April 21, 2020  |  

Whole-Genome Sequencing for Characterization of Capsule Locus and Prediction of Serogroup of Invasive Meningococcal Isolates

Invasive meningococcal disease is mainly caused by Neisseria meningitidis serogroups A, B, C, X, W, and Y. The serogroup is typically determined by slide agglutination serogrouping (SASG) and real-time PCR (RT-PCR). We describe a whole-genome sequencing (WGS)-based method to characterize the capsule polysaccharide synthesis (cps) locus, classify N. meningitidis serogroups, and identify mechanisms for nongroupability using 453 isolates from a global strain collection. We identified novel genomic organizations within functional cps loci, consisting of insertion sequence (IS) elements in unique positions that did not disrupt the coding sequence. Genetic mutations (partial gene deletion, missing genes, IS insertion, internal stop, and phase-variable off) that led to nongroupability were identified. The results of WGS and SASG were in 91% to 100% agreement for all serogroups, while the results of WGS and RT-PCR showed 99% to 100% agreement. Among isolates determined to be nongroupable by WGS (31 of 453), the results of all three methods agreed 100% for those without a capsule polymerase gene. However, 61% (WGS versus SASG) and 36% (WGS versus RT-PCR) agreements were observed for the isolates, particularly those with phase variations or internal stops in cps loci, which warrant further characterization by additional tests. Our WGS-based serogrouping method provides comprehensive characterization of the N. meningitidis capsule, which is critical for meningococcal surveillance and outbreak investigations.

April 21, 2020  |  

Phylogenetic relationships and regional spread of meningococcal strains in the meningitis belt, 2011-2016.

Historically, the major cause of meningococcal epidemics in the meningitis belt of sub-Saharan Africa has been Neisseria meningitidis serogroup A (NmA), but the incidence has been substantially reduced since the introduction of a serogroup A conjugate vaccine starting in 2010. We performed whole-genome sequencing on isolates collected post-2010 to assess their phylogenetic relationships and inter-country transmission.A total of 716 invasive meningococcal isolates collected between 2011 and 2016 from 11 meningitis belt countries were whole-genome sequenced for molecular characterization by the three WHO Collaborating Centers for Meningitis.We identified three previously-reported clonal complexes (CC): CC11 (n?=?434), CC181 (n?=?62) and CC5 (n?=?90) primarily associated with NmW, NmX, and NmA, respectively, and an emerging CC10217 (n?=?126) associated with NmC. CC11 expanded throughout the meningitis belt independent of the 2000 Hajj outbreak strain, with isolates from Central African countries forming a distinct sub-lineage within this expansion. Two major sub-lineages were identified for CC181 isolates, one mainly expanding in West African countries and the other found in Chad. CC10217 isolates from the large outbreaks in Nigeria and Niger were more closely related than those from the few cases in Mali and Burkina Faso.Whole-genome based phylogenies revealed geographically distinct strain circulation as well as inter-country transmission events. Our results stress the importance of continued meningococcal molecular surveillance in the region, as well as the development of an affordable vaccine targeting these strains. FUND: Meningitis Research Foundation; CDC’s Office of Advanced Molecular Detection; GAVI, the Vaccine Alliance. Copyright © 2019. Published by Elsevier B.V.

April 21, 2020  |  

Iron-associated protein interaction networks reveal the key functional modules related to survival and virulence of Pasteurella multocida.

Pasteurella multocida causes respiratory infectious diseases in a multitude of birds and mammals. A number of virulence-associated genes were reported across different strains of P. multocida, including those involved in the iron transport and metabolism. Comparative iron-associated genes of P. multocida among different animal hosts towards their interaction networks have not been fully revealed. Therefore, this study aimed to identify the iron-associated genes from core- and pan-genomes of fourteen P. multocida strains and to construct iron-associated protein interaction networks using genome-scale network analysis which might be associated with the virulence. Results showed that these fourteen strains had 1587 genes in the core-genome and 3400 genes constituting their pan-genome. Out of these, 2651 genes associated with iron transport and metabolism were selected to construct the protein interaction networks and 361 genes were incorporated into the iron-associated protein interaction network (iPIN) consisting of nine different iron-associated functional modules. After comparing with the virulence factor database (VFDB), 21 virulence-associated proteins were determined and 11 of these belonged to the heme biosynthesis module. From this study, the core heme biosynthesis module and the core outer membrane hemoglobin receptor HgbA were proposed as candidate targets to design novel antibiotics and vaccines for preventing pasteurellosis across the serotypes or animal hosts for enhanced precision agriculture to ensure sustainability in food security. Copyright © 2018. Published by Elsevier Ltd.

April 21, 2020  |  

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

April 21, 2020  |  

High quality reference genomes for toxigenic and non-toxigenic Vibrio cholerae serogroup O139.

Toxigenic Vibrio cholerae of the O139 serogroup have been responsible for several large cholera epidemics in South Asia, and continue to be of clinical and historical significance today. This serogroup was initially feared to represent a new, emerging V. cholerae clone that would lead to an eighth cholera pandemic. However, these concerns were ultimately unfounded. The majority of clinically relevant V. cholerae O139 isolates are closely related to serogroup O1, biotype El Tor V. cholerae, and comprise a single sublineage of the seventh pandemic El Tor lineage. Although related, these V. cholerae serogroups differ in several fundamental ways, in terms of their O-antigen, capsulation phenotype, and the genomic islands found on their chromosomes. Here, we present four complete, high-quality genomes for V. cholerae O139, obtained using long-read sequencing. Three of these sequences are from toxigenic V. cholerae, and one is from a bacterium which, although classified serologically as V. cholerae O139, lacks the CTXf bacteriophage and the ability to produce cholera toxin. We highlight fundamental genomic differences between these isolates, the V. cholerae O1 reference strain N16961, and the prototypical O139 strain MO10. These sequences are an important resource for the scientific community, and will improve greatly our ability to perform genomic analyses of non-O1 V. cholerae in the future. These genomes also offer new insights into the biology of a V. cholerae serogroup that, from a genomic perspective, is poorly understood.

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.