Wee, B. | bioRxiv Science

bioRxiv2023

Rapid metagenomic sequencing for diagnosis and antimicrobial sensitivity prediction of canine bacterial infections

Antimicrobial resistance is one of the greatest current threats to human and animal health. There is an urgent need to ensure that antimicrobials are used appropriately to limit the emergence and impact of resistance. In the human and veterinary healthcare setting, traditional culture and antimicrobial sensitivity testing is typically conducted, requiring 48-72 h to identify appropriate antibiotics for treatment. In the meantime, broad-spectrum antimicrobials are often used, which may be ineffective or impact non-target commensal bacteria. Here, we present a rapid diagnostics pipeline, involving metagenomic Nanopore sequencing directly from clinical urine and skin samples of dogs. We have optimised this pipeline to be versatile and easily implementable in a clinical setting, with the potential for future adaptation to different sample types and animals. Using our approach, we can identify the bacterial pathogen present in a sample with 100% sensitivity within 5 hours. For urine samples, we can predict antibiotic sensitivity with up to 95% accuracy. However, skin swabs which exhibited lower bacterial abundance and higher host DNA, were less amenable and an additional host depletion step may be required prior to DNA extraction. In summary, our pipeline represents an important step towards the design of individually tailored veterinary treatment plans on the same day as presentation, facilitating effective use of antibiotics and promoting antimicrobial stewardship. Impact statementAntimicrobial resistance (AMR) is a major threat to veterinary and human healthcare. It is a one-health problem, as humans and dogs are in close contact, require similar antibiotics, and share bacterial pathogens and AMR genes. Limited treatments options due to AMR would have a catastrophic effect. The risk of infection would render much of modern healthcare (including critical care, orthopaedic and complex surgeries, implants and oncology) impossible. In addition, routine infections could become life threatening. It is therefore critical to preserve the efficacy of these drugs for the future. Inappropriate antimicrobial use is the single biggest factor driving AMR. Antimicrobial stewardship involves reducing antimicrobial use, using first-line narrow-spectrum drugs, and avoiding overly long treatment. Delays in culture-based diagnosis lead clinicians to speculatively use broad-spectrum antibiotics and prolong courses of treatment beyond clinical cure. Our rapid diagnostic approach will have a major impact in reducing, refining and replacing antibiotic use. This will advance antimicrobial stewardship in veterinary and human healthcare. Data summaryAll sequencing data mentioned in this work is available from NCBI, BioProject PRJNA925092, Biosamples SAMN32880396 to SAMN32880438, run accessions SRR23195371 to SRR23195413. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2020

Population analysis of Legionella pneumophila reveals the basis of resistance to complement-mediated killing

Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires disease. L. pneumophila is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 900 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We found that although some clones are more commonly associated with clinical infections, the capacity for human disease is representative of the breadth of species diversity. To investigate the bacterial genetic basis for human disease potential, we carried out a genome-wide association study that identified a single gene (lag-1), to be most strongly associated with clinical isolates. Molecular evolutionary analysis showed that lag-1, which encodes an O-acetyltransferase responsible for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. Functional analysis revealed a correlation between the presence of a functional lag-1 gene and resistance to killing in human serum and bovine broncho-alveolar lavage. In addition, L. pneumophila strains that express lag-1 escaped complement-mediated phagocytosis by neutrophils. Importantly, we discovered that the expression of lag-1 confers the capacity to evade complement-mediated killing by inhibiting deposition of classical pathway molecules on the bacterial surface. In summary, our combined population and functional analyses identified L. pneumophila genetic traits linked to human disease and revealed the molecular basis for resistance to complement-mediated killing, a previously elusive trait of direct relevance to human disease pathogenicity. SignificanceLegionella pneumophila is an environmental bacterium associated with a severe pneumonia known as Legionnaires disease. A small number of L. pneumophila clones are responsible for a large proportion of human infections suggesting they have enhanced pathogenicity. Here, we employed a large-scale population analysis to investigate the evolution of human pathogenicity and identified a single gene (lag-1) that was more frequently found in clinical isolates. Functional analysis revealed that the lag-1-encoded O-acetyltransferase, involved in modification of lipopolysaccharide, conferred resistance to the classical pathway of complement in human serum. These findings solve a long-standing mystery in the field regarding L. pneumophila resistance to serum killing, revealing a novel mechanism by which L. pneumophila may avoid immune defences during infection.

microbiology↗