Diving Deep Into Hospital-Acquired Legionella pneumophila With Whole-Genome Sequencing

Background: Hospital-acquired infections pose a significant threat to patient health. Laboratories are starting to consider whole-genome sequencing (WGS) as a molecular method for outbreak detection and epidemiological surveillance. The objective of this study was to assess the use of the iSeq100 platform (Illumina, San Diego, CA) for accurate sequencing and WGS-based outbreak detection using the bioMérieux EPISEQ CS, a novel cloud-based software for sequence assembly and data analysis. Methods: In total, 25 isolates, including 19 MRSA isolates and 6 ATCC strains were evaluated in this study: A. baumannii ATCC 19606, B. cepacia ATCC 25416, E. faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. DNA extraction of all isolates was performed on the QIAcube (Qiagen, Hilden, Germany) using the DNEasy Ultra Clean Microbial kit extraction protocol. DNA libraries were prepared for WGS using the Nextera DNA Flex Library Prep Kit (Illumina) and sequenced at 2×150-bp on the iSeq100 according to the manufacturer’s instructions. The 19 MRSA isolates were previously characterized by the DiversiLab system (bioMérieux, France). Upon validation of the iSeq100 platform, a new outbreak analysis was performed using WGS analysis using EPISEQ CS. ATCC sequences were compared to assembled reference genomes from the NCBI GenBank to assess the accuracy of the iSeq100 platform. The FASTQ files were aligned via BowTie2 version 2.2.6 software, using default parameters, and FreeBayes version 1.1.0.46-0 was used to call homozygous single-nucleotide polymorphisms (SNPs) with a minimum coverage of 5 and an allele frequency of 0.87 using default parameters. ATCC sequences were analyzed using ResFinder version 3.2 and were compared in silico to the reference genome. Results: EPISEQ CS classified 8 MRSA isolates as unrelated and grouped 11 isolates into 2 separate clusters: cluster A (5 isolates) and cluster B (6 isolates) with similarity scores of ≥99.63% and ≥99.50%, respectively. This finding contrasted with the previous characterization by DiversiLab, which identified 3 clusters of 2, 8, and 11 isolates, respectively. The EPISEQ CS resistome data detected the mecA gene in 18 of 19 MRSA isolates. Comparative analysis of the ATCCsequences to the reference genomes showed 99.9986% concordance of SNPs and 100.00% concordance between the resistance genes present. Conclusions: The iSeq100 platform accurately sequenced the bacterial isolates and could be an affordable alternative in conjunction with EPISEQ CS for epidemiological surveillance analysis and infection prevention.Funding: NoneDisclosures: None

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Transmission of ESBL-producing Enterobacteriaceae and their mobile genetic elements—identification of sources by whole genome sequencing: study protocol for an observational study in Switzerland

BMJ Open ◽

10.1136/bmjopen-2018-021823 ◽

2018 ◽

Vol 8 (2) ◽

pp. e021823 ◽

Cited By ~ 12

Author(s):

Tanja Stadler ◽

Dominik Meinel ◽

Lisandra Aguilar-Bultet ◽

Jana S Huisman ◽

Ruth Schindler ◽

...

Keyword(s):

Observational Study ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bacterial Species ◽

Mobile Genetic Elements ◽

University Hospital ◽

Whole Genome ◽

Hospital Acquired Infections ◽

Genetic Elements ◽

Hospital Acquired

IntroductionExtended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae were first described in relation with hospital-acquired infections. In the 2000s, the epidemiology of ESBL-producing organisms changed as especially ESBL-producingEscherichia coliwas increasingly described as an important cause of community-acquired infections, supporting the hypothesis that in more recent years ESBL-producing Enterobacteriaceae have probably been imported into hospitals rather than vice versa. Transmission of ESBL-producing Enterobacteriaceae is complicated by ESBL genes being encoded on self-transmissible plasmids, which can be exchanged among the same and different bacterial species. The aim of this research project is to quantify hospital-wide transmission of ESBL-producing Enterobacteriaceae on both the level of bacterial species and the mobile genetic elements and to determine if hospital-acquired infections caused by ESBL producers are related to strains and mobile genetic elements predominantly circulating in the community or in the healthcare setting. This distinction is critical in prevention since the former emphasises the urgent need to establish or reinforce antibiotic stewardship programmes, and the latter would call for more rigorous infection control.Methods and analysisThis protocol presents an observational study that will be performed at the University Hospital Basel and in the city of Basel, Switzerland. ESBL-producing Enterobacteriaceae will be collected from any specimens obtained by routine clinical practice or by active screening in both inpatient and outpatient settings, as well as from wastewater samples and foodstuffs, both collected monthly over a 12-month period for analyses by whole genome sequencing. Bacterial chromosomal, plasmid and ESBL-gene sequences will be compared within the cohort to determine genetic relatedness and migration between humans and their environment.Ethics and disseminationThis study has been approved by the local ethics committee (Ethikkommission Nordwest-und Zentralschweiz) as a quality control project (Project-ID 2017–00100). The results of this study will be published in peer-reviewed medical journals, communicated to participants, the general public and all relevant stakeholders.

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Outbreak of Vancomycin-resistant Enterococcus faecium in Interventional Radiology: Detection Through Whole-genome Sequencing-based Surveillance

Clinical Infectious Diseases ◽

10.1093/cid/ciz666 ◽

2019 ◽

Vol 70 (11) ◽

pp. 2336-2343 ◽

Cited By ~ 7

Author(s):

Alexander J Sundermann ◽

Ahmed Babiker ◽

Jane W Marsh ◽

Kathleen A Shutt ◽

Mustapha M Mustapha ◽

...

Keyword(s):

Interventional Radiology ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Enterococcus Faecium ◽

Whole Genome ◽

Training Video ◽

Prevention Interventions ◽

Vancomycin Resistant ◽

Record Review ◽

Hospital Acquired

Abstract Background Vancomycin-resistant enterococci (VRE) are a major cause of hospital-acquired infections. The risk of infection from interventional radiology (IR) procedures is not well documented. Whole-genome sequencing (WGS) surveillance of clinical bacterial isolates among hospitalized patients can identify previously unrecognized outbreaks. Methods We analyzed WGS surveillance data from November 2016 to November 2017 for evidence of VRE transmission. A previously unrecognized cluster of 10 genetically related VRE (Enterococcus faecium) infections was discovered. Electronic health record review identified IR procedures as a potential source. An outbreak investigation was conducted. Results Of the 10 outbreak patients, 9 had undergone an IR procedure with intravenous (IV) contrast ≤22 days before infection. In a matched case-control study, preceding IR procedure and IR procedure with contrast were associated with VRE infection (matched odds ratio [MOR], 16.72; 95% confidence interval [CI], 2.01 to 138.73; P = .009 and MOR, 39.35; 95% CI, 7.85 to infinity; P < .001, respectively). Investigation of IR practices and review of the manufacturer’s training video revealed sterility breaches in contrast preparation. Our investigation also supported possible transmission from an IR technician. Infection prevention interventions were implemented, and no further IR-associated VRE transmissions have been observed. Conclusions A prolonged outbreak of VRE infections related to IR procedures with IV contrast resulted from nonsterile preparation of injectable contrast. The fact that our VRE outbreak was discovered through WGS surveillance and the manufacturer’s training video that demonstrated nonsterile technique raise the possibility that infections following invasive IR procedures may be more common than previously recognized.

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Whole-genome sequencing for identification of the source in hospital-acquired Legionnaires' disease

Journal of Hospital Infection ◽

10.1016/j.jhin.2017.04.020 ◽

2017 ◽

Vol 96 (4) ◽

pp. 392-395 ◽

Cited By ~ 7

Author(s):

A.M. Rosendahl Madsen ◽

A. Holm ◽

T.G. Jensen ◽

E. Knudsen ◽

H. Lundgaard ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Legionnaires Disease ◽

Hospital Acquired

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Comparison of the Legionella pneumophila population structure as determined by sequence-based typing and whole genome sequencing

BMC Microbiology ◽

10.1186/1471-2180-13-302 ◽

2013 ◽

Vol 13 (1) ◽

pp. 302 ◽

Cited By ~ 33

Author(s):

Anthony P Underwood ◽

Garan Jones ◽

Massimo Mentasti ◽

Norman K Fry ◽

Timothy G Harrison

Keyword(s):

Population Structure ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Legionella Pneumophila ◽

Whole Genome

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Using Whole-Genome Sequencing to Improve Surveillance Measures: Case Study of Methicillin-Resistant Staphylococcus aureus (MRSA) in a Florida Hospital

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.1186 ◽

2020 ◽

Vol 41 (S1) ◽

pp. s505-s506

Author(s):

Susanna Lamers ◽

David Nolan ◽

Yvette Simone McCarter ◽

Chad Neilsen ◽

Rebecca Rose ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Infection Control ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

The United States ◽

Whole Genome ◽

City Hospital ◽

Methicillin Resistant ◽

Clinical Records ◽

Hospital Acquired

Background: The CDC considers methicillin-resistant Staphylococcus aureus (MRSA) one of the most important hospital-acquired infections (HAIs) in the United States. However, infection control departments (ICDs) often rely on subjective data to determine whether multiple MRSA cases are a true outbreak and whether the hospital is responsible (community- vs hospital-acquired). Objective: Our objective was to determine whether whole-genome sequencing (WGS) of MRSA provided new insights into on transmission dynamics at large, inner-city hospital in Jacksonville, Florida. Methods: Over a 4-month period, MRSA samples were obtained from 44 infected patients at 3 campuses within a single hospital system. Limited nonpatient identifying information was obtained, including date of collection, campus, unit, reason for admission, and days post admission (DPA) of MRSA diagnosis. Whole-genome sequences were generated using the Illumina platform. Raw reads were processed, and genetic distances were calculated and used to identify genetically linked bacterial infections using FoxSeq version 1.0 software. Results: Based on their length of stay, 10 patients were reported by the ICD as obtaining an HAI. Three distinct “episodes” were evident in which >5 MRSA cases were observed within a 3–5-day period. Genomic analysis identified 5 clusters of linked infections: 4 clusters contained 2 patients and another contained 3. Of these clusters, only 1 contained multiple cases that were reported as HAIs; however, because these case were separated by 2 weeks, it is unlikely that they came from a source in the hospital. The results suggest that HAIs were overreported and that most MRSA present in the hospital likely came from community sources. Conclusions: WGS provided clear evidence that temporally clustered MRSA cases do not imply an outbreak is occurring. Furthermore, ongoing detection of the same community-acquired infections over several months is indicative of a shared source outside of the hospital, which could be uncovered through examination of clinical records. Considering the implications of HAIs, best approaches to combat them should include identifying their sources. As molecular surveillance approaches to infection control are rapidly becoming easier and less expensive, the methods can be used to bring objective clarity to the ICD.Funding: NoneDisclosures: Susanna L. Lamers reports salary from BioInfoExperts and contract research for the NIH, the University of California - San Francisco, and UMASS Medical School.

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Tracking Cefoperazone/Sulbactam Resistance Development In vivo in A. baumannii Isolated from a Patient with Hospital-Acquired Pneumonia by Whole-Genome Sequencing

Frontiers in Microbiology ◽

10.3389/fmicb.2016.01268 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 1

Author(s):

Xiaofen Liu ◽

Huajun Zheng ◽

Weipeng Zhang ◽

Zhen Shen ◽

Miao Zhao ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Resistance Development ◽

Hospital Acquired Pneumonia ◽

Hospital Acquired

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Genomic Resolution of Outbreak-Associated Legionella pneumophila Serogroup 1 Isolates from New York State

Applied and Environmental Microbiology ◽

10.1128/aem.00362-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3582-3590 ◽

Cited By ~ 23

Author(s):

Brian H. Raphael ◽

Deborah J. Baker ◽

Elizabeth Nazarian ◽

Pascal Lapierre ◽

Dianna Bopp ◽

...

Keyword(s):

New York ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Legionella Pneumophila ◽

New York State ◽

Whole Genome ◽

Content Type ◽

York State ◽

Laboratory Investigations ◽

Pfge Profiles

ABSTRACTA total of 30Legionella pneumophilaserogroup 1 isolates representing 10 separate legionellosis laboratory investigations (“outbreaks”) that occurred in New York State between 2004 and 2012 were selected for evaluation of whole-genome sequencing (WGS) approaches for molecular subtyping of this organism. Clinical and environmental isolates were available for each outbreak and were initially examined by pulsed-field gel electrophoresis (PFGE). Sequence-based typing alleles were extracted from WGS data yielding complete sequence types (ST) for isolates representing 8 out of the 10 outbreaks evaluated in this study. Isolates from separate outbreaks sharing the same ST also contained the fewest differences in core genome single nucleotide polymorphisms (SNPs) and the greatest proportion of identical allele sequences in a whole-genome multilocus sequence typing (wgMLST) scheme. Both core SNP and wgMLST analyses distinguished isolates from separate outbreaks, including those from two outbreaks sharing indistinguishable PFGE profiles. Isolates from a hospital-associated outbreak spanning multiple years shared indistinguishable PFGE profiles but displayed differences in their genome sequences, suggesting the presence of multiple environmental sources. Finally, thertxgene demonstrated differences in the repeat region sequence among ST1 isolates from different outbreaks, suggesting that variation in this gene may be useful for targeted molecular subtyping approaches forL. pneumophila. This study demonstrates the utility of various genome sequence analysis approaches forL. pneumophilafor environmental source attribution studies while furthering the understanding ofLegionellaecology.IMPORTANCEWe demonstrate that whole-genome sequencing helps to improve resolution ofLegionella pneumophilaisolated during laboratory investigations of legionellosis compared to traditional subtyping methods. These data can be important in confirming the environmental sources of legionellosis outbreaks. Moreover, we evaluated various methods to analyze genome sequence data to help resolve outbreak-related isolates.

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Clinical and Molecular Characteristics of Klebsiella pneumoniae Isolates Causing Bloodstream Infections in Japan: Occurrence of Hypervirulent Infections in Health Care

Journal of Clinical Microbiology ◽

10.1128/jcm.01206-19 ◽

2019 ◽

Vol 57 (11) ◽

Cited By ~ 11

Author(s):

Sohei Harada ◽

Kotaro Aoki ◽

Shungo Yamamoto ◽

Yoshikazu Ishii ◽

Noritaka Sekiya ◽

...

Keyword(s):

Health Care ◽

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Characteristics ◽

Bloodstream Infections ◽

Molecular Characteristics ◽

Whole Genome ◽

Content Type ◽

Hospital Acquired

ABSTRACT Although hypervirulent Klebsiella pneumoniae (hvKp) has been associated with severe community-acquired infections that occur among relatively healthy individuals, information about hvKp infections in health care settings remains limited. Here, we systematically analyzed the clinical and molecular characteristics of K. pneumoniae isolates causing bloodstream infections in a cross-sectional study. Clinical characteristics of K. pneumoniae bloodstream infections from hospitals across Japan were analyzed by a review of the medical records. Whole-genome sequencing of the causative isolates was performed. Bacterial species were confirmed and hvKp were identified using whole-genome sequencing data. Clinical characteristics of hvKp infections were compared with those of non-hvKp infections by bivariate analyses. Of 140 cases of K. pneumoniae bloodstream infections, 26 cases (18.6%) were caused by various clones of hvKp defined by the carriage of cardinal virulence genes. Molecular identification revealed that 24 (17.1%) and 14 (10%) cases were caused by Klebsiella variicola and Klebsiella quasipneumoniae, respectively. Patients with hvKp infections had higher proportions of diabetes mellitus (risk ratio [RR], 1.75; 95% confidence interval [CI], 1.05 to 2.94), and their infections had significantly higher propensity to involve pneumonia (RR, 5.85; 95% CI, 1.39 to 24.6), liver abscess (RR, 5.85; 95% CI, 1.39 to 24.6), and disseminated infections (RR, 6.58; 95% CI, 1.16 to 37.4) than infections by other isolates. More than one-half of hvKp infections were health care associated or hospital acquired, and a probable event of health care-associated transmission of hvKp was documented. hvKp isolates, which are significantly associated with severe and disseminated infections, are frequently involved in health care-associated and hospital-acquired infections in Japan.

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Monitoring meticillin resistant Staphylococcus aureus and its spread in Copenhagen, Denmark, 2013, through routine whole genome sequencing

Eurosurveillance ◽

10.2807/1560-7917.es2015.20.17.21112 ◽

2015 ◽

Vol 20 (17) ◽

Cited By ~ 19

Author(s):

M D Bartels ◽

H Larner-Svensson ◽

H Meiniche ◽

K Kristoffersen ◽

K Schønning ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Protein A ◽

Epidemiological Data ◽

Epidemiological Surveillance ◽

Whole Genome ◽

Staphylococcal Protein A ◽

Hospital Acquired ◽

Type 304

Typing of meticillin resistant Staphylococcus aureus (MRSA) by whole genome sequencing (WGS) is performed routinely in Copenhagen since January 2013. We describe the relatedness, based on WGS data and epidemiological data, of 341 MRSA isolates. These comprised all MRSA (n?=?300) identified in Copenhagen in the first five months of 2013. Moreover, because MRSA of staphylococcal protein A (spa)-type 304 (t304), sequence type (ST) 6 had been associated with a continuous neonatal ward outbreak in Copenhagen starting in 2011, 41 t304 isolates collected in the city between 2010 and 2012 were also included. Isolates from 2013 found to be of t304, ST6 (n=14) were compared to the 41 earlier isolates. In the study, isolates of clonal complex (CC) 22 were examined in detail, as this CC has been shown to include the hospital-acquired epidemic MRSA (EMRSA-15) clone. Finally, all MRSA ST80 were also further analysed, as representatives of an important community-acquired MRSA in Europe. Overall the analysis identified 85 spa-types and 35 STs from 17 CCs. WGS confirmed the relatedness of epidemiologically linked t304 neonatal outbreak isolates. Several non-outbreak related patients had isolates closely related to the neonatal isolates suggesting unrecognised community chains of transmission and insufficient epidemiological data. Only four CC22 isolates were related to EMRSA-15. No community spread was observed among the 13 ST80 isolates. WGS successfully replaced conventional typing and added information to epidemiological surveillance. Creation of a MRSA database allows clustering of isolates based on single nucleotide polymorphism (SNP) calling and has improved our understanding of MRSA transmission.

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