scholarly journals Multilocus Sequence Typing Methods for the Emerging Campylobacter Species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus

2012 ◽  
Vol 2 ◽  
Author(s):  
William G. Miller ◽  
Mary H. Chapman ◽  
Emma Yee ◽  
Stephen L. W. On ◽  
Desmond K. McNulty ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Typing Methods ◽  
Campylobacter Species

Distribution of virulence genes and the molecular epidemiology of Streptococcus pyogenes clinical isolates by emm and multilocus sequence typing methods

2020 ◽  
Author(s):  
Siti Nur Adila Hamzah ◽  
Mohd Nasir Mohd Desa ◽  
Azmiza Syawani Jasni ◽  
Niazlin Mohd Taib ◽  
Siti Norbaya Masri ◽  
...  
Keyword(s):  
Molecular Epidemiology ◽  
Virulence Factors ◽  
Streptococcus Pyogenes ◽  
Multilocus Sequence Typing ◽  
Virulence Genes ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Gold Standard Method ◽  
Non Invasive ◽  
Typing Methods

Abstract Background: Streptococcus pyogenes has a variety of virulence factors and the predominant invasive strains differ according to specific emm types and geographical orientation. Although emm typing is commonly used as the gold standard method for the molecular characterization, multilocus sequence typing (MLST) has become an important tool for comparing the genetic profiles globally. This study aimed to screen selected virulence genes from invasive and non-invasive clinical samples and to characterize the molecular epidemiology by emm typing and MLST methods. Methods: A total of 42 S. pyogenes isolates from invasive and non-invasive samples collected from 2014 to 2015 from two different tertiary hospitals were investigated for the distribution of virulence factors and their molecular epidemiology by emm and multilocus sequence typing methods. Detection of five virulence genes (speA , speB , speJ , ssa and sdaB) was performed using multiplex polymerase chain reaction (PCR) using the standard primers and established protocol. Results: Multiplex PCR analysis revealed that sdaB/speF (78.6%) and speB (61.9%) were the predominant virulence genes. Regardless of the type of invasiveness, diverse distribution of emm types/subtypes was noted which comprised of 27 different emm types/subtypes. The predominant emm types/subtypes were emm63 and emm18 with each gene accounted for 11.8% whereas 12% for each gene was noted for emm28, emm97.4 and emm91. The MLST revealed that the main sequence type (ST) in invasive samples was ST402 (17.7%) while ST473 and ST318 (12% for each ST) were the major types in non-invasive samples. Out of 18 virulotypes, Virulotype A (five genes, 55.6%) and Virulotype B (two genes, 27.8%) were the major virulotypes found in this study. Phylogenetic analysis indicated the presence of seven different clusters of S. pyogenes. Interestingly, Cluster VI showed that selected emm/ST types such as emm71/ST318 (n=2), emm70.1/ST318 (n=1), emm44/ST31 (n=1) and emm18/ST442 (n=1) have clustered within a common group (Virulotype A) for both hospitals studied. Conclusion: The present study showed that group A streptococci (GAS) are genetically diverse and possess virulence genes regardless of their invasiveness. Majority of the GAS exhibited no restricted pattern of virulotypes except for a few distinct clusters. Therefore, it can be concluded that virulotyping is partially useful for characterizing a heterogeneous population of GAS in hospitals.

Comparison of a newly developed binary typing with ribotyping and multilocus sequence typing methods for Clostridium difficile

2018 ◽  
Vol 147 ◽  
pp. 50-55
Author(s):  
Zhirong Li ◽  
Xiaolei Liu ◽  
Jianhong Zhao ◽  
Kaiyue Xu ◽  
Tiantian Tian ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Multilocus Sequence Typing ◽  
Typing Methods

Campylobacter Contamination during Poultry Slaughter in Belgium

2006 ◽  
Vol 69 (1) ◽  
pp. 27-33 ◽  
Author(s):  
G. RASSCHAERT ◽  
K. HOUF ◽  
J. VAN HENDE ◽  
L. de ZUTTER
Keyword(s):  
Alimentary Tract ◽  
Fragment Length Polymorphism ◽  
Pulsed Field Gel Electrophoresis ◽  
Species Level ◽  
Campylobacter Coli ◽  
Flagellin Gene ◽  
Thermophilic Campylobacter ◽  
Typing Methods ◽  
Campylobacter Species ◽  
Campylobacter Lari

The relation between internal carriage and surface contamination with thermophilic Campylobacter species in broilers was examined by molecular typing methods. Samples from 39 flocks were collected in three Belgian poultry slaughterhouses. From each flock, crop swabs before slaughter and intestines and neck skins during slaughter were collected. A total of 309 isolates were identified at species level and further characterized by flagellin gene A PCR/restriction fragment length polymorphism and pulsed-field gel electrophoresis. Isolates were identified as Campylobacter jejuni (90%), Campylobacter coli (8.7%), and Campylobacter lari (2.2%), and 27 genotypes could be distinguished by combining the two molecular methods. Seventy-two percent of the flocks arriving at the abattoir were colonized with campylobacters. After slaughter, 79% of the flocks had contaminated neck skins. In six flocks, genotypes isolated from the neck skins were also found in the alimentary tract from previously slaughtered flocks. Four of these flocks were initially free of Campylobacter. These four flocks might have had no contaminated carcasses after logistic slaughtering.

scholarly journals Molecular Differentiation of Mycoplasma gallisepticum Outbreaks: A Last Decade Study on Italian Farms Using GTS and MLST

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 665
Author(s):  
Andrea Matucci ◽  
Elisabetta Stefani ◽  
Michele Gastaldelli ◽  
Ilenia Rossi ◽  
Gelinda De Grandi ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Evolutionary Dynamics ◽  
Housekeeping Genes ◽  
Mycoplasma Gallisepticum ◽  
Economic Losses ◽  
Strain Identification ◽  
Molecular Differentiation ◽  
Typing Methods ◽  
Infection Monitoring ◽  
Monitoring Activity

Mycoplasma gallisepticum (MG) infects many avian species and leads to significant economic losses in the poultry industry. Transmission of this pathogen occurs both horizontally and vertically, and strategies to avoid the spread of MG rely on vaccination and the application of biosecurity measures to maintain breeder groups as pathogen-free. Two live attenuated MG vaccine strains are licensed in Italy: 6/85 and ts-11. After their introduction, the implementation of adequate genotyping tools became necessary to distinguish between field and vaccine strains and to guarantee proper infection monitoring activity. In this study, 40 Italian MG isolates collected between 2010–2019 from both vaccinated and unvaccinated farms were genotyped using gene-targeted sequencing (GTS) of the cythadesin gene mgc2 and multilocus sequence typing (MLST) based on six housekeeping genes. The discriminatory power of GTS typing ensures 6/85-like strain identification, but the technique does not allow the identification ts-11 strains; conversely, MLST differentiates both vaccine strains, describing more detailed interrelation structures. Our study describes MG genetic scenario within a mixed farming context. In conclusion, the use of adequate typing methods is essential to understand the evolutionary dynamics of MG strains in a particular area and to conduct epidemiological investigations in the avian population.

scholarly journals Development and validation of a Burkholderia pseudomallei core genome multilocus sequence typing scheme to facilitate molecular surveillance

2021 ◽  
Author(s):  
Sabine Lichtenegger ◽  
Trung T. Trinh ◽  
Karoline Assig ◽  
Karola Prior ◽  
Dag Harmsen ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Burkholderia Pseudomallei ◽  
Core Genome ◽  
Target Genes ◽  
Severe Disease ◽  
Recent Infection ◽  
Fast Analysis ◽  
Typing Scheme ◽  
Typing Methods ◽  
Good Concordance

Objectives: Burkholderia pseudomallei causes the severe disease melioidosis. Whole genome-sequencing (WGS) based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills and variable bioinformatic approaches, the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. Methods: A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,359 accessory targets. The scheme was validated with 320 WGS datasets. We compared our novel typing scheme with single nucleotide polymorphism based-approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. Results: The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigate not only closely related strains, but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugar cane field as the presumable source of a recent melioidosis case. Conclusion: We developed a robust cgMLST typing scheme which integrates high resolution, maximized standardization and fast analysis for the non-bioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

scholarly journals Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Steven J. Hancock ◽  
Minh-Duy Phan ◽  
Kate M. Peters ◽  
Brian M. Forde ◽  
Teik Min Chong ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Insertion Site ◽  
Epidemiological Studies ◽  
Multidrug Resistant ◽  
Complete Sequence ◽  
Putative Gene ◽  
Content Type ◽  
Group A ◽  
Typing Methods ◽  
Stable Maintenance

ABSTRACT Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae. They are associated with the dissemination of multiple clinically relevant resistance genes, including bla CMY and bla NDM. Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a bla NDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053. Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of bla NDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this bla NDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies.

Genetic analysis and pathogenicity of different sequence types of Streptococcus suis isolated from pigs in southern China

2020 ◽  
Vol 367 (6) ◽  
Author(s):  
Ling Peng ◽  
Mengting Lin ◽  
Zishu Huang ◽  
Sitao Guo ◽  
Hao Sun ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Associated Factors ◽  
Southern China ◽  
Streptococcus Suis ◽  
Genome Sequences ◽  
Swine Industry ◽  
Highly Pathogenic ◽  
Genetic Typing ◽  
Typing Methods ◽  
Sequence Types

ABSTRACT In this study, 52 Streptococcus suis isolates from pigs in southern China were divided into four known sequence types (STs) and six new STs, using multilocus sequence typing. Ten representative isolates were selected from 10 STs for the analysis of whole genome sequences. Virulence was assessed in 10 isolates, which were classified into three pathogenic groups. The prevalence of virulence-associated factors in the moderately pathogenic group was higher than that in the highly pathogenic group. The isolates from ST1 complex and serotype 2 were allocated to the moderately pathogenic group, while the isolates from the highly pathogenic group belonged to the non-ST1 complex and non-serotype 2. Three clusters were obtained based on multilocus sequence typing sequences: cluster III isolates from the nasal cavity of healthy pigs were classified into the highly pathogenic group and showed many peculiarities compared with cluster I and II isolates in virulence genotypes, genetic typing and pathogenesis, indicating a potential independent evolutionary line. Our results suggest that S. suis infections in China are becoming more complicated with constantly mutating isolates, which makes it difficult to distinguish their virulence by recognized typing methods. Thus, increased investigation and monitoring of these infections should be a priority for the swine industry in China.

scholarly journals Study of the population structure of Haemophilus parasuis by multilocus sequence typing

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3683-3690 ◽  
Author(s):  
Alex Olvera ◽  
Marta Cerdà-Cuéllar ◽  
Virginia Aragon
Keyword(s):  
Multilocus Sequence Typing ◽  
Group Method ◽  
Haemophilus Parasuis ◽  
Upper Respiratory Tract ◽  
Pair Group ◽  
Capsule Production ◽  
Clinical Background ◽  
Typing Methods ◽  
Almost All ◽  
Implementation Problems

Haemophilus parasuis is the aetiological agent of Glässer's disease in swine. In addition, this bacterium causes other clinical outcomes and can also be isolated from the upper respiratory tract of healthy pigs. Isolates of H. parasuis differ in phenotypic features (e.g. protein profiles, colony morphology or capsule production) and pathogenic capacity. Differences among strains have also been demonstrated at the genetic level. Several typing methods have been used to classify H. parasuis field strains, but they had resolution or implementation problems. To overcome these limitations, a multilocus sequence typing (MLST) system, using partial sequences of the house-keeping genes mdh, 6pgd, atpD, g3pd, frdB, infB and rpoB, was developed. Eleven reference strains and 120 field strains were included in this study. The number of alleles per locus ranged from 14 to 41, 6pgd being the locus with the highest diversity. The high genetic heterogeneity of this bacterium was confirmed with MLST, since the strains were divided into 109 sequence types, and only 13 small clonal complexes were detected by the Burst algorithm. Further analysis by unweighted-pair group method with arithmetic mean (UPGMA) identified six clusters. When the clinical background of the isolates was examined, one cluster was statistically associated with nasal isolation (putative non-virulent), while another cluster showed a significant association with isolation from clinical lesions (putative virulent). The remaining clusters did not show a statistical association with the clinical background of the isolates. Finally, although recombination among H. parasuis strains was detected, two divergent branches were found when a neighbour-joining tree was constructed with the concatenated sequences. Interestingly, one branch included almost all isolates of the putative virulent UPGMA cluster.

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