With PacBio long-read sequencing, scientists are making exciting new discoveries about the microbes that live around and within us. From viruses to bacteria to fungi, SMRT Sequencing is shedding light on how these organisms function and evolve.
In this AGBT plenary talk, Jonas Korlach presented a number of collaborative studies between PacBio and other institutions to make use of highly accurate, long-read sequence data, which has led to a revival of finished genomes. Examples from the infectious disease or pathogen realm included Pertussis, Salmonella, and Listeria, all of which now have closed genomes from PacBio-generated data. Korlach also reported on epigenomic information in Salmonella and Listeria, indicating potential new forms of DNA modifications.
UC Davis’s Bart Weimer describes foodborne pathogens and their proclivity for rapid genome rearrangement. The 100K Pathogen Genome Project he leads is using PacBio long-read sequencing to close genomes and analyze methylation; Weimer reports that his team has already discovered new epigenetic modifications in Salmonella and Listeria with the technology.
Jonas Korlach, CSO of PacBio, discusses the revival of finished genomes the microbial community will see with long read data, emphasizing that for certain organisms such as rapidly evolving microbes, having a de novo finished genome will be more useful than creating a draft based on a previous related reference genome. Korlach describes two bioinformatic methods from PacBio, a hierarchical genome assembly process (HGAP) and an consensus caller (Quiver), which are used to generate finished genomes from just long-read PacBio data, with final genome sequence accuracies over 99.999%. Korlach demonstrates the ability of PacBio data to generate closed, high-quality de…
For microbial sequencing on the PacBio Sequel System, the current yield per SMRT Cell is in excess relative to project requirements. Multiplexing offers a viable solution; greatly increasing throughput, efficiency, and reducing costs per genome. This approach is achieved by incorporating a unique barcode for each microbial sample into the SMRTbell adapters and using a streamlined library preparation process. To demonstrate performance,12 unique barcodes assigned to B. subtilis and sequenced on a single SMRT Cell. To further demonstrate the applicability of this method, we multiplexed the genomes of 16 strains of H. pylori. Each DNA was sheared to 10 kb,…
Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are…
Whole genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn3-like transposon in P. aeruginosa MRSN 12280. The isolate was non-susceptible to colistin by broth microdilution and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin non-susceptible P. aeruginosa.
Genome sequencing of carbapenem-resistant Klebsiella pneumoniae isolates from regional U.S. hospitals was used to characterize strain diversity and the bla(KPC) genetic context. A phylogeny based on core single-nucleotide variants (SNVs) supports a division of sequence type 258 (ST258) into two distinct groups. The primary differences between the groups are in the capsular polysaccharide locus (cps) and their plasmid contents. A strict association between clade and KPC variant was found. The bla(KPC) gene was found on variants of two plasmid backbones. This study indicates that highly similar K. pneumoniae subpopulations coexist within the same hospitals over time. Copyright © 2014, American…
The community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) epidemic in the United States is attributed to the spread of the USA300 clone. An epidemic of CA-MRSA closely related to USA300 has occurred in northern South America (USA300 Latin-American variant, USA300-LV). Using phylogenomic analysis, we aimed to understand the relationships between these 2 epidemics.We sequenced the genomes of 51 MRSA clinical isolates collected between 1999 and 2012 from the United States, Colombia, Venezuela, and Ecuador. Phylogenetic analysis was used to infer the relationships and times since the divergence of the major clades.Phylogenetic analyses revealed 2 dominant clades that segregated by geographical region, had…
Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a six-week period without epidemiological links. Detection of the blaKPC-2 gene in one isolate prompted inclusion of non-Enterobacteriaceae in our surveillance culture workup. Whole genome sequencing confirmed isolates were unrelated, and provided data for Aeromonas reference genomes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested…
We report complete genome sequences of fourblaNDM-1-harboring Gram-negative multidrug resistant (MDR) isolates from Colombia. TheblaNDM-1genes were located 193Kb-Inc FIA, 178Kb-Inc A/C2 and 47Kb (unknown Inc type) plasmids. MLST revealed that isolates belong to ST10 (Escherichia coli), ST392 (Klebsiella pneumoniae), and ST322 and ST464 (Acinetobacter baumanniiandA. nosocomialis, respectively). Our analysis identified that the Inc A/C2 plasmid inE. colicontained a novel complex transposon (Tn125and Tn5393with 3 copies ofblaNDM-1) and a recombination “hotspot” for the acquisition of new resistance determinants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
During 2010 and 2012, California and Vermont, respectively, experienced statewide epidemics of pertussis with differences seen in the demographic affected, case clinical presentation, and molecular epidemiology of the circulating strains. To overcome limitations of the current molecular typing methods for pertussis, we utilized whole-genome sequencing to gain a broader understanding of how current circulating strains are causing large epidemics. Through the use of combined next-generation sequencing technologies, this study compared de novo, single-contig genome assemblies from 31 out of 33 Bordetella pertussis isolates collected during two separate pertussis statewide epidemics and 2 resequenced vaccine strains. Final genome architecture assemblies were…
Whole-genome sequencing (WGS) has become an essential tool for public health surveillance and molecular epidemiology of infectious diseases and antimicrobial drug resistance. It provides precise geographical delineation of spread and enables incidence monitoring of pathogens at genotype level. Coupled with epidemiological and environmental investigations, it delivers ultimate resolution for tracing sources of epidemic infections. To ascertain the level of implementation of WGS-based typing for national public health surveillance and investigation of prioritized diseases in the European Union (EU)/European Economic Area (EEA), two surveys were conducted in 2015 and 2016. The surveys were designed to determine the national public health reference…
A scarlet fever outbreak began in mainland China and Hong Kong in 2011 (refs. 1-6). Macrolide- and tetracycline-resistant Streptococcus pyogenes emm12 isolates represent the majority of clinical cases. Recently, we identified two mobile genetic elements that were closely associated with emm12 outbreak isolates: the integrative and conjugative element ICE-emm12, encoding genes for tetracycline and macrolide resistance, and prophage FHKU.vir, encoding the superantigens SSA and SpeC, as well as the DNase Spd1 (ref. 4). Here we sequenced the genomes of 141 emm12 isolates, including 132 isolated in Hong Kong between 2005 and 2011. We found that the introduction of several ICE-emm12…