Olbermann P.,Institute for Medical Microbiology and Hospital Epidemiology |
Olbermann P.,University of Würzburg |
Josenhans C.,Institute for Medical Microbiology and Hospital Epidemiology |
Moodley Y.,Max Planck Institute for Infection Biology |
And 9 more authors.
PLoS Genetics | Year: 2010
The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI-carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown.© 2010 Olbermann et al.
PubMed | Erasme Hospital, Genoscreen, French Institute of Health and Medical Research, Onze Lieve Vrouw hospital and Applied Maths
Type: Journal Article | Journal: Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases | Year: 2016
We used a two-step whole genome sequencing analysis for resolving two concurrent outbreaks in two neonatal services in Belgium, caused by exfoliative toxin A-encoding-gene-positive (eta+) methicillin-susceptible Staphylococcus aureus with an otherwise sporadic spa-type t209 (ST-109). Outbreak A involved 19 neonates and one healthcare worker in a Brussels hospital from May 2011 to October 2013. After a first episode interrupted by decolonization procedures applied over 7months, the outbreak resumed concomitantly with the onset of outbreak B in a hospital in Asse, comprising 11 neonates and one healthcare worker from mid-2012 to January 2013. Pan-genome multilocus sequence typing, defined on the basis of 42 core and accessory reference genomes, and single-nucleotide polymorphisms mapped on an outbreak-specific de novo assembly were used to compare 28 available outbreak isolates and 19 eta+/spa-type t209 isolates identified by routine or nationwide surveillance. Pan-genome multilocus sequence typing showed that the outbreaks were caused by independent clones not closely related to any of the surveillance isolates. Isolates from only ten cases with overlapping stays in outbreak A, including four pairs of twins, showed no or only a single nucleotide polymorphism variation, indicating limited sequential transmission. Detection of larger genomic variation, even from the start of the outbreak, pointed to sporadic seeding from a pre-existing exogenous source, which persisted throughout the whole course of outbreak A. Whole genome sequencing analysis can provide unique fine-tuned insights into transmission pathways of complex outbreaks even at their inception, which, with timely use, could valuably guide efforts for early source identification.
PubMed | Centers for Disease Control and Prevention, Association of Public Health Laboratories, U.S. Food and Drug Administration, Applied Maths and 2 more.
Type: Journal Article | Journal: Clinical infectious diseases : an official publication of the Infectious Diseases Society of America | Year: 2016
Listeria monocytogenes (Lm) causes severe foodborne illness (listeriosis). Previous molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), were critical in detecting outbreaks that led to food safety improvements and declining incidence, but PFGE provides limited genetic resolution. A multiagency collaboration began performing real-time, whole-genome sequencing (WGS) on all US Lm isolates from patients, food, and the environment in September 2013, posting sequencing data into a public repository. Compared with the year before the project began, WGS, combined with epidemiologic and product trace-back data, detected more listeriosis clusters and solved more outbreaks (2 outbreaks in pre-WGS year, 5 in WGS year 1, and 9 in year 2). Whole-genome multilocus sequence typing and single nucleotide polymorphism analyses provided equivalent phylogenetic relationships relevant to investigations; results were most useful when interpreted in context of epidemiological data. WGS has transformed listeriosis outbreak surveillance and is being implemented for other foodborne pathogens.
Multilaboratory validation study of standardized multiple-locus variable-number tandem repeat analysis protocol for Shiga toxin-producing escherichia coli O157: A novel approach to normalize fragment size data between capillary electrophoresis platforms
Hyytia-Trees E.,Centers for Disease Control and Prevention |
Lafon P.,Centers for Disease Control and Prevention |
Vauterin P.,Applied Maths |
Ribot E.M.,Centers for Disease Control and Prevention
Foodborne Pathogens and Disease | Year: 2010
The PulseNet USA subtyping network recently established a standardized protocol for multiple-locus variable-number tandem repeat analysis (MLVA) to characterize Shiga toxin-producing Escherichia coli O157. To enable data comparisons from different laboratories in the same database, reproducibility and high quality of the data must be ensured. The aim of this study was to test the robustness and reproducibility of the proposed standardized protocol by subjecting it to a multilaboratory validation process and to address any discrepancies that may have arisen from the study. A set of 50 strains was tested in 10 PulseNet participating laboratories that used capillary electrophoresis instruments from two manufacturers. Six out of the 10 laboratories were able to generate correct MLVA types for 46 (92%) or more strains. The discrepancies in MLVA type assignment were caused mainly by difficulties in optimizing polymerase chain reactions that were attributed to technical inexperience of the staff and suboptimal quality of reagents and instrumentation. It was concluded that proper training of staff must be an integral part of technology transfer. The interlaboratory reproducibility of fragment sizing was excellent when the same capillary electrophoresis platform was used. However, sizing discrepancies of up to six base pairs for the same fragment were detected between the two platforms. These discrepancies were attributed to different dye and polymer chemistries employed by the manufacturers. A novel software script was developed to assign alleles based on two platform-specific (Beckman Coulter CEQ™8000 and Applied Biosystems Genetic Analyzer 3130xl) look-up tables containing fragment size ranges for all alleles. The new allele assignment method was validated at the PulseNet central laboratory using a diverse set of 502 Shiga toxin-producing Escherichia coli O157 isolates. The validation confirmed that the script reliably assigned the same allele for the same fragment regardless of the platform used to size the fragment. © Copyright 2010, Mary Ann Liebert, Inc.
PubMed | Centers for Disease Control and Prevention, World Health Organization, Public Health Agency of Canada, Public Health England and 4 more.
Type: | Journal: Nature microbiology | Year: 2016
Listeria monocytogenes (Lm) is a major human foodborne pathogen. Numerous Lm outbreaks have been reported worldwide and associated with a high case fatality rate, reinforcing the need for strongly coordinated surveillance and outbreak control. We developed a universally applicable genome-wide strain genotyping approach and investigated the population diversity of Lm using 1,696 isolates from diverse sources and geographical locations. We define, with unprecedented precision, the population structure of Lm, demonstrate the occurrence of international circulation of strains and reveal the extent of heterogeneity in virulence and stress resistance genomic features among clinical and food isolates. Using historical isolates, we show that the evolutionary rate of Lm from lineage I and lineage II is low (2.510
Bosch T.,National Institute for Public Health and the Environment RIVM |
Verkade E.,Amphia Hospital |
Verkade E.,St Elisabeth Hospital |
van Luit M.,National Institute for Public Health and the Environment RIVM |
And 9 more authors.
PLoS ONE | Year: 2013
After its emergence in 2003, a livestock-associated (LA-)MRSA clade (CC398) has caused an impressive increase in the number of isolates submitted for the Dutch national MRSA surveillance and now comprises 40% of all isolates. The currently used molecular typing techniques have limited discriminatory power for this MRSA clade, which hampers studies on the origin and transmission routes. Recently, a new molecular analysis technique named whole genome mapping was introduced. This method creates high-resolution, ordered whole genome restriction maps that may have potential for strain typing. In this study, we assessed and validated the capability of whole genome mapping to differentiate LA-MRSA isolates. Multiple validation experiments showed that whole genome mapping produced highly reproducible results. Assessment of the technique on two well-documented MRSA outbreaks showed that whole genome mapping was able to confirm one outbreak, but revealed major differences between the maps of a second, indicating that not all isolates belonged to this outbreak. Whole genome mapping of LA-MRSA isolates that were epidemiologically unlinked provided a much higher discriminatory power than spa-typing or MLVA. In contrast, maps created from LA-MRSA isolates obtained during a proven LA-MRSA outbreak were nearly indistinguishable showing that transmission of LA-MRSA can be detected by whole genome mapping. Finally, whole genome maps of LA-MRSA isolates originating from two unrelated veterinarians and their household members showed that veterinarians may carry and transmit different LA-MRSA strains at the same time. No such conclusions could be drawn based spa-typing and MLVA. Although PFGE seems to be suitable for molecular typing of LA-MRSA, WGM provides a much higher discriminatory power. Furthermore, whole genome mapping can provide a comparison with other maps within 2 days after the bacterial culture is received, making it suitable to investigate transmission events and outbreaks caused by LA-MRSA. © 2013 Bosch et al.
PubMed | Ghent University and Applied Maths
Type: Journal Article | Journal: Veterinary research | Year: 2017
The importance of diversity of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is not yet fully known. This study investigated the genetic diversity of M. hyopneumoniae strains in ten pig herds, and assessed associations between the presence of different strains of M. hyopneumoniae and lung lesions at slaughter. Within each herd, three batches of slaughter pigs were investigated. At slaughter, from each batch, 20 post mortem bronchoalveolar lavage fluid samples were collected for multiple locus variable-number tandem repeat analysis (MLVA), and lung lesions (Mycoplasma-like lesions, fissures) were examined. Multivariable analyses including potential risk factors for respiratory disease were performed to assess associations between the number of different strains per batch (three categories: one strain, two-six strains,seven strains), and the lung lesions as outcome variables. In total, 135 different M. hyopneumoniae strains were found. The mean (min.-max.) number of different strains per batch were 7 (1-13). Batches with two-six strains or more than six strains had more severe Mycoplasma-like lesions (P=0.064 and P=0.012, respectively), a higher prevalence of pneumonia [odds ratio (OR): 1.30, P=0.33 and OR: 2.08, P=0.012, respectively], and fissures (OR=1.35, P=0.094 and OR=1.70, P=0.007, respectively) compared to batches with only one strain. In conclusion, many different M. hyopneumoniae strains were found, and batches of slaughter pigs with different M. hyopneumoniae strains had a higher prevalence and severity of Mycoplasma-like lung lesions at slaughter, implying that reducing the number of different strains may lead to less lung lesions at slaughter and better respiratory health of the pigs.
Pouseele H.,Applied Maths |
Supply P.,Center for Infection and Immunity of Lille |
Supply P.,French Institute of Health and Medical Research |
Supply P.,French National Center for Scientific Research |
And 2 more authors.
Methods in Microbiology | Year: 2015
Efficient molecular-guided epidemiological control of tuberculosis is especially important, because of the complex epidemiology, the insidious transmission and the specific biological features of its etiologic agent Mycobacterium tuberculosis. The most used typing system for epidemiological tracing of the pathogen is MIRU-VNTR typing, which has been internationally standardised under two formats (standard 24-locus based and 4 hypervariable locus based), optionally combined with spoligotyping. This generalised use has been facilitated by the portable numerical genotypes that are generated, the precisely calibrated molecular cluster definition and the option to use published protocols or ready-to-go kits directly compatible with multifunctional web-based databases. However, such classical typing systems can inherently not capture all possible micro-variation that can occur in the genome. Near complete capture of the available genetic information is increasingly performed by whole-genome sequencing (WGS). Such approaches take advantage of the rapid advances and increasing affordability of next-generation sequencing technologies, including benchtop platforms meeting the needs of routine clinical microbiology. However, such technologies rely on a number of key technical steps that are important, including in particular the critical bioinformatics analysis step. This chapter will therefore provide an overview of analysis parameters that are determinant for accurate WGS-based epidemiological surveillance of M. tuberculosis, linked to its restricted genetic diversity and the particular richness of its genome in repetitive regions. © 2015 Elsevier Ltd.