Wenjuan Yin

Novel Plasmid-Mediated Colistin Resistance Gene mcr-3 in Escherichia coli

ABSTRACT The mobile colistin resistance gene mcr-1 has attracted global attention, as it heralds the breach of polymyxins, one of the last-resort antibiotics for the treatment of severe clinical infections caused by multidrug-resistant Gram-negative bacteria. To date, six slightly different variants of mcr-1, and a second mobile colistin resistance gene, mcr-2, have been reported or annotated in the GenBank database. Here, we characterized a third mobile colistin resistance gene, mcr-3. The gene coexisted with 18 additional resistance determinants in the 261-kb IncHI2-type plasmid pWJ1 from porcine Escherichia coli. mcr-3 showed 45.0% and 47.0% nucleotide sequence identity to mcr-1 and mcr-2, respectively, while the deduced amino acid sequence of MCR-3 showed 99.8 to 100% and 75.6 to 94.8% identity to phosphoethanolamine transferases found in other Enterobacteriaceae species and in 10 Aeromonas species, respectively. pWJ1 was mobilized to an E. coli recipient by conjugation and contained a plasmid backbone similar to those of other mcr-1-carrying plasmids, such as pHNSHP45-2 from the original mcr-1-harboring E. coli strain. Moreover, a truncated transposon element, TnAs2, which was characterized only in Aeromonas salmonicida, was located upstream of mcr-3 in pWJ1. This ΔTnAs2-mcr-3 element was also identified in a shotgun genome sequence of a porcine E. coli isolate from Malaysia, a human Klebsiella pneumoniae isolate from Thailand, and a human Salmonella enterica serovar Typhimurium isolate from the United States. These results suggest the likelihood of a wide dissemination of the novel mobile colistin resistance gene mcr-3 among Enterobacteriaceae and aeromonads; the latter may act as a potential reservoir for mcr-3. IMPORTANCE The emergence of the plasmid-mediated colistin resistance gene mcr-1 has attracted substantial attention worldwide. Here, we examined a colistin-resistant Escherichia coli isolate that was negative for both mcr-1 and mcr-2 and discovered a novel mobile colistin resistance gene, mcr-3. The amino acid sequence of MCR-3 aligned closely with phosphoethanolamine transferases from Enterobacteriaceae and Aeromonas species originating from both clinical infections and environmental samples collected in 12 countries on four continents. Due to the ubiquitous profile of aeromonads in the environment and the potential transfer of mcr-3 between Enterobacteriaceae and Aeromonas species, the wide spread of mcr-3 may be largely underestimated. As colistin has been and still is widely used in veterinary medicine and used at increasing frequencies in human medicine, the continuous monitoring of mobile colistin resistance determinants in colistin-resistant Gram-negative bacteria is imperative for understanding and tackling the dissemination of mcr genes in both the agricultural and health care sectors. IMPORTANCE The emergence of the plasmid-mediated colistin resistance gene mcr-1 has attracted substantial attention worldwide. Here, we examined a colistin-resistant Escherichia coli isolate that was negative for both mcr-1 and mcr-2 and discovered a novel mobile colistin resistance gene, mcr-3. The amino acid sequence of MCR-3 aligned closely with phosphoethanolamine transferases from Enterobacteriaceae and Aeromonas species originating from both clinical infections and environmental samples collected in 12 countries on four continents. Due to the ubiquitous profile of aeromonads in the environment and the potential transfer of mcr-3 between Enterobacteriaceae and Aeromonas species, the wide spread of mcr-3 may be largely underestimated. As colistin has been and still is widely used in veterinary medicine and used at increasing frequencies in human medicine, the continuous monitoring of mobile colistin resistance determinants in colistin-resistant Gram-negative bacteria is imperative for understanding and tackling the dissemination of mcr genes in both the agricultural and health care sectors.

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.

Chromosome-Mediated mcr-3 Variants in Aeromonas veronii from Chicken Meat.

ABSTRACT Two adjacent colistin resistance gene variants, termed mcr-3.3 and mcr-3-like, were identified in the chromosome of an Aeromonas veronii isolate obtained from retail chicken meat. The variants showed 95.20% and 84.19% nucleotide sequence identity, respectively, to mcr-3 from porcine Escherichia coli. Functional cloning indicated that only mcr-3.3 conferred polymyxin resistance in both E. coli and Aeromonas salmonicida. The mcr-3.3-mcr-3-like segment was also observed in other Aeromonas species, including A. media, A. caviae, and A. hydrophila.

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.

Characterization of NDM-1-producing carbapenemase in Acinetobacter spp. and E. coli isolates from diseased pigs

In recent years, the mobile metallo-β-lactamase (MBL) genes have been found to correspond to one of the most important resistance characters identified in Gram- negative bacteria, severely affecting clinical chemotherapy and threatening public health. The prevalence of mobile MBL genes and their flanking regions in Gram-negative bacteria from diseased pigs in China was investigated. A total of 334 lung samples from diseased pigs were screened for Gram-negative bacteria classified as non-susceptible to meropenem (MIC≥4mg

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

L -1 ). Six isolates, including three Escherichia coli, two Acinetobacter baumanii and one A. calcoaeticus, exhibited MBL production and carried the blaNDM-1 gene. S1-PFGE and Southern blot analysis showed that the blaNDM-1 gene was located on the chromosome of one A. baumanii isolate and on plasmids of various sizes in the other five isolates. MIC testing using broth microdilution revealed that all blaNDM-1-carrying isolates and some of their transconjugants exhibited resistance to almost all β-lactams tested. Whole genome sequencing revealed that the flanking region of the blaNDM-1 gene from all porcine isolates had high levels of similarity with the corresponding regions in human isolates. One porcine E. coli isolate carrying blaNDM-1 was typed as ST48, a common sequence type in human E. coli isolates. These results suggest the possibility of human-to- food animal transfer of blaNDM-1-producing E. coli, highlighting the need for surveillance of carbapenemase producers among bacteria from food animals. In addition, the prudent use of antimicrobial agents to decrease the opportunities for co-selection of carbapenemase genes in food animals is also urgently needed.

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

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.

Erratum for Yin et al., “Novel Plasmid-Mediated Colistin Resistance Gene mcr-3 in Escherichia coli”

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.

Reply to Cabello et al., “Aquaculture and mcr Colistin Resistance Determinants”

Wenjuan Yin,a Hui Li,a Yingbo Shen,a Zhihai Liu,a Shaolin Wang,a Zhangqi Shen,a Rong Zhang,b Timothy R. Walsh,c Jianzhong Shen,a Yang Wanga Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, Chinaa; The Second Affiliated Hospital of Zhejiang University, Zhejiang University, Hangzhou, Chinab; Department of Medical Microbiology and Infectious Disease, Institute of Infection and Immunity, Heath Park Hospital, Cardiff, United Kingdomc