Extended spectrum ß-lactamase-producing Escherichia coli among backyard poultry farms, farmers, and environments in Thailand

Food-producing animals, including poultry, have been considered as potential sources of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli. This study investigates the occurrence and dissemination of ESBL-producing E. coli among backyard poultry farms, farmers, and environments in Northern Thailand. Antimicrobial-resistant phenotypes, resistant determinants, genotypic characterizations, and spread of these isolates were studied. Fecal samples from poultry, farmers, and environments were captured from 27 farms. In total, 587 samples were collected and the overall 27.1% (159/587) of ESBL-producing E. coli isolates were obtained. Among these, ESBL-producing E. coli was isolated from 50% (farmers), 25.9% poultry (24.9% chicken and 36.6% duck) of the fecal samples, and 25.0% of the environmental samples. All isolates demonstrated multidrug resistance, most frequently to ≥ 10 different antimicrobial agents. Molecular analysis of ESBL-encoding genes showed that the predominant gene was blaCTX-M-55 (54.1%), followed by blaCTX-M-14 (28.3%), and blaCTX-M-15 (8.8%). blaCTX-M-27 (3.8%) and blaCTX-M-65 (0.6%) were also detected at low frequencies. Conjugation assays demonstrated that blaCTX-M could be transferred to E. coli J53 with the transfer frequencies ranging from 10−7 to 10−2. Pulsed field gel electrophoresis (PFGE) revealed diverse genotypes, however, identical and closely related PFGE profiles were detected among isolates within and between farms, suggesting the clonal transmission. In addition, our study identified 4 blaCTX-M-27-positive E. coli B2-ST131 isolates. Interestingly, two ST131 isolates, obtained from a farmer and chicken in the same area, showed closely related PFGE profiles. Our results suggest the presence and spread of ESBL-producing E. coli between backyard poultry farms, farmers, and environments in Thailand

Environmental dissemination of mcr-1 positive Enterobacteriaceae by Chrysomya spp. (common blowfly): An increasing public health risk

Until recently, the role of insects, and particularly flies, in disseminating antimicrobial resistance (AMR) has been poorly studied. In this study, we screened blowflies (Chrysomya spp.) from different areas near the city of Phitsanulok, Northern Thailand, for the presence of AMR genes and in particular, mcr-1, using whole genome sequencing (WGS). In total, 48 mcr-1-positive isolates were recovered, consisting of 17 mcr-1-positive Klebsiella pneumoniae (MCRPKP) and 31 mcr-1-positive Escherichia coli (MCRPEC) strains. The 17 MCRPKP were shown to be clonal (ST43) with few single poly nucleomorphs (SNPs) by WGS analysis. In in-vitro models, the MCRPKP were shown to be highly virulent. In contrast, 31 recovered MCRPEC isolates are varied, belonging to 12 different sequence types shared with those causing human infections. The majority of mcr-1 gene are located on IncX4 plasmids (29/48, 60.42%), sharing an identical plasmid backbone. These findings highlight the contribution of flies to the AMR contagion picture in low- and middle-income countries and the challenges of tackling global AMR

Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms

MCR-1 is a lipid A modifying enzyme that confers resistance to the antibiotic colistin. Here, we analyse the impact of MCR-1 expression on E. coli morphology, fitness, competitiveness, immune stimulation and virulence. Increased expression of mcr-1 results in decreased growth rate, cell viability, competitive ability and significant degradation in cell membrane and cytoplasmic structures, compared to expression of catalytically inactive MCR-1 (E246A) or MCR-1 soluble component. Lipopolysaccharide (LPS) extracted from mcr-1 strains induces lower production of IL-6 and TNF, when compared to control LPS. Compared to their parent strains, high-level colistin resistance mutants (HLCRMs) show reduced fitness (relative fitness is 0.41–0.78) and highly attenuated virulence in a Galleria mellonella infection model. Furthermore, HLCRMs are more susceptible to most antibiotics than their respective parent strains. Our results show that the bacterium is challenged to find a delicate equilibrium between expression of MCR-1-mediated colistin resistance and minimalizing toxicity and thus ensuring cell survival

Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from the Crystal Structure of the Catalytic Domain of MCR-1

The polymixin colistin is a "last line" antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism