Development and Validation of a High-throughput Short Sequence Typing Scheme for Serratia marcescens Pure Culture and Environmental DNA

Molecular typing methods are used to characterize the relatedness between bacterial isolates involved in infections. These approaches rely mostly on discrete loci or whole genome sequences (WGS) analyses of pure cultures. On the other hand, their application to environmental DNA profiling to evaluate epidemiological relatedness amongst patients and environments has received less attention. We developed a specific, high-throughput short sequence typing (HiSST) method for the opportunistic human pathogen Serratia marcescens . Genes displaying the highest polymorphism were retrieved from the core genome of 60 S. marcescens strains. Bioinformatics analyses showed that use of only three loci (within bssA , gabR and dhaM ) distinguished strains with a high level of efficiency. This HiSST scheme was applied to an epidemiological survey of S. marcescens in a neonatal intensive care unit (NICU). In a first case study, a strain responsible for an outbreak in the NICU was found in a sink drain of this unit, by using HiSST scheme and confirmed by WGS. The HiSST scheme was also applied to environmental DNA extracted from sink-environment samples. Diversity of S. marcescens was modest, with 11, 6 and 4 different sequence types (ST) of gabR , bssA and dhaM loci amongst 19 sink drains, respectively. Epidemiological relationships amongst sinks were inferred on the basis of pairwise comparisons of ST profiles. Further research aimed at relating ST distribution patterns to environmental features encompassing sink location, utilization and microbial diversity is needed to improve the surveillance and management of opportunistic pathogens. Importance Serratia marcescens is an important opportunistic human pathogen, multidrug resistant and often involved in outbreaks of nosocomial infections in neonatal intensive care unit. Here, we propose a quick and user-friendly method to select the best typing scheme for nosocomial outbreaks in relating environmental and clinical sources. This method, named high-throughput short sequence typing (HiSST), allows to distinguish strains and to explore the diversity profile of non-culturable S. marcescens . The application of HiSST profile analysis for environmental DNA offers new possibilities to track opportunistic pathogens, identify their origin and relate their distribution pattern with environmental features encompassing sink location, utilization and microbial diversity. Adaptation of the method to other opportunistic pathogens is expected to improve knowledge regarding their ecology, which of significant interest for epidemiological risk assessment and elaborate outbreak mitigation strategies.

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Development of a New High-Throughput Multilocus Sequence Typing Method to Monitor Causative Agents of Nosocomial Infections

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.726 ◽

2020 ◽

Vol 41 (S1) ◽

pp. s187-s187

Author(s):

Thibault Bourdin ◽

Emilie Bedard ◽

Marie-Ève Benoit ◽

Michèle Prévost ◽

Etienne Robert ◽

...

Keyword(s):

High Throughput ◽

Nosocomial Infections ◽

In Silico ◽

Multilocus Sequence Typing ◽

Phylogenetic Trees ◽

Culture Media ◽

Environmental Dna ◽

Opportunistic Pathogen ◽

Opportunistic Pathogens ◽

Bioinformatics Analyses

Background: Nosocomial infections cause 4%–56% mortality in newborns. Several epidemiological studies have shown that transmission of opportunistic pathogens from the sink to the patient, including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Serratia marcescens are associated with nosocomial infections in neonatal intensive care units (NICUs). In this project, we aimed to develop fast, accurate, and high-throughput multilocus sequence typing assays (HiMLST-Illumina) to detect opportunistic pathogens to assess their distribution in the sink environment of NICUs and their transfer to patients. Methods: Genome sequences of P. aeruginosa (n = 45), S. maltophilia (n = 23) and S. marcescens (n = 34) strains were retrieved from public genome databases to build their pangenomes, using the open-source PGAdb-builder server. The core genome was identified for each opportunistic pathogen and was searched for genes displaying the highest polymorphism. The minimal number of loci to include in a HiMLST-Illumina assay was determined by comparing topology of phylogenetic trees of concatenated loci based on genome similarity, computed as the average nucleotide identity (ANI) score. The primers used for HiMLST-Illumina schemes were designed in silico on a conserved domain and were tested on reference strains of each species. Results: Bioinformatics analyses showed that 3–4 loci (<300 base pairs per locus) distinguished strains with the same performances than ANI scores. The assays were tested using opportunistic pathogen isolates and environmental DNA originating from NICU sinks. The HiMLST-Illumina analysis of environmental DNA revealed the presence of at least 1 of the 3 studied opportunistic pathogens in 50% of sampled drains (n = 20). In a previous sampling, P. aeruginosa was isolated on selective culture media before and 48 hours after disinfection of a sink drain with chlorine. S. marcescens was also isolated from another sink 2 weeks after disinfection. Identification of the isolates was confirmed by HiMLST-Illumina analyses and will be typed to compare with clinical isolates. Conclusions: Initial in silico tests predict a high discriminating power of the HiMLST-Illumina method, suggesting that it would be possible to quickly identify strains of interest in a large number of samples. The power of this method is also in the possibility for molecular typing without a need for cultivation. Preliminary results suggest that sinks are readily colonized by opportunistic pathogens. This HiMLST-Illumina scheme will be applied in a 2-year intensive survey of NICUs in 3 hospitals in Montreal to evaluate the performance of new sink designs in limiting bioaerosol production and transmission of opportunistic pathogens to patients.Funding: NoneDisclosures: None

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RELATIONSHIPS BETWEEN DISTRIBUTION PATTERNS OF Plecoglossus altivelis altivelis ESTIMATED BY ENVIRONMENTAL DNA ANALYSIS AND STREAM TEMPARATURE

Journal of Japan Society of Civil Engineers Ser G (Environmental Research) ◽

10.2208/jscejer.74.iii_457 ◽

2018 ◽

Vol 74 (7) ◽

pp. III_457-III_462

Author(s):

Takanori KONO ◽

Yoshihisa AKAMATSU ◽

Ryutei INUI ◽

Masuji GOTO ◽

Kohei YAMAGUCHI

Keyword(s):

Dna Analysis ◽

Environmental Dna ◽

Distribution Patterns ◽

Plecoglossus Altivelis

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Serratia marcescens outbreak in a neonatal intensive care unit: crucial role of implementing hand hygiene among external consultants

BMC Infectious Diseases ◽

10.1186/s12879-014-0734-6 ◽

2015 ◽

Vol 15 (1) ◽

Cited By ~ 25

Author(s):

Carlotta Montagnani ◽

Priscilla Cocchi ◽

Laura Lega ◽

Silvia Campana ◽

Klaus Peter Biermann ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Hand Hygiene ◽

Neonatal Intensive Care Unit ◽

Serratia Marcescens ◽

Crucial Role ◽

Neonatal Intensive Care ◽

External Consultants

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Endemic Serratia marcescens infection in a neonatal intensive care nursery associated with gastrointestinal colonization

The Pediatric Infectious Disease Journal ◽

10.1097/00006454-198503000-00010 ◽

1985 ◽

Vol 4 (2) ◽

pp. 160-167 ◽

Cited By ~ 35

Author(s):

MARY T. NEWPORT ◽

JOSEPH F. JOHN ◽

YVONNE M. MICHEL ◽

ABNER H. LEVKOFF

Keyword(s):

Intensive Care ◽

Serratia Marcescens ◽

Neonatal Intensive Care ◽

Intensive Care Nursery ◽

Gastrointestinal Colonization

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Assessing high-throughput environmental DNA extraction methods for meta-barcode characterization of aquatic microbial communities

Journal of Water and Health ◽

10.2166/wh.2018.108 ◽

2018 ◽

Vol 17 (1) ◽

pp. 37-49 ◽

Cited By ~ 6

Author(s):

Abdolrazagh Hashemi Shahraki ◽

Subba Rao Chaganti ◽

Daniel Heath

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

High Throughput ◽

Environmentally Friendly ◽

Environmental Dna ◽

Magnetic Bead ◽

Extraction Methods ◽

Environmental Research ◽

Rrna Gene

Abstract The characterization of microbial community dynamics using genomic methods is rapidly expanding, impacting many fields including medical, ecological, and environmental research and applications. One of the biggest challenges for such studies is the isolation of environmental DNA (eDNA) from a variety of samples, diverse microbes, and widely variable community compositions. The current study developed environmentally friendly, user safe, economical, and high throughput eDNA extraction methods for mixed aquatic microbial communities and tested them using 16 s rRNA gene meta-barcoding. Five different lysis buffers including (1) cetyltrimethylammonium bromide (CTAB), (2) digestion buffer (DB), (3) guanidinium isothiocyanate (GITC), (4) sucrose lysis (SL), and (5) SL-CTAB, coupled with four different purification methods: (1) phenol-chloroform-isoamyl alcohol (PCI), (2) magnetic Bead-Robotic, (3) magnetic Bead-Manual, and (4) membrane-filtration were tested for their efficacy in extracting eDNA from recreational freshwater samples. Results indicated that the CTAB-PCI and SL-Bead-Robotic methods yielded the highest genomic eDNA concentrations and succeeded in detecting the core microbial community including the rare microbes. However, our study recommends the SL-Bead-Robotic eDNA extraction protocol because this method is safe, environmentally friendly, rapid, high-throughput and inexpensive.

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