Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips

sequencing of 6–8 housekeeping loci to assign unique sequence types. In this work we adapted MLST to a rapid microfluidics platform in order to enhance speed and reduce laboratory labor time. isolated in this study from one location in Rockville, Maryland (0.04 substitutions per site) was found to be as great as the global collection of isolates.Biogeographical investigation of pathogens is only one of a panoply of possible applications of microfluidics based MLST; others include microbiologic forensics, biothreat identification, and rapid characterization of human clinical samples

Multi-locus sequence typing of Escherichia coli isolates with acquired ampC genes and ampC promoter mutations

© 2016 Elsevier Inc. Multi-locus sequence typing was used to reveal a high degree of diversity amongst the E. coli isolates with AmpC plasmid genes, and a high prevalence of the −32 mutation present

First multi-locus sequence typing scheme for Arcobacter spp.

<p>Abstract</p> <p>Background</p> <p><it>Arcobacter </it>spp. are a common contaminant of food and water, and some species, primarily <it>A. butzleri </it>and <it>A. cryaerophilus</it>, have been isolated increasingly from human diarrheal stool samples. Here, we describe the first <it>Arcobacter </it>multilocus sequence typing (MLST) method for <it>A. butzleri</it>, <it>A. cryaerophilus</it>, <it>A. skirrowii, A. cibarius </it>and <it>A. thereius</it>.</p> <p>Results</p> <p>A sample set of 374 arcobacters, including 275 <it>A. butzleri</it>, 72 <it>A. cryaerophilus</it>, 15 <it>A. skirrowii </it>and 8 <it>A. cibarius </it>isolates from a wide variety of geographic locations and sources, was typed in this study. Additionally, this sample set contained four strains representing a new <it>Arcobacter </it>species, <it>A. thereius</it>. The seven loci used in the four-species <it>Arcobacter </it>MLST method are the same as those employed previously in <it>C. jejuni</it>, <it>C. coli</it>, <it>C. helveticus </it>and <it>C. fetus </it>(i.e. <it>aspA</it>, <it>atpA</it>(<it>uncA</it>), <it>glnA</it>, <it>gltA</it>, <it>glyA, pgm </it>and <it>tkt</it>). A large number of alleles were identified at each locus with the majority of isolates containing a unique sequence type. All <it>Arcobacter </it>isolates typed in this study contain two <it>glyA </it>genes, one linked to <it>lysS </it>(<it>glyA1</it>) and the other linked to <it>ada </it>(<it>glyA2</it>). <it>glyA1 </it>was incorporated into the <it>Arcobacter </it>MLST method while <it>glyA2 </it>was not because it did not increase substantially the level of discrimination.</p> <p>Conclusion</p> <p>No association of MLST alleles or sequence types with host or geographical source was observed with this sample set. Nevertheless, the large number of identified alleles and sequence types indicate that this MLST method will prove useful in both <it>Arcobacter </it>strain discrimination and in epidemiological studies of sporadic <it>Arcobacter</it>-related gastroenteritis. A new <it>Arcobacter </it>MLST database was created <url>http://pubmlst.org/arcobacter/</url>; allele and ST data generated in this study were deposited in this database and are available online.</p

The core genome multi-locus sequence typing of Mycoplasma anserisalpingitidis

Abstract Background: Mycoplasma anserisalpingitidis is a waterfowl pathogen that mainly infects geese, can cause significant economic losses and is present worldwide. With the advance of whole genome sequencing technologies, new methods are available for the researchers; one emerging methodology is the core genome Multi-Locus Sequence Typing (cgMLST). The core genome contains a high percentage of the coding DNA sequence (CDS) set of the studied strains. The cgMLST schemas are powerful genotyping tools allowing for the investigation of potential epidemics, and precise and reliable classification of the strains. Although whole genome sequences of M. anserisalpingitidis strains are available, to date, no cgMLST schema has been published for this species. Results: In this study, Illumina short reads of 81 M. anserisalpingitidis strains were used, including samples from Hungary, Poland, Sweden, and China. Draft genomes were assembled with the SPAdes software and analysed with the online available chewBBACA program. User made modifications in the program enabled analysis of mycoplasmas and provided similar results as the conventional SeqSphere+ software. The threshold of the presence of CDS in the strains was set to 93% due to the quality of the draft genomes, resulting in the most accurate and robust schema. Three hundred thirty-one CDSs constituted our cgMLST schema (representing 42,77% of the whole CDS set of M. anserisalpingitidis ATCC BAA-2147), and a Neighbor joining tree was created using the allelic profiles. The correlation was observed between the strains’ cgMLST profile and geographical origin; however, strains from the same integration but different locations also showed close relationship. Strains isolated from different tissue samples of the same animal revealed highly similar cgMLST profiles. Conclusions: The Neighbor joining tree from the cgMLST schema closely resembled the real-life spatial and temporal relationships of the strains. The incongruences between background data and the cgMLST profile in the strains from the same integration can be because of the higher probability of contacts between the flocks. This schema can help with the epidemiological investigation and can be used as a basis for further studies. Keywords: cgMLST, chewBBACA, Genotyping, Mycoplasma anserisalpingitidis, Waterfowl, Whole genome sequencin

First report on methicillin-resistant Staphylococcus aureus of Spa type T037, Sequence type 239, SCCmec type III/IIIA in Malaysia

Methicillin-resistant Staphylococcus aureus (MRSA) from Malaysia were shown to possess staphylococcal cassette chromosome mec (SCCmec)-III and IIIA. Spa sequencing and multi-locus sequence typing (MLST) documented t037 and ST 239 (CC8) for 83.3% of the isolates. This confirms observations in several other Far Eastern countries and corroborates the epidemicity of this clone

Comparison of classical multi-locus sequence typing software for next-generation sequencing data

Multi-locus sequence typing (MLST) is a widely used method for categorizing bacteria. Increasingly, MLST is being performed using next-generation sequencing (NGS) data by reference laboratories and for clinical diagnostics. Many software applications have been developed to calculate sequence types from NGS data; however, there has been no comprehensive review to date on these methods. We have compared eight of these applications against real and simulated data, and present results on: (1) the accuracy of each method against traditional typing methods, (2) the performance on real outbreak datasets, (3) the impact of contamination and varying depth of coverage, and (4) the computational resource requirements

Antimicrobial Resistance And Molecular Epidemiology Of Escherichia Coli Isolated From Urban And Rural River Systems

Antimicrobial resistance (AMR) is an emerging public health issue that threatens the efficacy of antibiotic treatment for bacterial infections and human health. Sources of antimicrobial resistance genes (ARGs) in the environment include wastewater treatment plants and animal feeding operations that discharge waste into waterways, such as rivers and streams. This retrospective descriptive study describes the presence of AMR, and specific ARGs in Escherichia coli isolates from two distinct watersheds, rural and urban, with the use of antimicrobial susceptibility testing, whole-genome sequencing (WGS) to detect ARGs, and multi-locus sequence typing. Antimicrobial susceptibility testing was performed for 143 E. coli isolates, 73 originating from a rural watershed and 70 originating from an urban watershed. E. coli isolates from the rural watershed had a significantly higher prevalence of phenotypic non-susceptibility and ARGs for tetracycline (21.9% vs. 2.9%, p \u3c 0.01) when compared to urban watershed isolates. Based on phenotypic-susceptibility testing, WGS data of 68 isolates were annotated for ARGs. These data were used for the prediction of antimicrobial susceptibility, demonstrating high accuracy for the prediction of non-susceptibility for tetracycline, trimethoprim-sulfamethoxazole, and cephalosporins. WGS multi-locus sequence typing (MLST) yielded 47 sequence types, dominated by ST58 (n=6), ST10 (n=5), and ST155 (n=4). Waterways are important reservoirs and disseminators of antimicrobial-resistant bacteria (ARB) and ARGs. The evaluation and monitoring of AMR and ARGs in aquatic environments will lead to improved health through better prevention and control of E. coli infections

An Assessment of Different Genomic Approaches for Inferring Phylogeny of Listeria monocytogenes

Background/objectives: Whole genome sequencing (WGS) has proven to be a powerful subtyping tool for foodborne pathogenic bacteria like L. monocytogenes. The interests of genome-scale analysis for national surveillance, outbreak detection or source tracking has been largely documented. The genomic data however can be exploited with many different bioinformatics methods like single nucleotide polymorphism (SNP), core-genome multi locus sequence typing (cgMLST), whole-genome multi locus sequence typing (wgMLST) or multi locus predicted protein sequence typing (MLPPST) on either core-genome (cgMLPPST) or pan-genome (wgMLPPST). Currently, there are little comparisons studies of these different analytical approaches. Our objective was to assess and compare different genomic methods that can be implemented in order to cluster isolates of L. monocytogenes.Methods: The clustering methods were evaluated on a collection of 207 L. monocytogenes genomes of food origin representative of the genetic diversity of the Anses collection. The trees were then compared using robust statistical analyses.Results: The backward comparability between conventional typing methods and genomic methods revealed a near-perfect concordance. The importance of selecting a proper reference when calling SNPs was highlighted, although distances between strains remained identical. The analysis also revealed that the topology of the phylogenetic trees between wgMLST and cgMLST were remarkably similar. The comparison between SNP and cgMLST or SNP and wgMLST approaches showed that the topologies of phylogenic trees were statistically similar with an almost equivalent clustering.Conclusion: Our study revealed high concordance between wgMLST, cgMLST, and SNP approaches which are all suitable for typing of L. monocytogenes. The comparable clustering is an important observation considering that the two approaches have been variously implemented among reference laboratories