Rapid detection and quantification of plasmid-mediated colistin resistance genes (mcr-1 to mcr-5) by real-time PCR in bacterial and environmental samples.

AIM: The aim of the study was to validate a rapid method to detect and quantify colistin resistance genes (mcr-1 to mcr-5) by real-time polymerase chain reaction (RT-PCR) in diverse matrices. METHODS AND RESULTS: The detection limit of two newly designed SYBR Green real-time PCR assays for mcr-4 and mcr-5 and of previously published protocols for mcr-1 to mcr-3 was assessed using serial dilutions of reference strains. The assays could detect all five mcr genes with the lower limit of 102 copy numbers. Escherichia coli isolates (n = 1062) and environmental samples (n = 93) were tested for the presence of mcr genes. The assays enabled the detection of colistin resistance genes both in bacterial isolates and in complex environmental samples. CONCLUSIONS: This method represents a set of sensitive, rapid and effective assays for the screening of colistin resistance directly from the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Colistin is an antimicrobial commonly used in animals and has recently emerged as a last-resort treatment in humans. Plasmid-mediated mcr genes confer resistance to colistin and represent a major threat for public health since they can be easily disseminated through horizontal gene transfer. The rapid and sensitive detection of mcr genes is of utmost necessity.

Effects of fenbendazole and triclabendazole on the expression of cytochrome P450 1A and flavin-monooxygenase isozymes in bovine precision-cut liver slices.

Combinations of the anthelmintics fenbendazole (FBZ) and triclabendazole (TCBZ) have shown enhanced efficacy against the liver fluke Fasciola hepatica. This study aimed to measuring the constitutive expression of CYP1A1, CYP1A2, FMO1 and FMO3, thought to be involved in the metabolism of those compounds, by using an absolute quantitative real time (RT)-PCR approach in bovine precision-cut liver slices (PCLS). It also aimed to characterize the effects of FBZ and TCBZ (alone and in combination) on the expression and activity of the aforementioned isozymes. Both FMO1 and FMO3 were equally represented in control PCLS, whereas CYP1A2 was expressed more than CYP1A1 (P<0.05). PCLS cultured in the presence of beta naphthoflavone (ß-NF; CYP1A inducer) had higher mRNA levels of CYP1A1, CYP1A2, FMO1 and FMO3 (P<0.05). No clear-cut evidence of transcriptional effects of the anthelmintics were recorded. After incubation of PCLS with FBZ, there was a significant increase (P<0.05) vs. controls and TBCZ was observed for CYP1A1. PCLS treated with FBZ showed a higher (P<0.05) expression of CYP1A2 compared to controls, TCBZ alone, and the combination FBZ+TCBZ. The gene expression profiles of FMO1 and FMO3 were not affected by the presence of the anthelmintics; the only exception was an upregulation of FMO3 by TCBZ alone. The observed transcriptional effects of the xenobiotics were not mirrored by increased enzyme activities using prototypical substrates of the isozymes under study. Although further confirmatory studies are needed, these results suggest that PCLS represent an alternative in vitro tool for studies on the expression, regulation and function of relevant xenobiotic-metabolizing enzymes in cattle.