Dataset Information

Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

ABSTRACT: Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

SUBMITTER: Alvaro A

PROVIDER: S-EPMC10952028 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Alvaro Alessandro A Piazza Aurora A Papaleo Stella S Perini Matteo M Pasala Ajay Ratan AR Panelli Simona S Nardi Tiago T Nodari Riccardo R Sterzi Lodovico L Pagani Cristina C Merla Cristina C Castelli Daniele D Olivieri Emanuela E Bracco Silvia S Ferrando Maria Laura ML Saluzzo Francesca F Rimoldi Sara Giordana SG Corbella Marta M Cavallero Annalisa A Prati Paola P Farina Claudio C Cirillo Daniela Maria DM Zuccotti Gianvincenzo G Comandatore Francesco F

iScience 20240301 4

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting ...[more]

PMID: 38510115

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Project description:Abstract Background Outbreaks of pathogenic agents within healthcare facilities may have sporadic or nosocomial sources. Effective intervention requires distinguishing between these sources, which in turn requires epidemiological investigation in conjunction with typing of pathogen isolates. For the latter, established methods range from ribotyping and pulse-field gel electrophoresis to multilocus sequence typing and whole genome sequencing. Due to technical complexities and costs associated with these methods, however, typing is rarely pursued. Polymorphic locus sequence typing (PLST) addresses these issue by employing conventional PCR and Sanger sequencing; the key to PLST is its focus on a tandem repeat-containing locus exhibiting maximal variation due to combinations of single nucleotide polymorphisms and insertions/deletions. Serratia marcescens has been implicated in numerous nosocomial outbreaks, and relatedly has the ability to colonize the hospital environment (e.g., sinks), although few studies have definitively established an outbreak-environment link. Methods To extend PLST to S. marcescens, tandem repeats were bioinformatically identified in the genome sequence of strain WW4 (accession CP003959) and used with flanking sequences as queries in BLASTN searches of the GenBank nr/nt Serratia genome database including 97 S. marcescens strains. One locus designated SerMT1 (bp 243212-244195) was present in all strains and exhibited high variability associated with 6-16 copies of a PVVEPE-encoding repeat within signal recognition particle receptor gene ftsY. Results SerMT1 was downloaded from all 97 strains, aligned using Clustal Omega, and phylogenetically analyzed using DNA parsimony. This analysis resolved 91 total alleles and yielded an impressively high Simpson’s Diversity Index of 0.998. Six clusters of 2 to 4 strains shared SerMT1 sequence; their epidemiologic relatedness was supported by their GenBank annotations (e.g., UMH-10 and UMH-11 isolated the same month at the same facility). Conclusion SerMT1 PLST provides an affordable, user-friendly new tool for epidemiologic investigation of S. marcescens outbreaks. Disclosures Tom Edlind, PhD, MicrobiType LLC: Ownership Interest.

Project description: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 sequencing (WGS) analyses of pure cultures. On the other hand, their application to environmental DNA profiling to evaluate epidemiological relatedness among 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 among 19 sink drains, respectively. Epidemiological relationships among 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, often multidrug resistant and involved in outbreaks of nosocomial infections in neonatal intensive care units. 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 nonculturable 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 is of significant interest for epidemiological risk assessment and elaborate outbreak mitigation strategies.

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Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Publications

Cultivation and sequencing-free protocol for <i>Serratia marcescens</i> detection and typing.

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