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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 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.

The inactivation of Cryptosporidium oocysts is a main driver in the selection of water treatment disinfection strategies, and microbial risk analysis provides a sound basis for optimizing water treatment processes. U.S. Environmental Protection Agency method 1622/23 provides an estimate of the total oocyst count; however, it cannot be used directly for risk assessment, as it does not determine the fraction of infectious oocysts. Improved assessment of the risk for designated sources or in treated water requires evaluation of the total number of oocysts and an estimate of their infectivity. We developed a dual direct detection method using differential immunofluorescent staining that allows detection of both oocysts and cell culture infection foci for each sample. Using Cryptosporidium parvum oocysts, various pH levels, proteases, and gastroenteric compounds and substrates were assessed to determine their abilities to enhance the number of infection foci. The results showed that the key trigger for oocyst stimulation was acidification. Addition of a low concentration of d -glucose (50 mM) to the infection media increased rates of infectivity, while a higher dose (300 mM) was inhibitory. The total number of oocysts in each sample was determined by counting the oocysts remaining on a cell monolayer and the oocysts recovered from cell monolayer washes during processing using a simple filtration technique. With the dual direct detection on cell culture with immunofluorescence assay method, it is now possible to determine the numbers of total and infectious oocysts for a given sample in a single analysis. Direct percentages of infectivity are then calculated, which allows more accurate assessments of risk.

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 10 8 nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.