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Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community.

Mycoplasmas are respiratory pathogens in humans and animals and due to their fastidious nature, they have been historically underdiagnosed. Lack of standardised diagnostic, typing and antimicrobial susceptibility testing methods makes clinical management and epidemiological studies challenging. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of respiratory mycoplasmas using a rapid long-read sequencing platform. Critical aspects of bacterial whole genome sequencing were explored using fastidious respiratory Mycoplasma (M. felis and M. cynos) isolated from animals including: (i) four solid and liquid-based media based on a specialized formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for sequencing purposes, and (iii) two de novo assembly platforms as key components of a bioinformatic pipeline including Flye and Canu assemblers. DNA quality and quantity compatible with long-read sequencing requirements were obtained with culture volumes of 160ml in modified Hayflick’s broth incubated for 96 hours. The other three culture approaches investigated did not meet the DNA quality criteria required for long-read sequencing. The use of bead-beating bacterial cell lysis in the extraction protocol resulted in smaller fragments and shorter reads compared to enzymatic lysis methods. Overall, Flye generated more contiguous assemblies than the Canu assembler. This novel study provides a step-by-step sequencing workflow including mycoplasma culture, DNA extraction and de novo assembly approaches for the characterization of highly fastidious respiratory mycoplasmas. This workflow will provide diagnosticians, epidemiologists, and researchers with a more comprehensive tool than the laborious conventional methods for a complete genomic characterization of respiratory mycoplasmas.

Listeria monocytogenesis an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model forL. monocytogenesinfection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 differentL. monocytogenesisolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 µg lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p=0.004 and p=0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT,virR) known to be involved in intracellular growth and virulence.In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum.