Development of a multiplex taqMan real-time PCR assay for typing of Mycoplasma pneumoniae based on type-specific indels identified through whole genome sequencing.

We developed a multiplex real-time PCR assay for simultaneously detecting M. pneumoniae and typing into historically-defined P1 types. Typing was achieved based on the presence of short type-specific indels identified through whole genome sequencing. This assay was 100% specific compared to existing methods and may be useful during epidemiologic investigations.

Complete Genome Sequence of Mycoplasma pneumoniae Type 2 Reference Strain FH Using Single-Molecule Real-Time Sequencing Technology.

Mycoplasma pneumoniae type 2 strain FH was previously sequenced with Illumina (FH-Illumina) and 454 (FH-454) technologies according to Xiao et al. (2015) and Krishnakumar et al. (2010). Comparative analyses revealed differences in genomic content between these sequences, including a 6-kb region absent from the FH-454 submission. Here, we present a complete genome sequence of FH sequenced with the Pacific Biosciences RSII platform.

Comprehensive bioinformatics analysis of Mycoplasma pneumoniae genomes to investigate underlying population structure and type-specific determinants.

Mycoplasma pneumoniae is a significant cause of respiratory illness worldwide. Despite a minimal and highly conserved genome, genetic diversity within the species may impact disease. We performed whole genome sequencing (WGS) analysis of 107 M. pneumoniae isolates, including 67 newly sequenced using the Pacific BioSciences RS II and/or Illumina MiSeq sequencing platforms. Comparative genomic analysis of 107 genomes revealed >3,000 single nucleotide polymorphisms (SNPs) in total, including 520 type-specific SNPs. Population structure analysis supported the existence of six distinct subgroups, three within each type. We developed a predictive model to classify an isolate based on whole genome SNPs called against the reference genome into the identified subtypes, obviating the need for genome assembly. This study is the most comprehensive WGS analysis for M. pneumoniae to date, underscoring the power of combining complementary sequencing technologies to overcome difficult-to-sequence regions and highlighting potential differential genomic signatures in M. pneumoniae.

Correction: Comprehensive bioinformatics analysis of Mycoplasma pneumoniae genomes to investigate underlying population structure and type-specific determinants.

[This corrects the article DOI: 10.1371/journal.pone.0174701.].

Genomic Analysis Reveals Novel Diversity among the 1976 Philadelphia Legionnaires' Disease Outbreak Isolates and Additional ST36 Strains.

Legionella pneumophila was first recognized as a cause of severe and potentially fatal pneumonia during a large-scale outbreak of Legionnaires' disease (LD) at a Pennsylvania veterans' convention in Philadelphia, 1976. The ensuing investigation and recovery of four clinical isolates launched the fields of Legionella epidemiology and scientific research. Only one of the original isolates, "Philadelphia-1", has been widely distributed or extensively studied. Here we describe the whole-genome sequencing (WGS), complete assembly, and comparative analysis of all Philadelphia LD strains recovered from that investigation, along with L. pneumophila isolates sharing the Philadelphia sequence type (ST36). Analyses revealed that the 1976 outbreak was due to multiple serogroup 1 strains within the same genetic lineage, differentiated by an actively mobilized, self-replicating episome that is shared with L. pneumophila str. Paris, and two large, horizontally-transferred genomic loci, among other polymorphisms. We also found a completely unassociated ST36 strain that displayed remarkable genetic similarity to the historical Philadelphia isolates. This similar strain implies the presence of a potential clonal population, and suggests important implications may exist for considering epidemiological context when interpreting phylogenetic relationships among outbreak-associated isolates. Additional extensive archival research identified the Philadelphia isolate associated with a non-Legionnaire case of "Broad Street pneumonia", and provided new historical and genetic insights into the 1976 epidemic. This retrospective analysis has underscored the utility of fully-assembled WGS data for Legionella outbreak investigations, highlighting the increased resolution that comes from long-read sequencing and a sequence type-matched genomic data set.

Complete Genome Sequences of Three Outbreak-Associated Legionella pneumophila Isolates.

We report here the complete genome sequences of three Legionella pneumophila isolates that are associated with a Legionnaires' disease outbreak in New York in 2012. Two clinical isolates (D7630 and D7632) and one environmental isolate (D7631) were recovered from this outbreak. A single isolate-specific virulence gene was found in D7632. These isolates were included in a large study evaluating the genomic resolution of various bioinformatics approaches for L. pneumophila serogroup 1 isolates.