Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

<p>Abstract</p> <p>Background</p> <p>Anthrax and plague are diseases caused by <it>Bacillus anthracis </it>and <it>Yersinia pestis </it>respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination.</p> <p>Results</p> <p>Thirty-nine <it>B. anthracis </it>and ten <it>Y. pestis </it>strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA). Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer) and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced.</p> <p>Conclusion</p> <p>In this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of <it>B. anthracis </it>and <it>Y. pesti</it>s. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.</p

A rapid allele variant discrimination method for Yersinia pestis strains based on high-resolution melting curve analysis

Yersinia pestis isolates were genotyped analyzing the polymorphic DNA regions named variable number tandem repeats (VNTR). Allele variants were studied by high-resolution melting analysis (HRMA) of polymerase chain reaction fragments obtained for 25 VNTR loci. After comparison with previous results, 14 loci gave distinguishable normalized melting curves and allowed to correctly assign alleles. This HRMA typing technique permits to differentiate Y. pestis isolates and turned out to be robust, reproducible, and cheap. (C) 2009 Elsevier Inc. All rights reserved

A rapid allele variant discrimination method for Yersinia pestis strains based on high-resolution melting curve analysis.

International audienceYersinia pestis isolates were genotyped analyzing the polymorphic DNA regions named variable number tandem repeats (VNTR). Allele variants were studied by high-resolution melting analysis (HRMA) of polymerase chain reaction fragments obtained for 25 VNTR loci. After comparison with previous results, 14 loci gave distinguishable normalized melting curves and allowed to correctly assign alleles. This HRMA typing technique permits to differentiate Y. pestis isolates and turned out to be robust, reproducible, and cheap

A new genotyping scheme based on MLVA for inter-laboratory surveillance of Streptococcus pyogenes.

International audienceA newly developed MLVA seven-loci scheme for Streptococcus pyogenes is described. The method can be successfully applied by using both agarose gel with visual inspections of bands and Lab on Chip technology. The potential of the present MLVA has been tested on a collection of 100 clinical GAS strains representing the most common emm types found in high-income countries plus 18 published gap-free genomes, in comparison to PFGE and MLST. The MLVA analysis defined 30 MLVA types with ten out of the considered 15 emm types exhibiting multiple and specific MLVA types. In only one occasion the same MLVA profile was shared between isolates belonging to two different emm types. A robust congruency between the methods was observed, with MLVA discriminating within clonal complexes as defined by PFGE or MLST. This new MLVA scheme can be adopted as a quick, low-cost and reliable typing method to track the short-term diffusion of GAS clones in inter-laboratory-based surveillance

Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1

Effective diagnostic and therapeutic strategies are needed to control and combat the highly pathogenic avian influenza virus (AIV) subtype H5N1. To this end, we developed human monoclonal antibodies (mAbs) in single chain fragment variable (scFv) format towards the H5N1 avian influenza virus to gain new insights for the development of immunotherapy against human cases of H5N1. Using a biopanning based approach a large array of scFvs against H5N1 virus were isolated from the human semi-synthetic ETH-2 phage antibody library. H5N1 ELISA-positive scFvs with unique variable heavy (VH) and light (VL) chain gene sequences showed different biochemical properties and neutralization activity across H5N1 viral strains. In particular, the scFv clones AV.D1 and AV.C4 exerted a significant inhibition of the H5N1 A/Vietnam/1194/2004 virus infection in a pseudotype-based neutralization assay. Interestingly, these two scFvs displayed a cross-clade neutralizing activity versus A/whoopingswan/Mongolia/244/2005 and A/Indonesia/5/2005 strains. These studies provide proof of the concept that human mAbs in scFv format with well-defined H5N1 recognition patterns and in vitro neutralizing activity can be easily and rapidly isolated by biopanning selection of an entirely artificial antibody repertoire using inactivated H5N1 virus as a bait

Whole genome sequencing for the molecular characterization of carbapenem-resistant Klebsiella pneumoniae strains isolated at the Italian ASST Fatebenefratelli Sacco Hospital, 2012–2014

Abstract Background The emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment. Methods Sixty-eight carbapenemase-producing K. pneumoniae strains isolated at Luigi Sacco University Hospital-ASST Fatebenefratelli Sacco (Milan, Italy) between 2012 and 2014 were characterised microbiologically and molecularly. They were tested for drug susceptibility and carbapenemase phenotypes, investigated by means of repetitive extra-genic palindromic polymerase chain reaction (REP-PCR), and fully sequenced by means of next-generation sequencing for the in silico analysis of multi-locus sequence typing (MLST), their resistome, virulome and plasmid content, and their core single nucleotide polymorphism (SNP) genotypes. Results All of the samples were resistant to carbapenems, other β-lactams and ciprofloxacin; many were resistant to aminoglycosides and tigecycline; and seven were resistant to colistin. Resistome analysis revealed the presence of blaKPC genes and, less frequently blaSHV, blaTEM, blaCTX-M and blaOXA, which are related to resistance to carbapenem and other β-lactams. Other genes conferring resistance to aminoglycoside, fluoroquinolone, phenicol, sulphonamide, tetracycline, trimethoprim and macrolide-lincosamide-streptogramin were also detected. Genes related to AcrAB-TolC efflux pump-dependent and pump-independent tigecycline resistance mechanisms were investigated, but it was not possible to clearly correlate the genomic features with tigecycline resistance because of the presence of a common mutation in susceptible, intermediate and resistant strains. Concerning colistin resistance, the mgrB gene was disrupted by an IS5-like element, and the mobile mcr-1 and mcr-2 genes were not detected in two cases. The virulome profile revealed type-3 fimbriae and iron uptake system genes, which are important during the colonisation stage in the mammalian host environment. The in silico detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX1, IncX3, IncFII(K), IncN, IncL/M(pMU407) and IncFIA(HI1). REP-PCR showed five major clusters, and MLST revealed six different sequence types: 512, 258, 307, 1519, 745 and 101. Core SNP genotyping, which led to four clusters, correlated with the MLST data. Isolates of the same sequencing type often had common genetic traits, but the SNP analysis allowed greater strain tracking and discrimination than either the REP-PCR or MLST analysis. Conclusion Our findings support the importance of implementing bacterial genomics in clinical medicine in order to complement traditional methods and overcome their limited resolution

UPGMA dendrogram showing clustering and linkage distances of strains used to validate Agilent Bioanalyzer 2100 based genotyping

<p><b>Copyright information:</b></p><p>Taken from "Fieldable genotyping of and based on 25-loci Multi Locus VNTR Analysis"</p><p>http://www.biomedcentral.com/1471-2180/8/21</p><p>BMC Microbiology 2008;8():21-21.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2257963.</p><p></p> The results obtained by previously described method [15] and Bioanalyzer genotyping are identical

UPGMA dendrogram showing clustering and linkage distances of strains used to validate the Bioanalyzer 25 MLVA genotyping

<p><b>Copyright information:</b></p><p>Taken from "Fieldable genotyping of and based on 25-loci Multi Locus VNTR Analysis"</p><p>http://www.biomedcentral.com/1471-2180/8/21</p><p>BMC Microbiology 2008;8():21-21.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2257963.</p><p></p> The results obtained by previously described method [16] and Bioanalyzer genotyping are identical