ487 (blaNDM-1-carrying), and pogostone in combination with colistin led to one or more order of magnitude decreases in the bacterial burdens in the liver, spleen and kidney compared with pogostone or colistin alone. Our results confirm that pogostone is a potential inhibitor of MCR-1 for use in combination with polymyxin for the treatment of severe infections caused by MCR-1-positive Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is a quintessential One Health issue, among the most serious 21st century global threats in human and veterinary medicine. Wild animals are usually not directly exposed to clinically relevant antibiotics; however, antibacterial resistance in wild animals has been increasingly reported worldwide in parallel to the situation in human and veterinary medicine. In this work, we collected 100 fecal samples from the crested ibis protected areas. A total of eight Escherichia coli (E. coli) isolates positive for mcr-1 were obtained, and all of them were analyzed using WGS (whole genome sequencing). The WGS analysis showed that the isolates were assigned to four different sequence types (ST) overall. The antibiotic susceptibility profile showed that of eight E. coli isolates, six strains exhibited resistance to tetracycline and all mcr-1 -positive E. coli (MCREC) strains showed resistance to colistin. Our findings importantly document the epidemic spread of MCREC in crested ibis in China. [ABSTRACT FROM AUTHOR]

Colistin is one of the last-line drugs against difficult to treat and multidrug-resistant Gram-negative bacteria. The emergence of mobile colistin resistance gene m cr-1 increased worldwide attention on colistin resistance. mcr-1 is the dominant gene that caused resistance to colistin in chicken-derived Escherichia coli (E. coli) in China; it has a broad resistance spectrum and causes multiple drug resistance problems. There are only few studies on mcr -positive E. coli (MCRPEC) from laying ducks and quails in China. Here, the molecular and epidemiological characteristics of MCRPEC from three kinds of poultry farms (laying duck, quail, and broiler) were investigated in Changsha, China. A total of 17 mcr -positive E. coli (MCRPEC) strains were screened in 690 samples from the three kinds of poultry farms. This is the first report on MCRPEC, to our best knowledge, derived from quail. All the MCRPEC strains were resistant to colistin, sulfamethoxazole-trimethoprim, florfenicol, tetracycline, and ciprofloxacin, and mildly resistant to tigecycline, gentamicin, piperacillin/tazobactam, cefotaxime, and ceftiofur. All the strains were sensitive to meropenem and amikacin. By bioinformatics analysis, 17 MCRPEC strains belonging to 11 MLST types were distributed in phylogroups A (58.8%), B1 (23.5%), and phylogroup D (17.6%). mcr-1 was located in IncI2 plasmid with typical plasmid conjugation transfer part, type IV secretory system, and tellurium-resistant protein, increasing transmission capacity to other bacteria. Monitoring of colistin-resistant bacteria in poultry farms should be strengthened. [ABSTRACT FROM AUTHOR]

Global dissemination of mobilized colistin resistance (mcr)-1-carrying plasmids has been reported. This study aimed to investigate the global dissemination of these plasmids using whole genome sequencing to provide better understanding on genetic characteristics. Sixty-seven complete plasmid genomes harboring mcr-1 were obtained. Phylogeny was built against full plasmid genomes. Different replicon types of plasmid were compared in terms of antimicrobial resistance genes (ARGs), insertion sequence, and other functional genes. Five different replicon types of plasmid (IncX4, IncI2, IncP1, IncHIA, and IncFIB) were found to harbor mcr-1. IncX4 and IncI2 types of plasmid were well clustered in accordance with the country where they were isolated (and not as IncHIA and IncFIB). Three insertion sequences (ISApl1, ISKpn26, and IS1294) were identified in up- and/or downstream of mcr-1. Plasmids IncX4 and IncI2 were observed across the sample origin. Plasmids IncX4 showed high uniformity regardless of the origin of isolates and harbored H–NS coding genes, a facilitator for successful plasmid transfer. All three insertion sequences were observed in IncI2 plasmids. IncHI2 plasmids harbored various ARGs in addition to mcr-1. Our results elucidate the characteristics and phylogenetic relationships of complete mcr-1-harboring plasmids, indicating that global dissemination of mcr-1 is primarily owing to plasmid transfer rather than clonal spread. [ABSTRACT FROM AUTHOR]

Colistin is considered the last-line antimicrobial for the treatment of multidrugresistant gram-negative bacterial infections. The emergence and spread of superbugs carrying the mobile colistin resistance gene (mcr) have become the most serious and urgent threat to healthcare. Here, we discover that silver (Ag+), including silver nanoparticles, could restore colistin efficacy against mcr-positive bacteria. We show that Ag+ inhibits the activity of the MCR-1 enzyme via substitution of Zn2+ in the active site. Unexpectedly, a tetra-silver center was found in the active-site pocket of MCR-1 as revealed by the X-ray structure of the Ag-bound MCR-1, resulting in the prevention of substrate binding. Moreover, Ag+ effectively slows down the development of higherlevel resistance and reduces mutation frequency. Importantly, the combined use of Ag+ at a low concentration with colistin could relieve dermonecrotic lesions and reduce the bacterial load of mice infected with mcr-1-carrying pathogens. This study depicts a mechanism of Ag+ inhibition of MCR enzymes and demonstrates the potentials of Ag+ as broad-spectrum inhibitors for the treatment of mcr-positive bacterial infection in combination with colistin. [ABSTRACT FROM AUTHOR]

Background: Since 2015, plasmid-borne mcr-1 has been reported in various bacterial strains in the clinical setting globally. However, the transmission mechanisms of this gene in Salmonella are not well defined. This study aimed to characterize the genomic features of a Salmonella enterica ST34 isolate, which carried a mcr-1, mapped to a carbapenemase and extended spectrum β-lactamase encoding gene located on the IncX4 plasmid. Methods: Salmonella enterica was recovered from a diarrheal paediatric patient in Shenzhen, China. Antimicrobial susceptibility testing was performed by using the VITEK 2 system. Drug resistance genes were identified using targeted primers and Sanger sequencing. The transferability and genome location of mcr-1 was determined by performing conjugation, S1-PFGE and Southern blot hybridization analysis. WGS was performed by Illumina MiSeq sequencing and was assembled using the A5-Miseq pipeline, and gene annotation was performed using RAST 2.0. The database Centre for Genomic Epidemiology's website was used to identify resistance genes and sequence types (STs). Results: We found that the isolate was extensively drug resistant and belonging to ST34, carrying an IncX4 plasmid with mcr-1, blaKPC-2 and blaCTX-M-15. We also noticed that genes blaPAO, fosA, catB, the mutation in oprD and mexT (MexEF-OprN efflux regulator), and exotoxin-encoding genes (exoS, exoY and exoT) were associated with resistance and virulence in the genome. In addition, heavy metal resistance genes as silP and silE were determined. Conclusion: This study highlights the potential risk of ST34 of Salmonella enterica serotype Typhimurium carrying multiple drug resistance encoding genes in a single IncX4 plasmid. [ABSTRACT FROM AUTHOR]

In response to the spread of colistin resistance gene mcr-1, China banned the use of colistin in livestock fodders. We used a time-series analysis of inpatient colonization data from 2011–2019 to accurately reveal the associated fluctuations of mcr-1 that occurred in inpatients in response to the ban.

Background: Antimicrobial resistance of Salmonella enterica is a major global concern. Recent findings suggest that colistin as a last resort treatment for multidrug-resistant gram-negative bacteria is seriously threatened by the report of plasmid-mediated colistin resistance gene mcr-1 in China. Methods: A total of 827 S. Typhimurium isolates were recovered from 4 cities of China, including Henan, Shanghai, Zhejiang, and Hubei provinces. Subsequently, mcr-1 presence was identified by PCR screening. Antimicrobial susceptibility testing was performed by broth microdilution using a 96-well microtiter plate. Plasmid conjugation transfer experiments were conducted using Escherichia coli J53 as the recipient. Results: Only one mcr-1 positive strain from the stool sample of an infant with acute diarrhea was isolated. Apart from colistin, the mcr-1-positive isolate showed co-resistance to the third-generation cephalosporins, ampicillin, nalidixic acid, tetracycline, chloramphenicol, sulfisoxazole, gentamicin, and cefotaxime revealing a multidrug-resistant phenotype. This strain harbored mcr-1 on a 227 kb IncHI2 plasmid, termed pJZ26, which could be transferred to E. coli J53. In addition to mcr-1, pJZ26 coharbored other resistance genes, including aph(4)-Ia, aac(3)-IVa, fosA, floR, sul2, and blaCTX-M-14. Compared with p2474-MCR1 and pHYEC7-IncHI2, pJZ26 contains an additional 4.6 kb fragment harboring the resistance gene tet(A) and its regulator tetR located on TnAs1 transposable element, which could mediate resistance to tetracycline. Conclusions: These findings highlight that the fact the mcr-1–harboring plasmid pJZ26 has a high potential to disseminate the mcr-1 gene and further challenge the clinical treatment.

Mobile colistin resistance gene mcr-1 and extended-spectrum β-lactamase gene blaCTX-M are highly prevalent in human – and pet-derived bacteria. Isolation of identical strains of mcr-1-positive Escherichia coli (MCRPEC) or blaCTX-M-positive E. coli (CTX-MPEC) from pets and humans highlighted the potential for co-colonization of antibiotic-resistant bacteria which can be a risk for dissemination of resistance genes. In this study, the prevalence of mcr-1 and blaCTX-M carriage from rectal swabs in 299 families (dogs and their owners) were 2.7 and 5.3%, respectively. We identified a significant association of mcr-1 carriage between dogs and their owners. Whilst antibiotic use in the previous three months was associated with blaCTX-M carriage in dogs. Only one instance of dog and owner carrying identical CTX-MPEC was observed. Although the prevalence of identical strains in one family is rare, the huge number of dog ownership worldwide suggest that this threat should not be underestimated.

Objectives: The aim of this study was to explore the genomic content of a blaKPC-2- and mcr-1-harbouring Escherichia coli strain and to clarify the molecular mechanism for the transmission of multidrug resistance. Methods: Antimicrobial susceptibility testing was conducted by the broth microdilution method to determine the resistance profile. Filter-mating conjugation assays were performed to confirm the plasmid transfer ability. Whole-genome sequence data were acquired by a combination of Illumina paired-end reads and Nanopore long-read sequencing. Results: Escherichia coli strain QE11−421 was an mcr-1-positive colistin-resistant isolate that co-harboured the blaKPC-2 gene conferring carbapenem resistance. Genome sequence data confirmed QE11−421 as a sequence type 48 (ST48) E. coli that harboured five large conjugative plasmids encoding several multidrug resistance genes. The blaKPC-2 gene was located on a Tn3–Tn4401 composite transposon, which is part of a 65-kb multidrug-resistant IncN plasmid. The mcr-1 gene was harboured on another 33-kb IncX4 plasmid that was more conserved and presented few antimicrobial resistance determinants. No copies of insertion sequence ISApl1 were found flanking the mcr-1 gene, decreasing the mobility of mcr-1 based on its original Tn6330 transposon. Conclusions: Horizontal transfer of multidrug resistance plasmids or resistance-related transposon units was responsible for the emergence of this notorious superbug. The coexistence of blaKPC-2–IncN and mcr-1–IncX4 plasmids in a ST48 E. coli strain in humans poses a great threat.

Novaria Sari Dewi Panjaitan,* Christina Safira Whinie Lestari* Center for Biomedical Research, Research Organization for Health, National Research and Innovation Agency (BRIN), Cibinong Science Center, Cibinong–Bogor, West Java, Indonesia*These authors contributed equally to this workCorrespondence: Novaria Sari Dewi Panjaitan, Center for Biomedical Research, Research Organization for Health, National Research and Innovation Agency (BRIN), Genomic Building, Cibinong Science Center, Jl. Raya Bogor No. 490, Km. 46, Cibinong–Bogor, West Java, Indonesia, Email nova014@brin.go.id

Research on resistance against polymyxins induced by the mcr-1 gene is gaining interest. In this study, using agar dilution method, polymerase chain reaction, and comparative genomic analysis, we investigated the colistin resistance mechanism of clinical E. coli isolates. The minimum inhibitory concentration (MIC) analysis results revealed that of the 515 isolates tested, bacteria with significantly increased MIC levels against colistin were isolated in 2019. Approximately one-fifth (17.14% to 19.65%) of the isolates showed MIC values ≥1 mg/L against colistin in 2015, 2016, and 2017. However, in 2019, up to three-quarters (74.11%, 146/197) of the isolates showed MIC values ≥1 mg/L against colistin indicating an increase in colistin resistance. Six isolates (EC7518, EC4968, EC3769, EC16, EC117, EC195, 1.13%, 6/515) were found to carry the mcr-1 gene and a novel mcr-1 variant with Met2Ile mutation was identified in EC3769. All six strains showed higher MIC levels (MIC=4 mg/L) than any mcr-1-negative strains (MIC ≤ 2 mg/L). Whole-genome sequencing of the six mcr-1-positive isolates revealed that EC195 carried the highest number of resistance genes (n = 28), nearly a half more than those of the following EC117 (n = 19). Thus, EC195 showed a wider resistance spectrum and higher MIC levels against the antimicrobials tested than the other five isolates. Multi-locus sequence typing demonstrated that these mcr-1-positive strains belonged to six different sequence types. The six mcr-1 genes were located in three different incompatibility group plasmids (IncI2, IncHI2 and IncX4). The genetic context of mcr-1 was related to a sequence derived from Tn6330 (ISApl1-mcr-1-pap2-ISApl1). Investigations into the colistin resistance mechanism and characterization of the molecular background of the mcr genes may help trace the development and spread of colistin resistance in clinical settings.

Plasmid-borne colistin resistance mediated by mcr-1 is a growing problem, which poses a serious challenge to the clinical application of colistin for Gram-negative bacterial infections. Drug combination is one of the effective strategies to treat colistin-resistant bacteria. Here, we found a guanidine compound, namely, isopropoxy benzene guanidine (IBG), which boosted the efficacy of colistin against mcr-1-positive Salmonella. This study aimed to develop a pharmacokinetics/pharmacodynamics (PK/PD) model by combining colistin with IBG against mcr-1-positive Salmonella in an intestinal infection model. Antibiotic susceptibility testing, checkerboard assays and time-kill curves were used to investigate the antibacterial activity of the synergistic activity of the combination. PK studies of colistin in the intestine were determined through oral gavage of single dose of 2, 4, 8, and 16 mg/kg of body weight in broilers with intestinal infection. On the contrary, PD studies were conducted over 24 h based on a single dose ranging from 2 to 16 mg/kg. The inhibitory effect Imax model was used for PK/PD modeling. The combination of colistin and IBG showed significant synergistic activity. The AUC0−24h/MIC index was used to evaluate the relationship between PK and PD, and the correlation was >0.9085. The AUC0−24h /MIC targets in combination required to achieve the bacteriostatic action, 3-log10 kill, and 4-log10 kill of bacterial counts were 47.55, 865.87, and 1894.39, respectively. These results can facilitate the evaluation of the use of IBG as a potential colistin adjuvant in the treatment of intestinal diseases in broilers caused by colistin-resistant Salmonella.

ABSTRACT Emergence of pathogens harboring multiple resistance genes incurs great concerns. Cooccurrence of mobile resistance genes conferring resistance to tigecycline, colistin, and carbapenems in Escherichia coli has not been investigated. This study aimed to characterize three E. coli isolates coharboring tet(X4), mcr-1, and blaNDM-5. Isolates coharboring tet(X4), mcr-1, and blaNDM-5 were identified and characterized by PCR, Sanger sequencing, antimicrobial susceptibility testing, conjugation assays, Illumina sequencing, nanopore sequencing, and bioinformatic analysis. Three E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 were identified from pigeons in China. They were resistant to almost all antimicrobials except enrofloxacin. tet(X4) and blaNDM-5 could be conjugated into E. coli C600, but mcr-1 was nontransferable in three isolates. Three isolates belonged to sequence type 6775 (ST6775), and clonal dissemination of isolates carrying tet(X4), mcr-1, and blaNDM-5 existed in the pigeon farm. Genetic analysis revealed that mcr-1 mediated by the Tn6330 was located on the chromosome, tet(X4) was located on the IncFII plasmid, and blaNDM-5 was located on the IncX3 plasmid. We first characterized the E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 simultaneously. Relevant measures should be taken to decrease the prevalence of pathogens carrying tet(X4), mcr-1, and blaNDM-5. IMPORTANCE Tigecycline and colistin are regarded as vital antimicrobials to treat multidrug-resistant (MDR) bacterial infections, such as that caused by carbapenemase-producing Enterobacteriaceae (CPE). Cooccurrence of mobile resistance genes conferring resistance to last-resort antimicrobials in E. coli remains unknown. Here, we characterized E. coli strains coharboring tet(X4), mcr-1, and blaNDM-5 phenotypically and genetically. Resistance genes tet(X4), mcr-1, and blaNDM-5 were located on transposons or plasmids that were mobile genetic elements related to the capture, accumulation, and dissemination of such important resistance genes. The emergence of E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 highlights the importance of monitoring the coexistence of novel mobile resistance genes in different settings with a One Health approach. Risk of transmission of such MDR pathogens from animals to humans should be evaluated comprehensively.

Colistin is one of the last-line drugs against difficult to treat and multidrug-resistant Gram-negative bacteria. The emergence of mobile colistin resistance gene mcr-1 increased worldwide attention on colistin resistance. mcr-1 is the dominant gene that caused resistance to colistin in chicken-derived Escherichia coli (E. coli) in China; it has a broad resistance spectrum and causes multiple drug resistance problems. There are only few studies on mcr-positive E. coli (MCRPEC) from laying ducks and quails in China. Here, the molecular and epidemiological characteristics of MCRPEC from three kinds of poultry farms (laying duck, quail, and broiler) were investigated in Changsha, China. A total of 17 mcr-positive E. coli (MCRPEC) strains were screened in 690 samples from the three kinds of poultry farms. This is the first report on MCRPEC, to our best knowledge, derived from quail. All the MCRPEC strains were resistant to colistin, sulfamethoxazole-trimethoprim, florfenicol, tetracycline, and ciprofloxacin, and mildly resistant to tigecycline, gentamicin, piperacillin/tazobactam, cefotaxime, and ceftiofur. All the strains were sensitive to meropenem and amikacin. By bioinformatics analysis, 17 MCRPEC strains belonging to 11 MLST types were distributed in phylogroups A (58.8%), B1 (23.5%), and phylogroup D (17.6%). mcr-1 was located in IncI2 plasmid with typical plasmid conjugation transfer part, type IV secretory system, and tellurium-resistant protein, increasing transmission capacity to other bacteria. Monitoring of colistin-resistant bacteria in poultry farms should be strengthened.

With the increasingly severe problem of bacterial resistance, colistin, as the last line of defense, has attracted attention again. Mobile colistin resistance (mcr-1) gene is involved in the horizontal transmission of colistin resistance in Gram-negative bacteria (GNB), which is a serious threat to human health. Therefore, rapid detection of mcr-1 gene presence in clinical samples is crucial. In this study, a Recombinase-aided amplification(RAA) method for mcr-1 was successfully constructed, with sensitivity of 20 copies/reaction. In addition, amplification signal could only be detected in the strain containing mcr-1 gene among 14 different bacterial species. The method was then used to test a total of 672 clinical samples from a pediatric hospital in Beijing. Five strains harbored mcr-1 genes were isolated from mcr-1-positive clinical samples and identified as Escherichia coli. Multi-locus sequence typing (MLST) analysis showed that the five E. coli belonged to different ST types. Notably, the mcr-1 gene from the isolates could be transferred conjugately to the recipient strain E. coli J53, with highest transfer efficiency up to 57–58%, suggesting that the mcr-1 gene was located on the plasmid. These findings showed that the RAA assay has potential to be a rapid and sensitive mcr-1 gene screening test for clinical samples, and mcr-1 could be transmitted vertically and horizontally between and within bacterial species in a plasmid-mediated manner.

Colistin resistance is a global health concern, with antibiotics being the last treatment for Gram-negative bacteria infections. We aimed to identify colistin-resistant enterobacteria on environmental surfaces of a long-term care facility (LTCF) for the elderly in southern Brazil. Samples were collected and screened on MacConkey agar plus colistin, followed by API20E identification and PCR. Two isolates were founded and identified as Klebsiella pneumoniae and Providencia stuartii harboring mcr-1 gene with MICs > 128 µg mL -1 for colistin. This is the first isolation of microorganisms resistant to colistin in the environment of a LTCF for the elderly in south Brazil, urging monitoring programs to reduce environmental contamination by multiresistant microorganisms.

The colistin resistance gene mcr-1 is emerging as a global public health concern, altering the regulation of colistin usage globally since 2017, especially in China. However, few studies have revealed the impact of policy change on the epidemiology of mcr -positive Enterobacteriaceae (MCRPE) in patients. Here, we describe a molecular epidemiological study to investigate the MCRPE in patients in China from 2009–2019. During the surveillance period, 26,080 non-duplicated Enterobacteriaceae isolates were collected in Beijing. Colistin-resistant isolates were screened by enrichment culture supplemented with colistin, and the presence of the mcr gene was determined by PCR amplification. MCRPE isolates were then analyzed by susceptibility testing, genotyping, and risk factor analysis. Of the 26,080 isolates, mcr-1 was detected in 171 (1.1%) of 15,742 Escherichia coli isolates and 7 (0.1%) of 10,338 Klebsiella pneumoniae isolates. The prevalence of mcr-1 -positive E. coli (MCRPEC) showed an increasing trend from 2009 to 2016, while a decreasing trend was observed since 2017. Multi-locus sequence typing analysis showed that MCRPEC isolates had extremely diverse genetic backgrounds, and most of these isolates were non-clonal. The prevalence of MCRPE in China remained at a low level, and even showed a declining trend over the last 3 years after the banning of colistin usage as feed additive in food animal in 2017. However, colistin permission in clinical therapy could still increase the risk of MCRPE transmission and intractable infections, active surveillance and monitoring strategies of MCRPE are recommended to prolong the clinical longevity of colistin. [ABSTRACT FROM AUTHOR]

Although the spread of plasmid-mediated antibiotic-resistant bacteria is a public health concern, food contamination with plasmid-mediated antibiotic-resistant Escherichia coli in Vietnam has not been well investigated. This study aimed to describe the prevalence of colistin-resistant, carbapenem-resistant, and endemic blaCTX-M in extended-spectrum β-lactamase (ESBL) producing E. coli isolates. Colistin and carbapenem-resistant ESBL-producing E. coli were isolated from chickens in Vietnam and Japan. Colistin-resistant and AmpC/ESBL-producing E. coli (52% and 93%, respectively) were detected in chickens from Vietnam, in comparison to 52.7%, AmpC/ESBL-producing E. coli found in chicken from Japan. Carbapenem-resistant E. coli has not been isolated in Vietnam and Japan. Genotyping revealed that colistin-resistant E. coli harboured mcr-1, and most of the AmpC/ESBL-related genes were blaCTX-M-55 and blaCTX-M-65 together with blaTEM in Vietnamese chickens and blaCMY-2 in Japanese chickens. Multi-drug resistance analysis showed that ESBL-producing E. coli isolates had greater resistance to quinolones, streptomycin, and chloramphenicol than colistin-resistant E. coli isolates from Vietnam, suggesting the selection of multiple antibiotic resistance genes in ESBL-producing E. coli. In conclusion, colistin-resistant E. coli was detected in approximately half of the chicken samples, the majority of which harboured mcr-1. The high prevalence of ESBL-producing E. coli has remained constant in the last 5 years. The predominant blaCTX-M in ESBL-producing E. coli was blaCTX-M-55 or blaCTX-M-65, with the coexistence of blaTEM in Vietnam. These results can be implemented in monitoring systems to overcome the development of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

This systematic review focuses on obtaining the most relevant information from multiple studies that detected a mobilized colistin resistance mcr gene in Salmonella for a better comprehension of its global distribution. A group of strategic and systematic keywords were combined to retrieve research data on the detection frequency of the mcr gene globally from four database platforms (Google Scholar, Science Direct, PubMed and Scielo). Forty‐eight studies attended all the eligibility criteria and were selected. China was the country with the highest frequency of Salmonella strains with the mcr gene, and Europe exhibited a wide diversity of countries with positive mcr strains. In addition, animals and humans carried the highest frequency of positive strains for the mcr gene. Salmonella Typhimurium was the most frequent serovar carrying the mcr gene. Apparently, colistin overuse in animal husbandry has increased the selective pressure of antimicrobial resistance, resulting in the emergence of a plasmid‐mediated colistin resistance mcr gene in China. The mcr‐positive Salmonella strains are recently predominant worldwide, which is probably due to the capacity of this gene to be swiftly horizontally transmissible. The transmission ability of mcr‐positive Salmonella strains to humans through the consumption of contaminated animal‐based food is a public health concern. [ABSTRACT FROM AUTHOR]

The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other β-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6′)-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies. [ABSTRACT FROM AUTHOR]

China banned the use of colistin as animal growth promoter in April 2017. Herein, we report the prevalence of mcr-1 in the intestine of healthy humans and risk factors associated with mcr-1 carriage after the implementation of the ban. We recruited 719 healthy volunteers from Shenzhen City from 1 March 2018 to 31 December 2019 to investigate the prevalence of mcr-1 in human intestine, and undertook a case–control study to ascertain the risk factors associated with the mcr-1 -positive population. A further comparative study was conducted to identify differences between genetic characteristics of mcr-1 -positive and mcr-1 -negative Escherichia coli. Overall, 56 (7.8%, 95% CI 5.9%–10.0%, n = 719) individual faecal samples were positive for mcr-1 , and prevalence of mcr-1 among individuals in 2019 (2.4%, 95% CI 8.7%–15.0%, 7/294) was significantly lower than that in 2018 (11.5%, 95% CI 1.0%–4.8%, 49/425) (p < 0.0001). After the colistin ban, animal-derived food (pork and chicken meat) was no longer a risk factor for mcr-1 carriage in human intestine, whereas a higher intake of fish and seafood (>75 g/day) and whole grains (>150 g/day) was associated with higher and lower risk of mcr-1 carriage, respectively (OR 2.175, 95% CI 1.047–4.517; OR 0.045, 95% CI 0.004–0.567). Compared with mcr-1 -negative E. coli , the mcr-1 -positive E. coli had different patterns of resistance genes and genetic heterogeneity. Our study implicates aquatic food as beeing associated with mcr-1 carriage in the healthy population, even after the ban on colistin. Dietary modification (e.g. whole grains) may help to combat mcr-1 -positive bacterial colonization of the gut. [ABSTRACT FROM AUTHOR]

Background: Mobile colistin resistance like gene (mcr-like gene) is a new type of polymyxin resistance gene that can be horizontally transferred in the Enterobacteriaceae. This has brought great challenges to the treatment of multidrug-resistant Escherichia coli and K. pneumoniae. Results: K. pneumoniae 16BU137 and E. coli 17MR471 were isolated from the bus and subway handrails in Guangzhou, China. K. pneumoniae 19PDR22 and KP20191015 were isolated from patients with urinary tract infection and severe pneumonia in Anhui, China. Sequence analysis indicated that the mcr-1.1 gene was present on the chromosome of E. coli 17MR471, and the gene was in the gene cassette containing pap2 and two copies of ISApl1.The mcr-1.1 was found in the putative IncX4 type plasmid p16BU137_mcr-1.1 of K. pneumoniae 16BU137, but ISApl1 was not found in its flanking sequence. Mcr-8 variants were found in the putative IncFIB/ IncFII plasmid pKP20191015_mcr-8 of K. pneumoniae KP20191015 and flanked by ISEcl1 and ISKpn26. Conclusion: This study provides timely information on Enterobacteriaceae bacteria carrying mcr-like genes, and provides a reference for studying the spread of mcr-1 in China and globally. [ABSTRACT FROM AUTHOR]

Colistin is a last-resort antibiotic in fighting severe infections caused by multidrug resistant Gram negative pathogens in hospitals. Zoonotic bacteria acquire colistin resistance in animal reservoirs and mediate its spread along the food chain. This is the case of non-typhoid serovars of Salmonella enterica. Colistin-resistant S. enterica in foods represents a threat to human health. Here, we assessed the prevalence of colistin-resistance in food-borne isolates of S. enterica (2014–2019; Asturias, Spain), and established the genetic basis and transferability of this resistance. Five out of 231 isolates tested (2.2%) were resistant to colistin. Four of them, belonging to the European monophasic ST34 clone of S. Typhimurium, were characterized in the present study. They were collected from pork or pork and beef meat-derived products, either in 2015 (three isolates) or 2019 (one isolate). Molecular typing with XbaI-PFGE and plasmid profiling revealed distinct patterns for each isolate, even though two of the 2015 isolates derived from the same sample. The MICs of colistin ranged from 8 to 16 mg/L. All isolates carried the mcr-1.1 gene located on conjugative plasmids of the incompatibility groups IncX4 (2015 isolates) or IncHI2 (2019 isolate). Apart from colistin resistance, the four isolates carried chromosomal genes conferring resistance to ampicillin, streptomycin, sulfonamides and tetracycline [ bla TEM–1, strA-strB , sul2 , and tet (B)] and heavy metals, including copper and silver (silESRCFBAGP and pcoGE1ABCDRSE2), arsenic (arsRSD2A2BCA1D1) ± mercury (merEDACPTR), which are characteristically associated with the European ST34 monophasic clone. The 2019 isolate was also resistant to other antibiotics, comprising third generation cephalosporins and cephamycins. The latter phenotype was conferred by the bla CMY–2 gene located on an IncI1-I(α)-ST2 plasmid. Results in the present study identified meat-derived products as a reservoir of a highly successful clone harboring transferable plasmids which confer resistance to colistin and other clinically important antibiotics. An important reduction in the number of food-borne S. enterica detected during the period of the study, together with the low frequency of colistin resistance, underlines the success of One Health initiatives, such as those implemented at the UE, to control zoonotic bacteria along the food chain and to halt the spread of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Escherichia fergusonii , an opportunistic zoonotic pathogen, has a greatly increased importance in public health with the discovery of cephalosporin- and colistin-resistant strains. The IncHI2 plasmid carrying mcr-1 has been reported in E. fergusonii , but the other types of plasmids carrying mcr-1 have never been reported. In this study, 268 samples of cecal contents or anal swabs were collected from slaughterhouses and farms in two cities in Zhejiang, China, where 54 E. fergusonii strains (18.88%) were isolated between 2020 and 2021. To our knowledge, this is the first report of the isolation of E. fergusonii in meat ducks (slaughterhouse). The minimum inhibitory concentration (MIC) of isolates was determined by the broth microdilution method, in which it was determined that the tetracycline resistance rate was the highest (83.33%) and the multidrug-resistance (MDR) rate was 75.93%. A total of four strains of colistin-resistant E. fergusonii were found and identified as mcr-1 -positive by PCR. Importantly, these strains could transfer the mcr-1 gene to strain E. coli J53 by conjugation. Genome sequencing revealed that the mcr-1 genes of the above four strains were all located on the Incl2 plasmid, and the mobile element IS Apl1 upstream of mcr-1 was missing. Moreover, the plasmid pEF45-4 (61 140 bp) harboring mcr-1 in strain EF20JDJ4045 was revealed by Oxford Nanopore Technology, showing high homology with the previously reported in E. coli. Taken together, the high antimicrobial resistance (AMR) rate of E. fergusonii may herald a novel reservoir of AMR genes and IncI2 plasmid may be an important factor affecting mcr-1 transfer in poultry. [ABSTRACT FROM AUTHOR]

The rise and global dissemination of extensively drug-resistant (XDR) bacteria are often related to plasmid-borne mobile antimicrobial resistance genes. Notably, isolates having multiple plasmids are often highly resistant to almost all the antibiotics available. In this study, we characterized an extensively drug-resistant Klebsiella pneumoniae 1678, which exhibited high-level resistance to almost all the available antibiotics. Through whole-genome sequencing (WGS), more than 20 resistant elements and 5 resistant plasmids were observed. Notably, the tigecycline resistance of K. pneumoniae 1678 was not related to the plasmid-borne tetA gene but associated with the overexpression of AcrAB and OqxAB efflux pumps, according to the susceptibility results of tetA -transformant and the related mRNA quantification of RND efflux pumps. Except for tigecycline resistance, three plasmids, mediating resistance to colistin, Fosfomycin, and ceftazidime–avibactam, respectively, were focused. Detailed comparative genetic analysis showed that all these plasmids belonged to dominated epidemic plasmids, and harbored completed conjugation systems. Results of conjugation assay indicated that these three plasmids not only could transfer to E. coli J53 with high conjugation frequencies, respectively, but also could co-transfer to E. coli J53 effectively, which was additionally confirmed by the S1-PFGE plasmids profile. Moreover, multiple insertion sequences (IS) and transposons (Tn) were also found surrounding the vital resistant genes, which may form several novel mechanisms involved in the resistant determinants' mobilization. Overall, we characterized and reported the uncommon co-existence and co-transferring of FosA3-, NDM-5, and MCR-1-encoding plasmids in a K. pneumoniae isolate, which may increase the risk of spread of these resistant phenotypes and needing great concern. [ABSTRACT FROM AUTHOR]

Abstract The emergence and transmission of the mobile colistin resistance gene (mcr-1) threatened the extensive use of polymyxin antimicrobials. Accumulated evidence showed that the banning of colistin additive in livestock feed efficiently reduce mcr-1 prevalence, not only in animals but also in humans and environments. However, our previous study has revealed that a small proportion of Escherichia coli could continually carry chromosomally-encoded mcr-1. The chromosomally-encoded events, indicated the existence of stabilized heritage of mcr-1 and revealed a potential threat in the antimicrobial stewardship interventions, are yet to be investigated. In this study, we systematically investigated the genetic basis of chromosomally-encoded mcr-1 in prevalence and potential mechanisms of lineage, plasmid, insertion sequence, and phage. Our results demonstrated that the emergence of chromosomally-encoded mcr-1 could originate from multiple mechanisms, but mainly derived through the recombination of ISApl1/Tn6330. We reported a specific transmission mechanism, which is a phage-like region without lysogenic components, could associate with the emergence and stabilization of chromosomally-encoded mcr-1. These results highlighted the potential origin and risks of chromosomally-encoded mcr-1, which could be a heritable repository and thrive again when confronted with new selective pressures. To the best of our knowledge, this is the first study to systematically reveal the genomic basis of chromosomally-encoded mcr-1, and report a specific transmission pattern involved in phage-like region. Overall, we demonstrate the origin mechanisms and risks of chromosomally-encoded mcr-1. It highlights the need of public attention on chromosome-encoded mcr-1 to prevent from its reemergence.