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      Species identification of Enterococcus spp: Whole genome sequencing compared to three biochemical test‐based systems and two Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry (MALDI‐TOF MS) systems

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          Abstract

          Aim

          Here, we evaluated the performance of two commercial MALDI‐TOF MS systems and three biochemical‐based systems and compared them to WGS as the gold standard for identifying isolates of vancomycin‐resistant enterococci (VRE).

          Methods

          A total of 87 VRE clinical isolates were included. The mass spectrometers were the Microflex system with Biotyper software 3.1 and the Vitek MS system. The biochemical‐based systems included the Vitek 2, Phoenix, and MicroScan WalkAway systems. WGS was performed on an Illumina MiSeq instrument using the MiSeq v3 reagent kit. Vancomycin resistance was determined according to CLSI criteria.

          Results

          Among the 87 VRE, 71 and 16 were identified as Enterococcus faecium and Enterococcus faecalis by WGS. All 71 E faecium were correctly identified by both mass spectrometers, as well as the Vitek 2 and Phoenix instruments. However, only 51 E faecium isolates were correctly identified by the MicroScan system. The most frequent misidentification was Enterococcus casseliflavus (n = 20). For vancomycin‐resistant E faecium, the Microflex Biotyper system had the highest sensitivity (85.54%), and all instruments (except for the Microscan) had a 100% specificity and PPV. Up to 87% of E faecalis isolates were misidentified by VITEK MS and VITEK2, 81% by Microscan and Phoenix, and 75% by Bruker biotyper.

          Conclusion

          As the coverage of type strain‐genome sequence database continues to grow and the cost of DNA sequencing continues to decrease, genome‐based identification can be a useful tool for diagnostic laboratories, with its superior accuracy even over MALDI‐TOF and database‐driven operations.

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          Most cited references12

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          Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

          Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic. We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured. Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%-32% cost of current methods of identification. MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in or =10 reference spectra per bacterial species and a 1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.
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            Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology.

            Nosocomial infections pose a significant threat to patient health; however, the gold standard laboratory method for determining bacterial relatedness (pulsed-field gel electrophoresis [PFGE]) remains essentially unchanged 20 years after its introduction. Here, we explored bacterial whole-genome sequencing (WGS) as an alternative approach for molecular strain typing. We compared WGS to PFGE for investigating presumptive outbreaks involving three important pathogens: vancomycin-resistant Enterococcus faecium (n=19), methicillin-resistant Staphylococcus aureus (n=17), and Acinetobacter baumannii (n=15). WGS was highly reproducible (average≤0.39 differences between technical replicates), which enabled a functional, quantitative definition for determining clonality. Strain relatedness data determined by PFGE and WGS roughly correlated, but the resolution of WGS was superior (P=5.6×10(-8) to 0.016). Several discordant results were noted between the methods. A total of 28.9% of isolates which were indistinguishable by PFGE were nonclonal by WGS. For A. baumannii, a species known to undergo rapid horizontal gene transfer, 16.2% of isolate pairs considered nonidentical by PFGE were clonal by WGS. Sequencing whole bacterial genomes with single-nucleotide resolution demonstrates that PFGE is prone to false-positive and false-negative results and suggests the need for a new gold standard approach for molecular epidemiological strain typing.
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              A Year of Infection in the Intensive Care Unit: Prospective Whole Genome Sequencing of Bacterial Clinical Isolates Reveals Cryptic Transmissions and Novel Microbiota

              Bacterial whole genome sequencing holds promise as a disruptive technology in clinical microbiology, but it has not yet been applied systematically or comprehensively within a clinical context. Here, over the course of one year, we performed prospective collection and whole genome sequencing of nearly all bacterial isolates obtained from a tertiary care hospital’s intensive care units (ICUs). This unbiased collection of 1,229 bacterial genomes from 391 patients enables detailed exploration of several features of clinical pathogens. A sizable fraction of isolates identified as clinically relevant corresponded to previously undescribed species: 12% of isolates assigned a species-level classification by conventional methods actually qualified as distinct, novel genomospecies on the basis of genomic similarity. Pan-genome analysis of the most frequently encountered pathogens in the collection revealed substantial variation in pan-genome size (1,420 to 20,432 genes) and the rate of gene discovery (1 to 152 genes per isolate sequenced). Surprisingly, although potential nosocomial transmission of actively surveilled pathogens was rare, 8.7% of isolates belonged to genomically related clonal lineages that were present among multiple patients, usually with overlapping hospital admissions, and were associated with clinically significant infection in 62% of patients from which they were recovered. Multi-patient clonal lineages were particularly evident in the neonatal care unit, where seven separate Staphylococcus epidermidis clonal lineages were identified, including one lineage associated with bacteremia in 5/9 neonates. Our study highlights key differences in the information made available by conventional microbiological practices versus whole genome sequencing, and motivates the further integration of microbial genome sequencing into routine clinical care.
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                Author and article information

                Contributors
                elvira_garza_gzz@yahoo.com
                Journal
                J Clin Lab Anal
                J. Clin. Lab. Anal
                10.1002/(ISSN)1098-2825
                JCLA
                Journal of Clinical Laboratory Analysis
                John Wiley and Sons Inc. (Hoboken )
                0887-8013
                1098-2825
                01 May 2020
                August 2020
                : 34
                : 8 ( doiID: 10.1002/jcla.v34.8 )
                : e23348
                Affiliations
                [ 1 ] Hospital Universitario “Dr. José Eleuterio González” Universidad Autónoma de Nuevo León Monterrey Mexico
                [ 2 ] Division of Infectious Diseases and Center for Antimicrobial Resistance and Microbial Genomics (CARMiG) McGovern Medical School UTHealth Houston TX USA
                [ 3 ] Centro Universitario de Ciencias de la Salud Hospital Civil de Guadalajara “Fray Antonio Alcalde” e Instituto de Patología Infecciosa y Experimental Universidad de Guadalajara Guadalajara Mexico
                [ 4 ] Facultad de Medicina Universidad de Colima Colima Mexico
                [ 5 ] Hospital General Dr. Manuel Gea González Mexico City Mexico
                [ 6 ] School of Public Health Center for Infectious Diseases UTHealth Houston TX USA
                [ 7 ] Molecular Genetics and Antimicrobial Resistance Unit and International Center for Microbial Genomics Universidad El Bosque Bogotá Colombia
                Author notes
                [*] [* ] Correspondence

                Elvira Garza‐González, Hospital Universitario ‘Dr. José Eleuterio González’, Universidad Autónoma de Nuevo León. Av. Francisco I. Madero S/N, Edificio Eugenio Clariond Garza, 2°Piso, Colonia Mitras Centro, C.P, Monterrey 64460, Mexico.

                Email: elvira_garza_gzz@ 123456yahoo.com

                Author information
                https://orcid.org/0000-0001-5831-9661
                https://orcid.org/0000-0003-1744-9173
                Article
                JCLA23348
                10.1002/jcla.23348
                7439347
                32358872
                cac2af27-5e6a-4d86-ad95-454af669f7af
                © 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 07 January 2020
                : 06 April 2020
                : 08 April 2020
                Page count
                Figures: 0, Tables: 2, Pages: 4, Words: 2238
                Categories
                Brief Report
                Brief Report
                Custom metadata
                2.0
                August 2020
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.7 mode:remove_FC converted:20.08.2020

                Clinical chemistry
                maldi tof,phoenix,vitek ms,whole genome sequenicng
                Clinical chemistry
                maldi tof, phoenix, vitek ms, whole genome sequenicng

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