scholarly journals Hidden Diversity within Common Protozoan Parasites Revealed by a Novel Genomotyping Scheme

2021 ◽  
Author(s):  
Matthew H. Seabolt ◽  
Konstantinos T. Konstantinidis ◽  
Dawn M. Roellig
Keyword(s):  
High Resolution ◽  
Molecular Typing ◽  
Giardia Duodenalis ◽  
Molecular Techniques ◽  
Microbial Pathogens ◽  
Whole Genome ◽  
Genome Sequences ◽  
Phylogenetic Resolution ◽  
Typing Methods ◽  
Assemblage B

Giardia duodenalis (syn. G. lamblia, G. intestinalis) is the causative agent of giardiasis, one of the most common diarrheal infections in humans. Evolutionary relationships among G. duodenalis genotypes (or subtypes) of assemblage B, one of two genetic assemblages causing the majority of human infections, remain unclear due to poor phylogenetic resolution of current typing methods. Here, we devised a methodology to identify new markers for a streamlined multi-locus sequence typing (MLST) scheme based on comparisons of all core genes against the phylogeny of whole-genome sequences (WGS). Our analysis identified three markers with comparable resolution to WGS data. Using newly designed PCR primers for our novel MLST loci, we typed an additional 68 strains of assemblage B. Analyses of these strains and previously determined genome sequences showed that genomes of this assemblage can be assigned to 16 clonal complexes, each with unique gene content that is apparently tuned to differential virulence and ecology. Obtaining new genomes of Giardia spp. and other eukaryotic microbial pathogens remains challenging due to difficulties in culturing the parasites in the laboratory. Hence, the methods described here are expected to be widely applicable to other pathogens of interest and advance understanding of their ecology and evolution. IMPORTANCE Giardia duodenalis assemblage B is a major waterborne pathogen and the most commonly identified genotype causing human giardiasis worldwide. The lack of morphological characters for classification requires the use of molecular techniques for strain differentiation, however, the absence of scalable and affordable NGS-based typing methods has prevented meaningful advancements in high resolution molecular typing for further understanding of the evolution and epidemiology of Assemblage B. Prior studies have reported high sequence diversity but low phylogenetic resolution at standard loci in Assemblage B, highlighting the necessity of identifying new markers for accurate and robust molecular typing. Data from comparative analyses of available genomes in this study identified three loci that together form a novel high-resolution typing scheme with high concordance to whole-genome-based phylogenomics and which should aid in future public health endeavors related to this parasite. In addition, data from newly characterized strains suggest evidence of biogeographic and ecologic endemism.

Molecular Epidemiology in the Care of Patients

1999 ◽  
Vol 123 (11) ◽  
pp. 1007-1010
Author(s):  
Michael A. Pfaller
Keyword(s):  
Molecular Typing ◽  
Restriction Endonuclease Analysis ◽  
Microbial Pathogens ◽  
Chromosomal Dna ◽  
Typing Method ◽  
Epidemiologic Surveillance ◽  
Term Evaluation ◽  
Typing Methods ◽  
Plasmid Profiling

Abstract Several different epidemiologic typing methods have been applied in studies of microbial pathogens. These methods include the more traditional nonmolecular approaches as well as the more sophisticated molecular typing methods. Application of traditional epidemiologic typing methods, such as antibiogram, serotyping, biotyping, and phage typing, have occasionally been useful in describing the epidemiology of infectious diseases. However, these methods have generally been considered to be too variable, labor intensive, and slow to be of practical value in epidemiologic investigations. In response to these limitations, several techniques have been adopted from the molecular biology field for use as epidemiologic typing methods and have been applied in studies of bacteria, fungi, viruses, and protozoa. The most widely used molecular typing methods are the DNA-based methods, such as plasmid profiling, restriction endonuclease analysis of plasmid and genomic DNA, Southern hybridization analysis using specific DNA probes, and chromosomal DNA profiling using either pulsed-field gel electrophoresis or polymerase chain reaction–based methods. The various molecular typing methods may be applied to the investigation of outbreaks of infections or may be used in the context of epidemiologic surveillance. For outbreak investigation, typing methods are used to compare isolates from a suspected outbreak to delineate clonally related and unrelated strains with the goal of short-term control of transmission. In the context of epidemiologic surveillance, molecular typing methods may be used to monitor geographic spread and prevalence shifts of epidemic and endemic clones with the goal of long-term evaluation of preventive strategies or for the detection and monitoring of emerging and reemerging infections. The specific typing method selected may vary with the task at hand; however, the typing studies must always be used to supplement, rather than replace, careful epidemiologic investigation.

scholarly journals Whole genome sequencing for typing and characterisation of Listeria monocytogenes isolated in a rabbit meat processing plant

2017 ◽  
Vol 6 (3) ◽  
Author(s):  
Federica Palma ◽  
Frédérique Pasquali ◽  
Alex Lucchi ◽  
Alessandra De Cesare ◽  
Gerardo Manfreda
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Molecular Typing ◽  
Virulence Genes ◽  
Processing Plant ◽  
Whole Genome ◽  
Meat Processing ◽  
Typing Methods ◽  
Rabbit Meat

Listeria monocytogenes is a food-borne pathogen able to survive and grow in different environments including food processing plants where it can persist for month or years. In the present study the discriminatory power of Whole Genome Sequencing (WGS)-based analysis (cgMLST) was compared to that of molecular typing methods on 34 L. monocytogenes isolates collected over one year in the same rabbit meat processing plant and belonging to three genotypes (ST14, ST121, ST224). Each genotype included isolates indistinguishable by standard molecular typing methods. The virulence potential of all isolates was assessed by Multi Virulence-Locus Sequence Typing (MVLST) and the investigation of a representative database of virulence determinant genes. The whole genome of each isolate was sequenced on a MiSeq platform. The cgMLST, MVLST, and in silico identification of virulence genes were performed using publicly available tools. Draft genomes included a number of contigs ranging from 13 to 28 and N50 ranging from 456298 to 580604. The coverage ranged from 41 to 187X. The cgMLST showed a significantly superior discriminatory power only in comparison to ribotyping, nevertheless it allows the detection of two singletons belonging to ST14 that were not observed by other molecular methods. All ST14 isolates belonged to VT107, which 7-loci concatenated sequence differs for only 4 nucleotides to VT1 (Epidemic clone III). Analysis of virulence genes showed the presence of a fulllength inlA version in all ST14 isolates and of a mutated version including a premature stop codon (PMSC) associated to attenuated virulence in all ST121 isolates.

Molecular typing ofGiardia duodenalisin humans in Queensland – first report of Assemblage E

Parasitology ◽  
2017 ◽  
Vol 144 (9) ◽  
pp. 1154-1161 ◽  
Author(s):  
ALIREZA ZAHEDI ◽  
DANIEL FIELD ◽  
UNA RYAN
Keyword(s):  
Molecular Typing ◽  
Quantitative Polymerase Chain Reaction ◽  
Giardia Duodenalis ◽  
Protozoan Parasite ◽  
Mixed Infections ◽  
Chain Reaction ◽  
Specific Primers ◽  
First Report ◽  
Polymerase Chain ◽  
Assemblage B

SUMMARYLittle is known about the genetic diversity of the protozoan parasite,Giardia duodenalis,infecting humans in Queensland, Australia. The present study typed 88 microscopicallyGiardia-positive isolates using assemblage-specific primers at the triose phosphate isomerase (tpi) gene and sequenced a subset of isolates at the glutamate dehydrogenase (gdh) gene (n= 30) andtpilocus (n= 27). Using thetpi-assemblage specific primers,G. duodenalisassemblage A and assemblage B were detected in 50% (44/88) and 38·6% (34/88) of samples, respectively. Mixed infections with assemblages A and B were identified in 4·5% (4/88) and assemblage E was identified in 6·8% (6/88) of samples. Sequence analysis at thegdhandtpiloci also confirmed the presence of assemblage E in these isolates. Cyst numbers per gram of feces (g−1) were determined using quantitative polymerase chain reaction and of the isolates that were typed as assemblage E, cyst numbers ranged 13·8–68·3 × 106cysts g−1. This is the first report of assemblage E in humans in Australia, indicating that in certain settings, this assemblage may be zoonotic.

scholarly journals A whole genome-based gene-by-gene typing system for standardised high resolution strain typing of Bacillus anthracis

2021 ◽  
Author(s):  
Mostafa Y. Abdel-Glil ◽  
Alexandra Chiaverini ◽  
Giuliano Garofolo ◽  
Antonio Fasanella ◽  
Antonio Parisi ◽  
...  
Keyword(s):  
High Resolution ◽  
Bacillus Anthracis ◽  
Genetic Distances ◽  
Pathogen Transmission ◽  
Snp Analysis ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Routine Surveillance ◽  
Typing Methods ◽  
Definition Of

Whole-genome-sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high resolution strain characterisation. This study aimed at developing a novel cgMLST scheme for Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide. The scheme comprises 3,803 genes that were conserved in 57 B. anthracis genomes spanning the whole phylogeny. The scheme has been evaluated and applied to 584 genomes from 50 countries. On average, 99.5% of the cgMLST targets were detected. The cgMLST results confirmed the classical canonical Single-Nucleotide-Polymorphisms (SNPs) grouping of B. anthracis into major clades and subclades. Genetic distances calculated based on cgMLST were comparable to distances from whole-genome-based SNP analysis with similar phylogenetic topology and comparable discriminatory power. Additionally, the application of the cgMLST scheme to anthrax outbreaks from Germany and Italy led to a definition of a cut-off threshold of five allele differences to trace epidemiologically linked strains for cluster typing and transmission analysis. Finally, the association of two clusters of B. anthracis to human cases of injectional anthrax in four European countries was confirmed using cgMLST. In summary, this study presents a novel cgMLST scheme that provides a high-resolution strain genotyping for B. anthracis. This scheme can be used in parallel with SNP typing methods to facilitate rapid and harmonised inter-laboratory comparisons, essential for global surveillance and outbreak analysis. The scheme is publicly-available for application from users including those with little bioinformatics knowledge.

scholarly journals Draft Whole-Genome Sequences of 25 Salmonella enterica Strains Representing 24 Serovars: TABLE 1 

2016 ◽  
Vol 4 (2) ◽  
Author(s):  
Catherine Yoshida ◽  
Stephanie L. Brumwell ◽  
Erika J. Lingohr ◽  
Aaminah Ahmad ◽  
Travis M. Blimkie ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Molecular Typing ◽  
Salmonella Enterica ◽  
Selection Process ◽  
Draft Genome ◽  
Whole Genome ◽  
Genome Sequences ◽  
Public Repositories

We report the draft genome sequences of 25 Salmonella enterica strains representing 24 different serotypes, many of which were not available in public repositories during our selection process. These draft genomes will provide useful reference for the genetic variation between serotypes and aid in the development of molecular typing tools.

scholarly journals Draft Genome Sequences of 510 Listeria monocytogenes Strains from Food Isolates and Human Listeriosis Cases from Northern Italy

2018 ◽  
Vol 6 (3) ◽  
Author(s):  
Sara Lomonaco ◽  
Silvia Gallina ◽  
Virginia Filipello ◽  
Maria Sanchez Leon ◽  
George John Kastanis ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Genome Sequencing ◽  
Molecular Typing ◽  
Large Scale ◽  
Draft Genome ◽  
Northern Italy ◽  
Whole Genome ◽  
Genome Sequences ◽  
Sequencing Studies ◽  
Different Sources

ABSTRACT Listeriosis outbreaks are frequently multistate/multicountry outbreaks, underlining the importance of molecular typing data for several diverse and well-characterized isolates. Large-scale whole-genome sequencing studies on Listeria monocytogenes isolates from non-U.S. locations have been limited. Herein, we describe the draft genome sequences of 510 L. monocytogenes isolates from northern Italy from different sources.

scholarly journals Mycobacterium abscessus infection in cystic fibrosis: molecular typing and clinical outcomes

2014 ◽  
Vol 63 (10) ◽  
pp. 1241-1246 ◽  
Author(s):  
Kathryn A. Harris ◽  
Dervla T. D. Kenna
Keyword(s):  
Cystic Fibrosis ◽  
Genome Sequencing ◽  
Molecular Typing ◽  
Patient Population ◽  
Patient Management ◽  
Pcr Amplification ◽  
Mycobacterium Abscessus ◽  
Clinical Impact ◽  
Molecular Techniques ◽  
Whole Genome

Mycobacterium abscessus is a significant pathogen in the cystic fibrosis patient population. PCR amplification and sequencing can provide accurate subspecies identification, and can predict macrolide susceptibility, which is becoming increasingly important for patient management. Molecular techniques for further typing of isolates provide tools for the ongoing investigations into the clinical impact of particular M. abscessus strains. Whole-genome sequencing is likely to be the only technique that provides sufficient resolution for investigating transmission events between patients.

scholarly journals Beaver Fever: Whole-Genome Characterization of Waterborne Outbreak and Sporadic Isolates To Study the Zoonotic Transmission of Giardiasis

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Clement K.-M. Tsui ◽  
Ruth Miller ◽  
Miguel Uyaguari-Diaz ◽  
Patrick Tang ◽  
Cedric Chauve ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Surface Water ◽  
Diarrheal Disease ◽  
Genetic Relationships ◽  
Giardia Duodenalis ◽  
Zoonotic Transmission ◽  
Whole Genome ◽  
Content Type ◽  
Human Infections ◽  
Assemblage B

ABSTRACT Giardia causes the diarrheal disease known as giardiasis; transmission through contaminated surface water is common. The protozoan parasite’s genetic diversity has major implications for human health and epidemiology. To determine the extent of transmission from wildlife through surface water, we performed whole-genome sequencing (WGS) to characterize 89 Giardia duodenalis isolates from both outbreak and sporadic infections: 29 isolates from raw surface water, 38 from humans, and 22 from veterinary sources. Using single nucleotide variants (SNVs), combined with epidemiological data, relationships contributing to zoonotic transmission were described. Two assemblages, A and B, were identified in surface water, human, and veterinary isolates. Mixes of zoonotic assemblages A and B were seen in all the community waterborne outbreaks in British Columbia (BC), Canada, studied. Assemblage A was further subdivided into assemblages A1 and A2 based on the genetic variation observed. The A1 assemblage was highly clonal; isolates of surface water, human, and veterinary origins from Canada, United States, and New Zealand clustered together with minor variation, consistent with this being a panglobal zoonotic lineage. In contrast, assemblage B isolates were variable and consisted of several clonal lineages relating to waterborne outbreaks and geographic locations. Most human infection isolates in waterborne outbreaks clustered with isolates from surface water and beavers implicated to be outbreak sources by public health. In-depth outbreak analysis demonstrated that beavers can act as amplification hosts for human infections and can act as sources of surface water contamination. It is also known that other wild and domesticated animals, as well as humans, can be sources of waterborne giardiasis. This study demonstrates the utility of WGS in furthering our understanding of Giardia transmission dynamics at the water-human-animal interface. IMPORTANCE Giardia duodenalis causes large numbers of gastrointestinal illness in humans. Its transmission through the contaminated surface water/wildlife intersect is significant, and the water-dwelling rodents beavers have been implicated as one important reservoir. To trace human infections to their source, we used genome techniques to characterize genetic relationships among 89 Giardia isolates from surface water, humans, and animals. Our study showed the presence of two previously described genetic assemblages, A and B, with mixed infections detected from isolates collected during outbreaks. Study findings also showed that while assemblage A could be divided into A1 and A2, A1 showed little genetic variation among animal and human hosts in isolates collected from across the globe. Assemblage B, the most common type found in the study surface water samples, was shown to be highly variable. Our study demonstrates that the beaver is a possible source of human infections from contaminated surface water, while acknowledging that theirs is only one role in the complex cycle of zoonotic spread. Mixes of parasite groups have been detected in waterborne outbreaks. More information on Giardia diversity and its evolution using genomics will further the understanding of the epidemiology of spread of this disease-causing protozoan.

scholarly journals Giardia spp. Are Commonly Found in Mixed Assemblages in Surface Water, as Revealed by Molecular and Whole-Genome Characterization

2015 ◽  
Vol 81 (14) ◽  
pp. 4827-4834 ◽  
Author(s):  
Natalie Prystajecky ◽  
Clement K.-M. Tsui ◽  
William W. L. Hsiao ◽  
Miguel I. Uyaguari-Diaz ◽  
Jordan Ho ◽  
...  
Keyword(s):  
Surface Water ◽  
Castor Canadensis ◽  
Giardia Duodenalis ◽  
Whole Genome ◽  
Gastrointestinal Infections ◽  
Canadian Province ◽  
Content Type ◽  
Waterborne Outbreak ◽  
Surface Water Samples ◽  
Assemblage B

ABSTRACTGiardiais the most common parasitic cause of gastrointestinal infections worldwide, with transmission through surface water playing an important role in various parts of the world.Giardia duodenalis(synonyms:G. intestinalisandG. lamblia), a multispecies complex, has two zoonotic subtypes, assemblages A and B. When British Columbia (BC), a western Canadian province, experienced several waterborne giardiasis outbreaks due to unfiltered surface drinking water in the late 1980s, collection of isolates from surface water, as well as from humans and beavers (Castor canadensis), throughout the province was carried out. To better understandGiardiain surface water, 71 isolates, including 29 from raw surface water samples, 29 from human giardiasis cases, and 13 from beavers in watersheds from this historical library were characterized by PCR. Study isolates also included isolates from waterborne giardiasis outbreaks. Both assemblages A and B were identified in surface water, human, and beavers samples, including a mixture of both assemblages A and B in waterborne outbreaks. PCR results were confirmed by whole-genome sequencing (WGS) for one waterborne outbreak and supported the clustering of human, water, and beaver isolates within both assemblages. We concluded that contamination of surface water byGiardiais complex, that the majority of our surface water isolates were assemblage B, and that both assemblages A and B may cause waterborne outbreaks. The higher-resolution data provided by WGS warrants further study to better understand the spread ofGiardia.

scholarly journals Classic and molecular study of Giardia duodenalis in children from a daycare center in the region of Presidente Prudente, São Paulo, Brazil

2009 ◽  
Vol 51 (1) ◽  
pp. 19-24 ◽  
Author(s):  
Nair Toshiko Tashima ◽  
Maria Jacira Silva Simões ◽  
Clarice Queico Fujimura Leite ◽  
Antonio Fluminhan ◽  
Marco Antonio Nogueira ◽  
...  
Keyword(s):  
Molecular Typing ◽  
Genomic Dna ◽  
Giardia Duodenalis ◽  
Molecular Study ◽  
Epidemiological Studies ◽  
Molecular Techniques ◽  
Parasite Transmission ◽  
Randomly Amplified Polymorphic Dna ◽  
Enterobius Vermicularis ◽  
Daycare Center

Epidemiological studies on giardiasis by using molecular techniques such as RAPD (Randomly Amplified Polymorphic DNA) may give information on factors related to the transmission of Giardia duodenalis. The aim of this work was to assess the epidemiology of G. duodenalis in 101 children attended at a daycare center in Presidente Bernardes, SP, Brazil. After parasitological examinations in feces samples, 15 children presented cysts of G. duodenalis. Their respective parents, brothers and pets, besides the daycare center workers, also had their feces submitted to parasitological analysis. Seven mothers and nine brothers also presented G. duodenalis cysts, while fathers, daycare workers and pets (dogs) did not presented the parasite. Besides the 15 cases with G. duodenalis, other 23 children presented other enteroparasites (Entamoeba coli, Endolimax nana, Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura). Samples of G. duodenalis cysts from children and their relatives were submitted to molecular typing by RAPD after genomic DNA extraction and amplification of a fragment of the 18S rDNA region by PCR. After examining 31 isolates of G. duodenalis (children and their respective mothers and brothers), it was concluded that the parasite transmission occurred in children, probably during daily cohabitation at the daycare center, but not at home among their relatives or pets.

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