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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The bacterium Serratia marcescens is an important opportunistic human pathogen that thrives in many environments, can become multidrug resistant, and is often involved in nosocomial outbreaks in neonatal intensive care units (NICU). We evaluated the role of sinks during five suspected S. marcescens outbreaks in a NICU.

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 the same level of efficiency than average nucleotide identity scores of whole genomes. 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.

Background Sink drains are known reservoirs of pathogens and have been associated with multiple nosocomial outbreaks. In this study, the distribution of Serratia marcescens was investigated within the sink environment of a NICU and in colonized or infected newborns. The effect of different types of drain disinfection on bacterial concentration and on the detection of Serratia marcescens in sink drains was also investigated. Methods Sink drains from a NICU were sampled: 20 drains sampled for 6 weeks in the first year and 28 drains sampled for 5 months in the second year. S. marcescens isolated from positive patients were collected. A high-throughput short sequence typing (HiSST) method was developed to identify S. marcescens and compare strains and environmental DNA from sink drains with 56 clinical strains from 5 nosocomial outbreaks. Five interventions were tested: self-disinfecting drains, hot water disinfection, chlorine disinfection, steam disinfection and hot tap water flushing. Bacterial concentration of samples was measured in culture and flow cytometry and the HiSST method was used to identify S. marcescens. Results During the first and second sampling campaigns, 40% and 60% of sink drain samples were positive for S. marcescens with a moderate genotype diversity (1 to 11 different STs). The genotype profile of the 56 clinical strains was heterogeneous (26 STs). Four distinct STs were retrieved in 8 sinks after detection in patients whereas 1 ST was detected in a sink before detection in patient. Some environmental and clinical strains were found in drains for up to a year after the first sampling campaign. Each tested drain intervention reduced culturable bacteria (4-8 log) and viable bacteria (2-3 log), except for chlorine. The self-disinfecting drains, hot water and steam disinfection were able to remove temporarily S. marcescens from the drains. Conclusion The high genotypic diversity of the clinical strains suggests diffuse sources of S. marcescens within the NICU likely to cause nosocomial infection outbreaks. The self-disinfecting drains, hot water and steam disinfection seem to be the best methods to reduce bacterial concentration in drains and eliminate S. marcescens in the short term, thus limiting the risk of pathogens spread from the sink environment to patients. Disclosures All Authors: No reported disclosures.