Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

A Validated Multiplex Real-Time PCR Assay for the Diagnosis of Infectious Leptospira spp.: A Novel Assay for the Detection and Differentiation of Strains From Both Pathogenic Groups I and II.

Leptospirosis is recognized as the most globally widespread reemerging zoonosis and represents a serious threat for both human and animal health. Indeed, leptospirosis is linked to more than 60,000 human deaths per year and to incalculable economic burden as consequence of medical treatment costs and livestock loss. The increasing number of reports from species of pathogenic Leptospira spp. group II causing disease in both humans and animals constitutes an additional concern to the complex epidemiology of this zoonotic agent. Diagnostic methods based on qPCR have improved the diagnosis of Leptospira spp. in terms of cost, time, and reliability, but most of the validated assays fail to detect species from the pathogenic group II. Hence, the current study was aimed to develop and validate a novel multiplex qPCR to enable the specific and selective detection of the whole group of infectious Leptospira spp., including both pathogenic groups I and II and moreover, selectively discriminate between them. To fit the "fitness of purpose" for the specific detection of infectious Leptospira spp. and further discrimination between both pathogenic groups three target regions on the 16S RNA gene were selected. These targets facilitated a broad and selective spectrum for the detection of all infectious Leptospira spp. with the exclusion of all saprophytic groups and the novel clade of environmental Leptospira spp. The analytical sensitivity (ASe) showed by the new assay also enables a wide window of detection for the agent at different stages of infection since the assay was able to efficiently detect at 95% of confidence ∼5 leptospires/reaction. From the evaluation of the analytical specificity (ASp) by in silico and in vitro approaches, it was congruently revealed that the primers and probes selected only recognized the specific targets for which the assay was intended. Bayesian latent class analysis of performance of the new assay on 684 clinical samples showed values of diagnostic sensitivity of 99.8% and diagnostic specificity of 100%. Thus, from the evaluation of the analytical and diagnostic parameters, the new multiplex qPCR assay is a reliable method for the diagnosis of Leptospira spp.