Jiyun Li

Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production

By 2030, the global population will be 8.5 billion, placing pressure on international poultry production, of which China is a key producer1. From April 2017, China will implement the withdrawal of colistin as a growth promoter, removing over 8,000 tonnes per year from the Chinese farming sector2. To understand the impact of banning colistin and the epidemiology of multi-drug-resistant (MDR) Escherichia coli (using blaNDM and mcr-1 as marker genes), we sampled poultry, dogs, sewage, wild birds and flies. Here, we show that mcr-1, but not blaNDM, is prevalent in hatcheries, but blaNDM quickly contaminates flocks through dogs, flies and wild birds. We also screened samples directly for resistance genes to understand the true breadth and depth of the environmental and animal resistome. Direct sample testing for blaNDM and mcr-1 in hatcheries, commercial farms, a slaughterhouse and supermarkets revealed considerably higher levels of positive samples than the blaNDM- and mcr-1-positive E. coli, indicating a substantial segment of unseen resistome—a phenomenon we have termed the ‘phantom resistome’. Whole-genome sequencing identified common blaNDM-positive E. coli shared among farms, flies, dogs and farmers, providing direct evidence of carbapenem-resistant E. coli transmission and environmental contamination.

Plasmid-Mediated Novel blaNDM-17 Gene Encoding a Carbapenemase with Enhanced Activity in a Sequence Type 48 Escherichia coli Strain

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) have spread worldwide, leaving very few treatment options available. New Delhi metallo-beta-lactamase (NDM) is the main carbapenemase mediating CRE resistance and is of increasing concern. NDM-positive Enterobacteriaceae of human origin are frequently identified; however, the emergence of NDM, and particularly novel variants, in bacteria of food animal origin has never been reported. Here, we characterize a novel NDM variant (assigned NDM-17) identified in a β-lactam-resistant sequence type 48 (ST48) Escherichia coli strain that was isolated from a chicken in China. Compared to NDM-1, NDM-17 had three amino acid substitutions (V88L, M154L, and E170K) that confer significantly enhanced carbapenemase activity. Compared to NDM-5, NDM-17 had only one amino acid substitution (E170K) and slightly increased isolate resistance to carbapenem, as indicated by increased MIC values. The gene encoding NDM-17 (blaNDM-17) was located on an IncX3 plasmid, which was readily transferrable to recipient E. coli strain J53 by conjugation, suggesting the possibility of the rapid dissemination of blaNDM-17. Enzyme kinetics showed that NDM-17 could hydrolyze all β-lactams tested, except for aztreonam, and had a significantly higher affinity for all β-lactams tested than did NDM-5. The emergence of this novel NDM variant could pose a threat to public health because of its transferability and enhanced carbapenemase activity.

Emergence of a novel mobile colistin resistance gene, mcr-8 , in NDM-producing Klebsiella pneumoniae

The rapid increase in carbapenem resistance among gram-negative bacteria has renewed focus on the importance of polymyxin antibiotics (colistin or polymyxin E). However, the recent emergence of plasmid-mediated colistin resistance determinants (mcr-1, -2, -3, -4, -5, -6, and -7), especially mcr-1, in carbapenem-resistant Enterobacteriaceae is a serious threat to global health. Here, we characterized a novel mobile colistin resistance gene, mcr-8, located on a transferrable 95,983-bp IncFII-type plasmid in Klebsiella pneumoniae. The deduced amino-acid sequence of MCR-8 showed 31.08%, 30.26%, 39.96%, 37.85%, 33.51%, 30.43%, and 37.46% identity to MCR-1, MCR-2, MCR-3, MCR-4, MCR-5, MCR-6, and MCR-7, respectively. Functional cloning indicated that the acquisition of the single mcr-8 gene significantly increased resistance to colistin in both Escherichia coli and K. pneumoniae. Notably, the coexistence of mcr-8 and the carbapenemase-encoding gene bla NDM was confirmed in K. pneumoniae isolates of livestock origin. Moreover, BLASTn analysis of mcr-8 revealed that this gene was present in a colistin- and carbapenem-resistant K. pneumoniae strain isolated from the sputum of a patient with pneumonia syndrome in the respiratory intensive care unit of a Chinese hospital in 2016. These findings indicated that mcr-8 has existed for some time and has disseminated among K. pneumoniae of both animal and human origin, further increasing the public health burden of antimicrobial resistance.

A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples

The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 102 cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry.

Presence ofmcr-3variant inAeromonas caviae,Proteus mirabilis, andEscherichia colifrom one domestic duck

The rapid rise and dissemination of multidrug-resistant (MDR) bacteria are a major threat to public health worldwide and have narrowed the treatment options for infections caused by these bacteria (1). Colistin is one of the last-resort drugs for the treatment of infections caused by MDR Gram-negative bacteria. However, within the past 2 years, shortly after the report of the first plasmid-mediated colistin resistance gene, mcr-1, in 2015, four other mobile colistin resistance genes (mcr-2, mcr-3, mcr-4, and mcr-5) and multiple variants have been reported (2–6). Compared to mcr-2, mcr-4, and mcr-5, which have been detected solely in Europe so far, both mcr-1 and mcr-3 have been identified globally. Here, we report a novel mcr-3 variant in Aeromonas caviae, Proteus mirabilis, and Escherichia coli from a single domestic duck. A total of 15 cloacal samples were obtained from free-range domestic ducks near a river in a suburban area of Qingdao City, Shandong Province, China, in March 2017. Direct sample testing was then performed on all 15 samples to detect the mcr-3 gene, and only one sample was identified as mcr-3 positive. The sample positive for mcr-3 was further isolated on CHROMagar orientation agar plates (bioMérieux, Lyon, France) containing 2 mg/liter colistin. Three mcr-3-positive strains, A. caviae 17AC, P. mirabilis 17PM, and E. coli 17EC, were obtained, and their sequences were confirmed by using 16S rRNA gene sequencing and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry analysis. A. caviae 17AC, P. mirabilis 17PM, and E. coli 17EC were then subjected to 150-bp paired-end whole-genome sequencing (WGS) using the Illumina HiSeq 2500 platform (Annoroad, Beijing, China). The draft genomes were assembled using CLC Genomics Workbench 9.0 (CLC Bio, Aarhus, Denmark), and all contigs were searched for mcr-3 by standalone BLAST analysis. WGS analysis identified mcr-3-carrying fragments in each genome, including a 26.2-kb contig from 17AC, a 17.8-kb contig from 17PM, and a 13.0-kb contig from 17EC. An mcr-3 variant gene in three contigs showed 98.83% nucleotide sequence identity to mcr-3 from porcine E. coli. The deduced protein sequence differed from MCR-3.1 by seven amino acid substitutions, one of which (V122G) was located in a putative transmembrane region, while the remaining six (R297L, I313V, E337K, H341Y, D358E, and Q468K) were in the catalytic domain. This novel mcr-3 variant was designated mcr-3.10. A. caviae 17AC and E. coli 17EC belong to novel sequence types (STs), ST513 and ST457, respectively. E. coli of ST457 carrying mcr-1 has been reported from humans in the United States and Vietnam (7, 8). Plasmid replicon typing revealed that the mcr-3.10-carrying plasmid in both 17EC and its transconjugant T-17EC belongs to the IncI2 replicon. Accepted manuscript posted online 4 December 2017 Citation Wang X, Zhai W, Li J, Liu D, Zhang Q, Shen Z, Wang S, Wang Y. 2018. Presence of an mcr-3 variant in Aeromonas caviae, Proteus mirabilis, and Escherichia coli from one domestic duck. Antimicrob Agents Chemother 62:e02106-17. https://doi.org/10.1128/AAC .02106-17. Copyright

Presence of VIM-Positive Pseudomonas Species in Chickens and Their Surrounding Environment

ABSTRACT Metallo-β-lactamase gene blaVIM was identified on the chromosome of four Pseudomonas sp. isolates from a chicken farm, including one Pseudomonas aeruginosa isolate from a swallow (Yanornis martini), one Pseudomonas putida isolate from a fly, and two P. putida isolates from chickens. The four isolates shared two variants of blaVIM-carrying genomic contexts that resemble the corresponding regions of clinical metallo-β-lactamase-producing Pseudomonas spp. Our study suggests that the surveillance of carbapenemase-producing bacteria in livestock and their surrounding environment is urgently needed.

Novel Variant of New Delhi Metallo-β-lactamase, NDM-20, in Escherichia coli

The spread of carbapenem-resistant Enterobacteriaceae (CRE) mediated by New Delhi metallo-β-lactamase (NDM) poses a serious challenge to clinicians and has become a major public health concern. NDM has been evolving into variants that possess different hydrolysis activity toward antibiotics, so as to affect treatment strategy. In addition, very few studies on NDM variants have focused on animal-derived bacterial isolates. Our study reports a novel NDM variant, NDM-20, in an isolate of Escherichia coli CCD1 recovered from the food animal swine in China. The isolate that was assigned to ST1114, exhibited high level resistance to all β-lactams tested, including aztreonam and carbapenems. The gene of blaNDM-20 was located on an IncX3-type plasmid, surrounded by multiple insertion sequences. Sequencing analysis demonstrated that blaNDM-20 contained three point mutations at positions 262 (G→T), 460 (A→C), and 809 (G→A), compared with blaNDM-1, and just one point mutation at position 809 (G→A), relative to blaNDM-5. Functional analysis revealed that the blaNDM-20 transformant, DH5α+pHSG398/NDM-20, exhibited a higher resistance to ertapenem than that of blaNDM-1 transformant DH5α+pHSG398/NDM-1. Kinetic parameter analysis showed that NDM-20 had increased enzymatic activity against some penicillins and cephalosporins but decreased carbapenemase activity relative to NDM-5. The identification of NDM-20 further confirms the evolution and prevalence of NDM variants in bacteria of food-animal origin.

Occurrence of the mobile colistin resistance gene mcr-3 in Escherichia coli from household pigs in rural areas

Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China; Public Health Agency of Sweden, Stockholm, Sweden; Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Department of Animal Health and Antimicrobial Strategies, National Veterinary Institute (SVA), Uppsala, Sweden; Shandong Center for Disease Control and Prevention, Jinan, China; Institute of Microbiology and Epizootics, Centre for Infection Medicine, Department of Veterinary Medicine, Freie Universit€ at Berlin, Berlin, Germany

Mobile colistin resistance gene mcr-5 in porcine Aeromonas hydrophila

Abstract Objectives To characterize the mobile colistin resistance gene mcr-5 in Aeromonas hydrophila from backyard pigs in rural areas of China. Methods Pig faecal samples from 194 households were directly tested for the presence of mcr-5 by PCR assay and the phenotypic antimicrobial susceptibility profiles of the mcr-5-positive isolates were determined using the broth dilution method. The genomic location and transferability of mcr-5 were analysed by S1-PFGE with Southern blotting and DNA hybridization, and natural transformation, respectively. One strain isolated from an mcr-5-positive sample was subjected to WGS and the stability of the mcr-5-harbouring plasmid over successive generations was examined by subculturing. Results One mcr-5-positive A. hydrophila isolate showing resistance, with a colistin MIC of 4 mg/L, was isolated from a backyard pig faecal sample. mcr-5 was located on a 7915 bp plasmid designated pI064-2, which could naturally transform into a colistin-susceptible A. hydrophila strain of porcine origin and mediated colistin resistance in both the original isolate and its transformants. The plasmid backbone (3790 bp) of pI064-2 showed 81% nucleotide sequence identity to the corresponding region of the ColE2-type plasmid pAsa1 from Aeromonas salmonicida, while similar replication primases are widely distributed among aeromonads, Enterobacteriaceae and Pseudomonas species. Conclusions To the best of our knowledge, this is the first identification of the novel colistin resistance gene mcr-5 in an A. hydrophila isolate from the faeces of a backyard pig. mcr-5 is expected to be able to disseminate among different bacterial species and genera.

Corrigendum: Novel Variant of New Delhi Metallo-β-lactamase, NDM-20, in Escherichia coli

[This corrects the article on p. 248 in vol. 9, PMID: 29515538.].