No abstract

Objectives WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. Methods The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. Results Genotype–phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype–phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. Conclusions WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

Since the 1960s, methicillin-resistant Staphylococcus aureus (MRSA) has emerged, disseminated globally and become a leading cause of bacterial infections in both health-care and community settings. However, there is marked geographical variation in MRSA burden owing to several factors, including differences in local infection control practices and pathogen-specific characteristics of the circulating clones. Different MRSA clones have resulted from the independent acquisition of staphylococcal cassette chromosome mec (SCCmec), which contains genes encoding proteins that render the bacterium resistant to most β-lactam antibiotics (such as methicillin), by several S. aureus clones. The success of MRSA is a consequence of the extensive arsenal of virulence factors produced by S. aureus combined with β-lactam resistance and, for most clones, resistance to other antibiotic classes. Clinical manifestations of MRSA range from asymptomatic colonization of the nasal mucosa to mild skin and soft tissue infections to fulminant invasive disease with high mortality. Although treatment options for MRSA are limited, several new antimicrobials are under development. An understanding of colonization dynamics, routes of transmission, risk factors for progression to infection and conditions that promote the emergence of resistance will enable optimization of strategies to effectively control MRSA. Vaccine candidates are also under development and could become an effective prevention measure.

We identified a novel plasmid-mediated colistinresistance gene in porcine and bovine colistin-resistant Escherichia coli that did not contain mcr-1. The gene, termed mcr-2, a 1,617 bp phosphoethanolamine transferase harboured on an IncX4 plasmid, has 76.7% nucleotide identity to mcr-1. Prevalence of mcr-2 in porcine colistin-resistant E. coli (11/53) in Belgium was higher than that of mcr-1 (7/53). These data call for an immediate introduction of mcr-2 screening in ongoing molecular epidemiological surveillance of colistinresistant Gram-negative pathogens. . mcr-1 was detected in 12.4% (n = 13) of the E. coli isolates, of which six and seven were from calves and piglets, respectively [3,4]. In the present study, we analysed porcine and bovine colistin-resistant Escherichia coli isolates that did not show presence of mcr-1 and identified a novel plasmid-mediated colistin resistance-conferring gene, mcr-2.

Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.