separating-bacteria-capsule-amount-using-discontinuous-density

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

Wellcome Sanger Institute, Wellcome Genome Campus

Sequence-based typing (SBT), analogous to multilocus sequence typing (MLST), is the current "gold standard" typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila However, as common sequence types (STs) cause many infections, some investigations remain unresolved. In this study, various whole-genome sequencing (WGS)-based methods were evaluated according to published guidelines, including (i) a single nucleotide polymorphism (SNP)-based method, (ii) extended MLST using different numbers of genes, (iii) determination of gene presence or absence, and (iv) a kmer-based method. L. pneumophila serogroup 1 isolates (n = 106) from the standard "typing panel," previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI), were tested together with another 229 isolates. Over 98% of isolates were considered typeable using the SNP- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50 genes) to 86.8% (1,455 genes), while only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP based), and all values were higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ∼50 genes provides optimal epidemiological concordance while substantially improving the discrimination offered by SBT and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila.

Evaluation of an Optimal Epidemiological Typing Scheme for Legionella pneumophila with Whole-Genome Sequence Data Using Validation Guidelines.

 is a human commensal and opportunistic pathogen that has become a leading causative agent of hospital-based infections over the past few decades. The emergence and global expansion of hypervirulent and multidrug-resistant (MDR) clones of  have been increasingly reported in community-acquired and nosocomial infections. Despite this, the population genomics and epidemiology of MDR  at the national level are still poorly understood. To obtain insights into these, we analyzed a systematic large-scale collection of invasive MDR  isolates from hospitals across the United Kingdom and Ireland. Using whole-genome phylogenetic analysis, we placed these in the context of previously sequenced  populations from geographically diverse countries and identified their virulence and drug resistance determinants. Our results demonstrate that United Kingdom and Ireland MDR isolates are a highly diverse population drawn from across the global phylogenetic tree of  and represent multiple recent international introductions that are mainly from Europe but in some cases from more distant countries. In addition, we identified novel genetic determinants underlying resistance to beta-lactams, gentamicin, ciprofloxacin, and tetracyclines, indicating that both increased virulence and resistance have emerged independently multiple times throughout the population. Our data show that MDR  isolates in the United Kingdom and Ireland have multiple distinct origins and appear to be part of a globally circulating  population. is a major human pathogen that has been implicated in infections in healthcare settings over the past few decades. Antimicrobial treatment of  infections has become increasingly difficult as a consequence of the emergence and spread of strains that are resistant to multiple antimicrobials. To better understand the spread of resistant , we studied the genomes of a large-scale population of extensively antimicrobial-resistant  in the United Kingdom and Ireland by utilizing the fine resolution that whole-genome sequencing of pathogen genomes provides. Our results indicate that the  population is highly diverse and that, in some cases, resistant strains appear to have spread across the country over a few years. In addition, we found evidence that some strains have acquired antimicrobial resistance genes independently, presumably in response to antimicrobial treatment.

Julian Parkhill

Wellcome Sanger Institute, Wellcome Genome Campus

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient