Xianhui Huang

Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study

BACKGROUND Until now, polymyxin resistance has involved chromosomal mutations but has never been reported via horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae. METHODS The mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model. FINDINGS Polymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10(-1) to 10(-3) cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa. In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011-14, and 16 (1%) of 1322 samples from inpatients with infection. INTERPRETATION The emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria. FUNDING Ministry of Science and Technology of China, National Natural Science Foundation of China.

Prevalence and Dissemination of oqxAB in Escherichia coli Isolates from Animals, Farmworkers, and the Environment

ABSTRACT OqxAB has recently been identified as one of the mechanisms of plasmid-mediated quinolone resistance (PMQR). Compared to what is observed for other PMQR determinants, there is a paucity of data with regard to the prevalence and epidemiology of OqxAB and its contribution to resistance to different antimicrobials. In this study, the prevalence and dissemination of oqxAB and other PMQR genes in Escherichia coli isolates from animals, farmworkers, and the environment in 2002 in China were investigated. Of the 172 E. coli isolates, 39.0% carried oqxA, while only 4.1%, 2.9%, and 0.6% carried qnr (1 qnrB6 isolate, 5 qnrS1 isolates, and 1 qnrD isolate), qepA, and aac(6′)-Ib-cr, respectively. Among the 33 isolates from farmworkers, 10 (30.3%) were positive for oqxA. oqxAB was associated with IS26 and was carried on the 43- to 115-kb IncF transferable plasmid. Transconjugants carrying oqxAB showed 4- to 16-fold increases in the MICs of quinolones, 16- to 64-fold increases in the MICs of quinoxalines, 8- to 32-fold increases in the MICs of chloramphenicol and trimethoprim-sulfamethoxazole, and 4- to 8-fold increases in the MICs of florfenicol compared to the levels for the recipient. The pulsed-field gel electrophoresis (PFGE) analysis showed that the high levels of prevalence and dissemination of oqxAB in E. coli in animal farms were primarily due to the transmission of plasmids carrying oqxAB, although clonal transmission between human and swine E. coli isolates was observed. It is concluded that oqxAB was widespread in animal farms in China, which may be due to the overuse of quinoxalines in animals. This study warrants the prudent use of quinoxalines in food animals.

Dissemination of the Fosfomycin Resistance Gene fosA3 with CTX-M β-Lactamase Genes and rmtB Carried on IncFII Plasmids among Escherichia coli Isolates from Pets in China

ABSTRACT The presence and characterization of plasmid-mediated fosfomycin resistance determinants among Escherichia coli isolates collected from pets in China between 2006 and 2010 were investigated. Twenty-nine isolates (9.0%) were positive for fosA3, and all of them were CTX-M producers. The fosA3 genes were flanked by IS26 and were localized on F2:A−:B− plasmids or on very similar F33:A−:B− plasmids carrying both blaCTX-M-65 and rmtB. These findings indicate that the fosA3 gene may be coselected by antimicrobials other than fosfomycin.

Novel cyromazine imprinted polymer applied to the solid-phase extraction of melamine from feed and milk samples

A water compatible molecularly imprinted polymer (MIP) using cyromazine as a mimic template, methacrylic acid as the functional polymer and ethylene glycol dimethacrylate as the cross-linker was synthesized and used to extract melamine from feed and milk samples via a molecularly imprinted solid-phase extraction (MISPE) protocol. Optimum retention of melamine on the MISPE cartridge was achieved using methanol, and the interferences in the samples were effectively washed out. The binding capacity of the polymer toward melamine was found to be about 500 microg of melamine/g of polymer. The recoveries of 2 microg and 20 microg melamine standard spiked into water extract of blank feeds and milk samples were between 83.4% and 103%, with relative standard deviation <5.6%. The cyromazine-MIP demonstrated high cross-reactivity for melamine and low affinity to cyanuric acid. The ionic bond interaction was regarded as the main factors that dominated the retention of the melamine on the MISPE cartridge.

Use of a Monte Carlo analysis within a physiologically based pharmacokinetic model to predict doxycycline residue withdrawal time in edible tissues in swine

The pharmacokinetics of doxycycline were studied following a single intravenous (I.V.) and intramuscular (I.M.) injection of 10 mg/kg into eight healthy pigs. The steady-state tissue/plasma partition coefficients were obtained via a 3-h constant rate infusion (CRI) in four pigs. Based on the results of in vivo studies and the parameters derived from published work, a physiologically based pharmacokinetic (PBPK) model was developed to predict the drug concentration in edible tissues. The predicted values were then compared with those derived from a previous study. To account for individual differences in the processes of drug metabolism and/or diffusion, a Monte Carlo (MC) run of 1000 simulations was incorporated into the PBPK model to predict the doxycycline residue withdrawal times in edible tissues in swine. The withdrawal periods were compared with those derived from linear regression analysis. The PBPK model presented here provided accurate predictions of the observed concentrations in all tissues except for the injection site. The withdrawal times in all edible tissues derived from the MC analysis were longer than those from linear regression analysis. Based on the residues in the injection site and muscle tissue, the MC analysis predicted a withdrawal time of 33 days. Here, we illustrate that MC analysis can be incorporated into the PBPK model to accurately predict doxycycline residue withdrawal time in edible tissues in swine.

Metabolism of mequindox and its metabolites identification in chickens using LC-LTQ-Orbitrap mass spectrometry

Mequindox (MEQ), 3-methyl-2-quinoxalinacetyl-1,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial and feed additive. Its toxicities have been reported to be closely related to its metabolism. To understand more clearly the metabolic pathways of MEQ, its metabolism in chickens was studied using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC-LTQ-Orbitrap) mass spectrometry. The structures of the MEQ metabolites and their product ions were easily and reliably characterized based on the accurate MS-squared spectra and known structure of MEQ. Twenty-four metabolites were detected in chicken plasma, bile, faeces, and tissues, of which 12 were detected in vivo for the first time. The major metabolic pathways reported previously for in vitro metabolism of MEQ in chicken microsomes were confirmed in this study, including N→O group reduction, carbonyl reduction, and methyl mono-hydroxylation. In addition, deacetylation and acetyl-hydroxylation of MEQ were shown to be important metabolic pathways. Collectively, these data contribute to our understanding of the in vivo metabolism of MEQ.

High Prevalence of Colistin Resistance and mcr-1 Gene in Escherichia coli Isolated from Food Animals in China

The objective of this study was to determine the minimal inhibitory concentration of colistin for Escherichia coli from food animals and the possible underlying colistin resistance mechanisms. During 2007–2014, 4,438 E. coli isolates of food animal origins were collected. The susceptibility of colistin was tested by the agar dilution method. Mutations in pmrA, pmrB, and mgrB and the presence of mcr-1 gene were determined by PCR and DNA sequencing. Complementation experiments were carried out to evaluate the contribution of the mutations to colistin resistance. There was a high frequency of colistin resistance in E. coli from pigs on farm (24.1%) and at slaughter (24.3%) in 2013–2014, followed by chickens on farm (14.0%) and at slaughter (9.5%). The resistance frequency of E. coli in cow isolates was the lowest (0.9%). MIC distribution for colistin showed that most isolates (75.2%) were distributed at 0.25 mg/L–0.5 mg/L, followed by 4 mg/L–8 mg/L (16.8%). Compared with the isolates from pigs and chickens recovered during 2013–2014, E. coli isolates collected during 2007–2008 (5.5%) and 2010–2011 (12.4%) showed significantly lower frequency of colistin resistance (P < 0.05). DNA sequencing and complementation experiments failed to detect any insertion inactivation or mutation in pmrA, pmrB, and mgrB associated with colistin resistance. However, 91.0% colistin-resistant isolates were positive for mcr-1. The high frequency of colistin resistance and mcr-1 gene among E. coli isolates from food animals in China urged the need to minimize potential risks of colistin resistance development and the spread of mcr-1 gene.

Identification and elucidation of the structure of in vivo metabolites of diaveridine in chicken

Diaveridine (DVD) is a popular antibacterial synergist that is widely used in combination with sulfonamide. It has been reported to be genotoxic to mammalian cells, but more studies are required to clarify this. Moreover, there is very little information on its pharmacokinetics, metabolic elimination and mechanism of toxicity. Therefore, in order to gain a better understanding of the metabolism of DVD, we performed high-performance liquid chromatography linear ion trapped orbitrap mass spectrometer (LC-LTQ-Orbitrap). With this approach, we identified 15 metabolites of DVD in chicken after a single oral administration of DVD; 10 of these metabolites have been identified in vivo for the first time. Nine phase I and five phase II metabolites were detected in the plasma, and eight phase I and six phase II metabolites were found in feces. The major phase I metabolites were formed via the O-demethylation and N-oxidation pathways, and the major phase II metabolites were glucuronide conjugates. These results are essential for understanding this compound more clearly and lay the basis for further studies about the metabolism of DVD. Therefore, using this approach, we were able to identify and characterize metabolites of DVD with high sensitivity and resolution. We were able to detect a broad range of metabolites, even some trace ones and some so far unknown metabolites.

Metabolism of nitazoxanide in rats, pigs, and chickens: Application of liquid chromatography coupled to hybrid linear ion trap/Orbitrap mass spectrometer.

Nitazoxanide (NTZ) is a nitrothiazole benzamide compound with a broad activity spectrum against parasites, Gram-positive and Gram-negative anaerobic bacteria, and viruses. In this study, hybrid linear ion trap/Orbitrap mass spectrometer providing a high mass resolution and accuracy was used to investigate the metabolism of NTZ in rats, pigs, and chickens. The results revealed that acetylation and glucuronidation were the main metabolic pathways in rats and pigs, whereas acetylation and sulfation were the major metabolic pathways in chickens, which indicated interspecies variations in drug metabolism and elimination. With the accurate mass data and the characteristic MS(n) product ions, we identified six metabolites in which tizoxanide and hydroxylated tizoxanide were phase I metabolites and tizoxanide glucuronide, tizoxanide glucose, tizoxanide sulfate and hydroxyl tizoxanide sulfate were phase II metabolites. Hydroxylated tizoxanide and tizoxanide glucose were identified for the first time. All the comprehensive data were provided to make out the metabolism of NTZ in rats, pigs and chickens more clearly.

Estimating tulathromycin withdrawal time in pigs using a physiologically based pharmacokinetics model.

A physiologically based pharmacokinetics model was developed to predict tulathromycin concentrations in edible swine tissues. Physiological parameters included volumes of and plasma flows through different tissues which were obtained from the literatures. The tissue/plasma partition coefficient was calculated according to the area method, and the model was validated through a comparison of predicted and observed concentrations. Withdrawal times in different tissues were predicted. The physiologically based pharmacokinetics model presented here provided accurate predictions of the observed concentrations in all tissues. The results showed that the injection site had the longest withdrawal time (21 days), followed by skin together with fat (19 days) and then kidney (10 days), lung (6 days), liver (4 days) and muscle (1 day). A withdrawal time of 21 days was finally predicted for tulathromycin in swine after a single intramuscular injection at 2.5 mg/kg body weight.