Development of a Novel mcr-6 to mcr-9 Multiplex PCR and Assessment of mcr-1 to mcr-9 Occurrence in Colistin-Resistant Salmonella enterica Isolates From Environment, Feed, Animals and Food (2011–2018) in Germany

Abstract

The polymyxin antibiotic colistin has been used in decades for treatment and prevention of infectious diseases in livestock. Nowadays, it is even considered as last-line treatment option for severe human infections caused by multidrug- and carbapenem-resistant Gram-negative bacteria. Therefore, the discovery of plasmid-mediated mobile colistin resistance (<i>mcr</i>) genes raised major public health concern. The aim of our study was to analyze colistin-resistant <i>Salmonella enterica</i> strains from animals, food, feed and the environment collected at the National Reference Laboratory for <i>Salmonella</i> in Germany on the presence of <i>mcr-1</i> to <i>mcr-9</i> genes. Altogether 407 colistin-resistant (MIC >2 mg/L) <i>Salmonella</i> isolates received between 2011 and 2018 were selected and screened by PCR using a published <i>mcr-1</i> to <i>mcr-5</i> as well as a newly developed <i>mcr-6</i> to <i>mcr-9</i> multiplex PCR protocol. 254 of 407 (62.4%) isolates harbored either <i>mcr-1</i> (<i>n</i> = 175), <i>mcr-4</i> (<i>n</i> = 53), <i>mcr-5</i> (<i>n</i> = 18) or <i>mcr-1</i> and <i>mcr-9</i> (<i>n</i> = 8). The number of <i>mcr</i>-positive isolates ranged from 19 (2017) to 64 (2012) per year. WGS revealed that none of our isolates harbored the <i>mcr-9.1</i> gene. Instead, two novel <i>mcr-9</i> variants were observed, which both were affected by frameshift mutations and are probably non-functional. The <i>mcr</i>-harboring isolates were mainly derived from animals (77.2%) or food (20.1%) and could be assigned to ten different <i>Salmonella</i> serovars. Many of the isolates were multidrug-resistant. Co-occurrence of <i>mcr-1</i> and AmpC or ESBL genes was observed in eight isolates. Our findings suggest that <i>mcr</i> genes are widely spread among colistin-resistant <i>Salmonella</i> isolates from livestock and food in Germany. Potential transfer of <i>mcr</i>-harboring isolates along the food chain has to be considered critically.