31 research outputs found

    Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis

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    <p>Abstract</p> <p>Background</p> <p>Brucellosis is an important zoonosis caused by the genus <it>Brucella</it>. In addition <it>Brucella </it>represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for <it>Brucella </it>spp. genotyping.</p> <p>Results</p> <p>Seventeen DNA samples of <it>Brucella </it>strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA <it>Brucella </it>VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results.</p> <p>Conclusion</p> <p>In this paper we described a rapid and specific detection method for the characterization of <it>Brucella </it>isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.</p

    High throughput MLVA-16 typing for Brucella based on the microfluidics technology

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    <p>Abstract</p> <p>Background</p> <p>Brucellosis, a zoonosis caused by the genus <it>Brucella</it>, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of <it>Brucella </it>field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being <it>Brucella </it>a potential biological warfare agent. In the last years MLVA-16 has been described for <it>Brucella </it>spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for <it>Brucella </it>spp. by using of the microfluidics technology.</p> <p>Results</p> <p>The Caliper LabChip 90 equipment was evaluated for MLVA-16 typing of sixty-three <it>Brucella </it>samples. Furthermore, in order to validate the system, DNA samples previously resolved by sequencing system and Agilent technology, were <it>de novo </it>genotyped. The comparison of the MLVA typing data obtained by the Caliper equipment and those previously obtained by the other analysis methods showed a good correlation. However the outputs were not accurate as the Caliper DNA fragment sizes showed discrepancies compared with real data and a conversion table from observed to expected data was created.</p> <p>Conclusion</p> <p>In this paper we described the MLVA-16 using a rapid, sophisticated microfluidics technology for detection of amplification product sizes. The comparison of the MLVA typing data produced by Caliper LabChip 90 system with the data obtained by different techniques showed a general concordance of the results. Furthermore this platform represents a significant improvement in terms of handling, data acquiring, computational efficiency and rapidity, allowing to perform the strain genotyping in a time equal to one sixth respect to other microfluidics systems as e.g. the Agilent 2100 bioanalyzer.</p> <p>Finally, this platform can be considered a valid alternative to standard genotyping techniques, particularly useful dealing with a large number of samples in short time. These data confirmed that this technology represents a significative advancement in high-throughput accurate <it>Brucella </it>genotyping.</p

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

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    <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

    Antimicrobial susceptibility profiles and genotyping of Neisseria meningitidis of serogroup C, Italy, 2000–2020

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    BackgroundIn Italy the introduction of meningococcal C conjugate vaccine in 2005 has led to a significant reduction of invasive meningococcal disease (IMD) caused by Neisseria meningitidis of serogroup C (MenC). However, this serogroup is still responsible of sporadic cases, clusters and local outbreaks. The study aims to investigate the genotype and antimicrobial susceptibility profile of MenC isolates collected in Italy from 2000 to 2020.MethodsBacterial isolates and biological samples (blood or cerebrospinal fluid) from invasive meningococcal cases are collected and characterized at the National Reference Laboratory for IMD of Istituto Superiore di Sanità. Antimicrobial susceptibility was determined by MIC Test Strip Method and interpreted according to the EUCAST breakpoints guideline. Genotypic characteristics, including multi locus sequence typing (MLST), finetype, and antimicrobial resistance target genes were performed and analyzed using the PubMLST database. Genomic comparison of core genome MLST (cgMLST) of MenC genomes was also carried out.ResultsFrom 2000 to 2020, a total of 665 MenC isolates were investigated for antimicrobial susceptibility and 301 for genotyping. Over two decades, almost all MenC isolates resulted susceptible to antimicrobials with few isolates resulting resistant to ciprofloxacin (N = 2), penicillin G (N = 13), and rifampicin (N = 9), respectively. Molecular typing of MenC obtained from isolates or clinical specimens identified mostly the genotype C:P1.5-1,10-8:F3-6:ST-11(cc11). However, phylogenetic analysis, performed on genomes from MenC isolates, identified two sub lineages, 11.1 and 11.2, among cc11, of which the sub lineage 11.2 was the predominant.ConclusionWider application of the genomic analysis and monitoring of antimicrobial susceptibility represent key aspects of IMD surveillance and to monitor the continued evolution of these hyperinvasive strains

    Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

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    BACKGROUND: The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. RESULTS: Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. CONCLUSION: In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology

    Genome analysis of Legionella pneumophila ST23 from various countries reveals highly similar strains

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    © 2022 Ricci et al. This article is available under a CreativeCommons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks.info:eu-repo/semantics/publishedVersio

    In Vitro Processing of the 16S rRNA of the Thermophilic Archaeon Sulfolobus solfataricus

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    In this paper we have analyzed the processing in vitro of the 16S rRNA of the thermophilic archaeon Sulfolobus solfataricus, using pre-rRNA substrates transcribed in vitro and different protein preparations as the source of processing enzymes. We show that the 5′ external transcribed spacer of the S. solfataricus pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also existing in the early A0 and 0 processing sites of Saccharomyces cerevisiae and vertebrates. This site is present in other members of the kingdom Crenarchaeota but apparently not in the Euryarchaeota. Furthermore, S. solfataricus pre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S RNA sequence, in correspondence with a bulge-helix-bulge motif. The endonuclease responsible for this cleavage is present in both the Crenarchaeota and the Euryarchaeota. The processing pattern remained the same when the substrate was a 30S ribonucleoprotein particle instead of the naked RNA. Maturation of either the 5′ or the 3′ end of the 16S RNA molecule was not observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts

    A bifunctional archaeal protein that is a component of 30S ribosomal subunits and interacts with C/D box small RNAs

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    We have identified a novel archaeal protein that apparently plays two distinct roles in ribosome metabolism. It is a polypeptide of about 18 kDa (termed Rbp18) that binds free cytosolic C/D box sRNAs in vivo and in vitro and behaves as a structural ribosomal protein, specifically a component of the 30S ribosomal subunit. As Rbp18 is selectively present in Crenarcheota and highly thermophilic Euryarchaeota, we propose that it serves to protect C/D box sRNAs from degradation and perhaps to stabilize thermophilic 30S subunits
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