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      Isolation of five Enterobacteriaceae species harbouring bla NDM-1 and mcr-1 plasmids from a single paediatric patient

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          Abstract

          In Argentina, NDM metallo-β-lactamase was first reported in 2013. By now, it has disseminated throughout the country in diverse Gram negative bacteria. Here, we report the case of a paediatric patient that underwent a 1-year hospitalisation due to erythrodermic psoriasis in 2014 and received multiple antimicrobial treatments. During his stay, five isolates were obtained from rectal swabs (rs) or blood culture (bc) suspicious of carbapenemase production: a K. quasipneumoniae subsp. quasipneumoniae (rs), Citrobacter freundii (rs), Escherichia coli (bc), Enterobacter cloacae (rs), and a Serratia marcescens (bc). The isolates were studied with broth microdilution, biparental conjugation and plasmid and whole genome sequencing (Illumina). All isolates harboured an 138,998-bp type 1 IncC plasmid that carried bla NDM-1, ble MBL, bla CMY-6, rmtC, aac(6’)-Ib, and sul1 resistance genes. Additionally, the bla NDM-plasmids contained IS Kpn8 an insertion sequence previously described as associated only to bla KPC. One isolate, a colistin-resistant E. coli, also carried a mcr-1-containing an IncI2 plasmid, which did not harbour additional resistance. The whole genome of K. quasipneumoniae subsp. quasipneumoniae isolate was fully sequenced. This isolate harboured, additionally to bla NDM, three plasmid-mediated quinolone resistance genes: qnrB4, qnrB52 and aac(6’)-Ib-cr1. The E. cloacae isolate also harboured qnrA1. These findings alert to the underestimated horizontal dissemination of multidrug-resistant plasmids limiting treatment options with last resort antimicrobials.

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          SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments

          Rapidly decreasing genome sequencing costs have led to a proportionate increase in the number of samples used in prokaryotic population studies. Extracting single nucleotide polymorphisms (SNPs) from a large whole genome alignment is now a routine task, but existing tools have failed to scale efficiently with the increased size of studies. These tools are slow, memory inefficient and are installed through non-standard procedures. We present SNP-sites which can rapidly extract SNPs from a multi-FASTA alignment using modest resources and can output results in multiple formats for downstream analysis. SNPs can be extracted from a 8.3 GB alignment file (1842 taxa, 22 618 sites) in 267 seconds using 59 MB of RAM and 1 CPU core, making it feasible to run on modest computers. It is easy to install through the Debian and Homebrew package managers, and has been successfully tested on more than 20 operating systems. SNP-sites is implemented in C and is available under the open source license GNU GPL version 3.
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            Resistance plasmid families in Enterobacteriaceae.

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              Rapid Detection of Carbapenemase-producing Enterobacteriaceae

              To rapidly identify carbapenemase producers in Enterobacteriaceae, we developed the Carba NP test. The test uses isolated bacterial colonies and is based on in vitro hydrolysis of a carbapenem, imipenem. It was 100% sensitive and specific compared with molecular-based techniques. This rapid (<2 hours), inexpensive technique may be implemented in any laboratory.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Software
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: Visualization
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: ValidationRole: Visualization
                Role: Data curationRole: MethodologyRole: Visualization
                Role: Data curationRole: MethodologyRole: Visualization
                Role: Data curationRole: Formal analysisRole: ValidationRole: Writing – review & editing
                Role: ResourcesRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Formal analysisRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                9 September 2019
                2019
                : 14
                : 9
                : e0221960
                Affiliations
                [1 ] Servicio Antimicrobianos (National Reference Laboratory on Antimicrobial Resistance), Instituto Nacional de Enfermedades Infecciosas-ANLIS “Dr. Carlos G. Malbrán”, Ciudad Autónoma de Buenos Aires, Argentina
                [2 ] Public Health Ontario Laboratories, Toronto, Ontario, Canada
                [3 ] The Welcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom
                [4 ] Liverpool School of Tropical Medicine, Liverpool, United Kingdom
                [5 ] Hospital de Niños “Dr. Ricardo Gutiérrez”, Ciudad Autónoma de Buenos Aires, Argentina
                [6 ] London School of Hygiene and Tropical Medicine, London, United Kingdom
                Leibniz-Institute DSMZ, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-9106-1762
                Article
                PONE-D-19-07438
                10.1371/journal.pone.0221960
                6733481
                31498841
                5e86f365-3a69-4499-894e-ccfa89c9f277
                © 2019 Martino et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 March 2019
                : 19 August 2019
                Page count
                Figures: 2, Tables: 1, Pages: 13
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100006668, Fondo para la Investigación Científica y Tecnológica;
                Award ID: PICT2012-0145
                Award Recipient : Sonia Alejandra Gomez
                This research was funded by Agencia Nacional de Promoción Científica y Tecnológica - Fondo para la Investigación Científica y Tecnológica, PICT-2012-0145 to SAG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Bacteria
                Klebsiella
                Klebsiella Pneumoniae
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Klebsiella
                Klebsiella Pneumoniae
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Klebsiella
                Klebsiella Pneumoniae
                Biology and life sciences
                Genetics
                DNA
                Forms of DNA
                Plasmids
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                Forms of DNA
                Plasmids
                Biology and Life Sciences
                Genetics
                Genetic Elements
                Mobile Genetic Elements
                Plasmids
                Biology and Life Sciences
                Genetics
                Genomics
                Mobile Genetic Elements
                Plasmids
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobial Resistance
                Medicine and Health Sciences
                Pharmacology
                Antimicrobial Resistance
                Biology and Life Sciences
                Organisms
                Bacteria
                Enterobacteriaceae
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Biology and Life Sciences
                Taxonomy
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Computer and Information Sciences
                Data Management
                Taxonomy
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Biology and Life Sciences
                Anatomy
                Digestive System
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                Rectum
                Medicine and Health Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
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                Physical Sciences
                Chemistry
                Analytical Chemistry
                Mass Spectrometry
                Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
                Research and Analysis Methods
                Spectrum Analysis Techniques
                Mass Spectrometry
                Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
                Medicine and Health Sciences
                Pharmacology
                Drugs
                Antimicrobials
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobials
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