Implications of ad-hoc molecular typing for infection control measures in a multi-cluster, multi-phenotypic Serratia marcescens outbreak in a neonatal intensive care unit.

BACKGROUND: Serratia marcescens is known to cause outbreaks in neonatal intensive care units (NICUs). Traditionally epidemiological data, antimicrobial resistance patterns and epidemiological typing have been used to guide infection prevention methods. Whole-genome sequencing (WGS) applications such as core-genome multi-locus sequence typing (cgMLST) applied during an outbreak would potentially yield more information. AIM: To use cgMLST to acquire detailed information on the source and spread of bacteria, enabling more efficient control measures during an S. marcescens outbreak at a NICU. METHODS: Neonates admitted to the NICU of the Leiden University Medical Center (LUMC) during an outbreak between September 2023 and January 2024, with S. marcescens being cultured, were included. Environmental samples were taken to search for a common source, antibiotic susceptibility testing was performed, and antimicrobial resistance genes were analysed. FINDINGS: S. marcescens strains from 17 of the 20 positive patients were available for molecular typing. The cgMLST scheme revealed five different complex types consisting of four separate clusters. Multiple clusters made an unidentified persistent environmental source as cause of the outbreak less likely, leading to a quick downscaling of infection prevention measures. Differences were shown in aminoglycoside resistance patterns of isolates within the same complex types and patients. CONCLUSION: The use of ad-hoc cgMLST provided timely data for rational decision-making during an S. marcescens outbreak at the NICU. Antibiotic phenotyping alone was found not to be suitable for studying clonal spread during this outbreak with S. marcescens.

The ICU environment contributes to the endemicity of the "Serratia marcescens complex" in the hospital setting.

Serratia marcescens is an opportunistic pathogen historically associated with sudden outbreaks in intensive care units (ICUs) and the spread of carbapenem-resistant genes. However, the ecology of S. marcescens populations in the hospital ecosystem remains largely unknown. We combined epidemiological information of 1,432 Serratia spp. isolates collected from sinks of a large ICU that underwent demographic and operational changes (2019-2021) and 99 non-redundant outbreak/non-outbreak isolates from the same hospital (2003-2019) with 165 genomic data. These genomes were grouped into clades (1-4) and subclades (A and B) associated with distinct species: Serratia nematodiphila (1A), S. marcescens (1B), Serratia bockelmannii (2A), Serratia ureilytica (2B), S. marcescens/Serratia nevei (3), and S. nevei (4A and 4B). They may be classified into an S. marcescens complex (SMC) due to the similarity between/within subclades (average nucleotide identity >95%-98%), with clades 3 and 4 predominating in our study and publicly available databases. Chromosomal AmpC ß-lactamase with unusual basal-like expression and prodigiosin-lacking species contrasted classical features of Serratia. We found persistent and coexisting clones in sinks of subclades 4A (ST92 and ST490) and 4B (ST424), clonally related to outbreak isolates carrying blaVIM-1 or blaOXA-48 on prevalent IncL/pB77-CPsm plasmids from our hospital since 2017. The distribution of SMC populations in ICU sinks and patients reflects how Serratia species acquire, maintain, and enable plasmid evolution in both "source" (permanent, sinks) and "sink" (transient, patients) hospital patches. The results contribute to understanding how water sinks serve as reservoirs of Enterobacterales clones and plasmids that enable the persistence of carbapenemase genes in healthcare settings, potentially leading to outbreaks and/or hospital-acquired infections.IMPORTANCEThe "hospital environment," including sinks and surfaces, is increasingly recognized as a reservoir for bacterial species, clones, and plasmids of high epidemiological concern. Available studies on Serratia epidemiology have focused mainly on outbreaks of multidrug-resistant species, overlooking local longitudinal analyses necessary for understanding the dynamics of opportunistic pathogens and antibiotic-resistant genes within the hospital setting. This long-term genomic comparative analysis of Serratia isolated from the ICU environment with isolates causing nosocomial infections and/or outbreaks within the same hospital revealed the coexistence and persistence of Serratia populations in water reservoirs. Moreover, predominant sink strains may acquire highly conserved and widely distributed plasmids carrying carbapenemase genes, such as the prevalent IncL-pB77-CPsm (pOXA48), persisting in ICU sinks for years. The work highlights the relevance of ICU environmental reservoirs in the endemicity of certain opportunistic pathogens and resistance mechanisms mainly confined to hospitals.

Comprehensive pan-genomic, resistome and virulome analysis of clinical OXA-48 producing carbapenem-resistant Serratia marcescens strains.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) have been thoroughly studied as the pathogens associated with hospital acquired infections. However, data on Serratia marcescens are not enough. S. marcescens is now becoming a propensity for its highly antimicrobial-resistant clinical infections. METHODS: Four carbapenem-resistant S. marcescens (CR-SM) isolates were obtained from hospitalized patients through routine microbiological experiments. We assembled the isolates genomes using whole genome sequencing (WGS) and compared their resistome and virulome patterns. RESULTS: The average length and CG content of chromosomes was 5.33 Mbp and 59.8%, respectively. The number of coding sequences (CDSs) ranged from 4,959 to 4,989. All strains had one single putative conjugative plasmid with IncL incompatibility (Inc) group. The strains harbored blaCTX-M-15, blaTEM-1 and blaSHV-134. All plamsids were positive for blaOXA-48. No blaNDM-1, blaKPC, blaVIM and blaIMP were identified. The blaSRT-2 and aac(6')-Ic genes were chromosomally-encoded. Class 1 integron was detected in strains P8, P11 and P14. The Escher_RCS47 and Salmon_SJ46 prophages played major role in plasmid-mediated carraige of extended spectrum ß-lactamases (ESBLs). The CR-SM strains were equipt with typical virulence factors of oppotunistic pathogens including biofilm formation, adhesins, secretory systems and siderophores. The strains did not have ability to produce prodigiosin but were positive for chitinase and EstA. CONCLUSION: The presence of conjugative plasmids harboring major ß-lactamases within prophage and class 1 integron structures highlights the role of different mobile genetic elements (MGEs) in distribution of AMR factors and more specifically carbapenemases. More molecular studies are required to determine the status of carbapenem resistance in clinical starins. However, appropriate strategies to control the global dissemination of CR-SM are urgent.

Production of the biosurfactant serrawettin W1 by Serratia marcescens S-1 improves hydrocarbon degradation.

With the frequent occurrence of oil spills, the bioremediation of petroleum hydrocarbons pollution has attracted more and more attention. In this study, we investigated the biodegradation of crude oil by the biosurfactant-producing strain S-1. The strain was isolated from petroleum-contaminated soil and identified as Serratia marcescens according to partial 16S rDNA gene analysis. It was able to effectively degrade hydrocarbons with the concomitant production of biosurfactants at 20-30 °C, while there was no biosurfactant production and the degradation rate was lower at 37 °C. The biosurfactant was identified as serrawettin W1 by UPLC-ESI-MS, and was found to reduce the surface tension of water to 30 mN/m, with stable surface activity and emulsion activity at temperatures from 20 to 100 °C, pH of 2-10 and NaCl concentrations of 0-50 g/L. Serrawettin W1 significantly increased the cell surface hydrophobicity (CSH) and enhanced the bioavailability of hydrocarbon pollutants, which was conducive to the degradation of crude oil, including long-chain alkanes and aromatic hydrocarbons. Serratia marcescens S-1 has potential applications in bioremediation at low temperature.

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 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.

Genomic surveillance, characterization and intervention of a polymicrobial multidrug-resistant outbreak in critical care.

Background. Infections caused by carbapenem-resistant Acinetobacter baumannii (CR-Ab) have become increasingly prevalent in clinical settings and often result in significant morbidity and mortality due to their multidrug resistance (MDR). Here we present an integrated whole-genome sequencing (WGS) response to a persistent CR-Ab outbreak in a Brisbane hospital between 2016-2018.Methods. A. baumannii, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa isolates were sequenced using the Illumina platform primarily to establish isolate relationships based on core-genome SNPs, MLST and antimicrobial resistance gene profiles. Representative isolates were selected for PacBio sequencing. Environmental metagenomic sequencing with Illumina was used to detect persistence of the outbreak strain in the hospital.Results. In response to a suspected polymicrobial outbreak between May to August of 2016, 28 CR-Ab (and 21 other MDR Gram-negative bacilli) were collected from Intensive Care Unit and Burns Unit patients and sent for WGS with a 7 day turn-around time in clinical reporting. All CR-Ab were sequence type (ST)1050 (Pasteur ST2) and within 10 SNPs apart, indicative of an ongoing outbreak, and distinct from historical CR-Ab isolates from the same hospital. Possible transmission routes between patients were identified on the basis of CR-Ab and K. pneumoniae SNP profiles. Continued WGS surveillance between 2016 to 2018 enabled suspected outbreak cases to be refuted, but a resurgence of the outbreak CR-Ab mid-2018 in the Burns Unit prompted additional screening. Environmental metagenomic sequencing identified the hospital plumbing as a potential source. Replacement of the plumbing and routine drain maintenance resulted in rapid resolution of the secondary outbreak and significant risk reduction with no discernable transmission in the Burns Unit since.Conclusion. We implemented a comprehensive WGS and metagenomics investigation that resolved a persistent CR-Ab outbreak in a critical care setting.

Complete genome sequence of the biocontrol agent Serratia marcescens strain N4-5 uncovers an assembly artefact.

Serratia marcescens are gram-negative bacteria found in several environmental niches, including the plant rhizosphere and patients in hospitals. Here, we present the genome of Serratia marcescens strain N4-5 (=NRRL B-65519), which has a size of 5,074,473 bp (664-fold coverage) and contains 4840 protein coding genes, 21 RNA genes, and an average G + C content of 59.7%. N4-5 harbours a plasmid of 11,089 bp and 43.5% G + C content that encodes six unique CDS repeated 2.5× times totalling 13 CDS. Our genome assembly and manual curation uncovered the insertion of two extra copies of the 5S rRNA gene in the assembled sequence, which was confirmed by PCR and Sanger sequencing to be a misassembly. This artefact was subsequently removed from the final assembly. The occurrence of extra copies of the 5S rRNA gene was also observed in most complete genomes of Serratia spp. deposited in public databases in our comparative analysis. These elements, which also occur naturally, can easily be confused with true genetic variation. Efforts to discover and correct assembly artefacts should be made in order to generate genome sequences that represent the biological truth underlying the studied organism. We present the genome of N4-5 and discuss genes potentially involved in biological control activity against plant pathogens and also the possible mechanisms responsible for the artefact we observed in our initial assembly. This report raises awareness about the extra copies of the 5S rRNA gene in sequenced bacterial genomes as they may represent misassemblies and therefore should be verified experimentally.

Isolation and Characterization of Serratiopeptidase Producing Bacteria from Mulberry Phyllosphere.

Serratiopeptidase (EC 3.4.24.40), a proteolytic enzyme, is one of the most promising enzymes being used in biopharmaceutical industry. Mulberry phyllosphere, being an unexplored niche for exploration of protease production, was chosen for the present study. Protease producing bacteria were isolated from the tissues of mulberry plant as well as its rhizospheric soil. Two protease producing bacteria belonging to Serratia genus were found to be potential serratiopeptidase producers. Among them, the endophyte, i.e., Serratia marcescens MES-4 presented 95 Units/mL activity, while the soil isolate i.e., Serratia marcescens MRS-11 presented 156 Units/mL activity.

Is there a widespread clone of Serratia marcescens producing outbreaks worldwide?

BACKGROUND: Serratia marcescens frequently causes outbreaks in healthcare settings. There are few studies using high-throughput sequencing (HTS) that analyse S. marcescens outbreaks. We present the analysis of two outbreaks in neonatal intensive care units (NICUs) in hospitals from the Comunitat Valenciana (CV, Spain) and the impact of using different reference genomes. METHODS: DNA from cultured isolates was extracted and sequenced by HTS using Illumina NextSeq. Reads were mapped against two reference genomes, strains UMH9 and Db11, and the unmapped fraction of the genomes was assembled to fully genetically characterize the isolates. FINDINGS: Isolates from the first outbreak were identical to the UMH9 reference, an unrelated isolate obtained three years earlier in the USA. This did not occur when the Db11 strain, a standard reference for S. marcescens, was used as the reference for mapping. To check whether UMH9 was a widely distributed clone spreading in the CV, the second outbreak isolates were mapped against this reference. They were not closely related to this strain, and this outbreak could be defined as such regardless of the reference used for mapping the reads. CONCLUSIONS: The choice of the reference for genomic analysis of outbreaks is a critical decision. In the case of the first outbreak, this choice changed the interpretation of the results drastically, allowing or preventing the definition of the outbreak according to the reference used. Although HTS is a powerful tool for epidemiological analysis, it is still essential to collect microbiological and epidemiological data for the correct interpretation of the results.

Isolation and identification of two Serratia marcescens strains from silkworm, Bombyx mori.

Bacterial septicemia commonly occurs and usually cause huge losses in sericulture industry. Here, two pathogenic bacterial strains were isolated from dead silkworm and named as ZJ-1 and ZJ-2. Phenotypic and genotypic analysis results revealed that both of these two strains are closely related to Serratia marcescens (S. marcescens). The morphological as well as physiological and biochemical characteristics of ZJ-1 were accordant with S. marcescens mentioned in Bergey's manual of determinative bacteriology, whereas ZJ-2 showed some discrepancies such as the utilization of malonate and starch, fermentation of maltose and sucrose, and tests of urease, etc. Surprisingly, ZJ-2 could produce red pigment at high temperature (37°) but ZJ-1 could not. Besides, by analyzing the lethal concentration 50 (LC50) of ZJ-1 and ZJ-2, it was found that the virulence of ZJ-2 was lower than that of ZJ-1. These results revealed that ZJ-1 and ZJ-2 were two different strains of S. marcescens and that ZJ-2 was expected to be a safe (low-toxicity) and efficient strain for the production of red pigment. Nonetheless, further research in molecular level is needed to understand the regulation mechanism of pigment production and infection of ZJ-2.

Outbreak investigation of Serratia marcescens neurosurgical site infections associated with a contaminated shaving razors.

BACKGROUND: Surgical site infection (SSI) is the most common healthcare-associated infection. We report an outbreak of neurosurgical site infections caused by Serratia marcescens after craniotomy in a tertiary care hospital. METHODS: Between August 6 and 21, 2018, five cases of early-onset SSI caused by S. marcescens after craniotomy were recorded in a 1786-bed tertiary care hospital. Cultures were collected from potential environmental sources and healthcare workers. Whole-genome sequencing (WGS) was used to investigate the genetic relationships among S. marcescens isolates. RESULTS: The outbreak involved five patients; S. marcescens was isolated from the cerebrospinal fluid, pus, tissue, and blood samples from these patients. S. marcescens was also isolated from shaving razors and brushes. All S. marcescens isolates from the infected patients and razors showed the same resistance patterns on antibiotic-susceptibility tests. WGS revealed close clustering among four of five isolates from the patients and among three of four isolates from the razors. No additional patient developed S. marcescens infection after we stopped using the razors for scalp shaving. CONCLUSIONS: We report an outbreak of neurosurgical site infections after craniotomy, which was associated with shaving razors contaminated by S. marcescens. Shaving scalps with razors should be avoided to prevent SSI.

Identification of Serratia marcescens isolated from Antheraea pernyi eggs and determination of bacterial pathogenicity and transmission pathway.

The Chinese oak silkworm, Antheraea pernyi, is an important alternate host for mass production of Trichogramma parasitoids, which play a vital role in the biological control of lepidopterous pests in China. The quality of A. pernyi eggs is particularly important for mass production of these parasitoids. In this study, a pathogenic bacterium, isolated and purified from A. pernyi eggs that had turned gray in color, was identified as Serratia marcescens. We used morphology, biochemistry and 16S rDNA analysis to characterize the strain, which was named "APE strain". Serratia marcescens APE strain was determined to be the causal bacterium associated with the disease in the eggs, verified by a test based on Koch's Postulates. We tested the pathogenicity of S. marcescens APE strain on A. pernyi eggs; the percentage of diseased (gray) eggs reached 57.78% when uninfected eggs were exposed to a concentration of 1 × 109 cfu/mL bacterial suspension for 7 h. S. marcescens was transmitted mechanically by Trichogramma parasitoids. The transmission rate was 25.56%. In a horizontal transmission test, the highest percentage of uninfected eggs that developed infections was 51.43% after being treated with contents of diseased eggs for 12 h. In a vertical transmission test, the number of infected eggs per treated adult female was 63.8-92.3 after treatment with different S. marcescens concentrations, significantly lower than the 304.3 eggs per female in the control group. Furthermore, the percentage of infected (gray) eggs produced by adult moths exposed to bacteria in the larval stage was 80.5-85.3%.

Genome-based analyses indicate that Serratia marcescens subsp. marcescens and Serratia marcescens subsp. sakuensis do not merit separation to subspecies status.

The species Serratia marcescens currently comprises two subspecies, viz. Serratia marcescenssubsp. marcescens and Serratia marcescenssubsp. sakuensis. The latter species was created by considering the differentiating endospore-formation capability of isolate KREDT. A member of the Subcommittee on the Taxonomy of Enterobacteriaceae for the International Committee on Systematics of Prokaryotes conducted a re-examination of S. marcescens KREDT and failed to detect its endospore-forming capability, thereby questioning the validity of S. marcescenssubsp. sakuensis as a separate subspecies. Nevertheless, the taxonomic allocation of S. marcescenssubsp. sakuensis has been followed till date. To obtain clarity for taxonomic allocation based on genome-based analysis, we sequenced the genome of type strain S. marcescenssubsp. sakuensis KREDT=DSM 17174T and carried out the analyses recommended to define the subspecies. As a result, DSM 17174T does not satisfy the genomic criteria for defining a subspecies: i.e. does not form a distinguishable clade by overall genomic related indexing and phylogenomic treeing. Also, there is currently no other rationale for separate recognition of this species. We propose the removal of the S. marcescenssubsp. sakuensis designation to avoid further confusion in the literature.

Serratia marcescens colonization in preterm neonates during their neonatal intensive care unit stay.

Background: Nosocomial sepsis is the main problem that preterms have to face during their stay at neonatal intensive care units (NICU). Serratia marcescens is an emerging cause of preterm sepsis but its epidemiology is still largely unknown. Consequently, the aims of this study were to know the rate of preterms colonized by S. marcescens during their stay at the NICU and the characteristics and evolution of the S. marcescens population, including the susceptibility to clinically relevant antibiotics. Methods: Twenty-six preterm infants born with a gestational age ≤ 32 weeks and/or weigh ≤1500 g were included in the study. Samples of meconium and feces (n = 92) were collected during their first month of life of the infants, together with feeding samples after their pass through enteral feeding tubes (n = 37). Samples were inoculated on MacConkey agar plates. The isolates identified as S. marcescens were genotyped using RAPD and PFGE; and antibiotics susceptibility was performed in a Vitek 2 system. Results: A total of 179 S. marcescens isolates were obtained from the samples. PFGE profiling and cluster analysis allowed the classification of the isolates into 7 different S. marcescens clones. PFGE patterns 1 and 3 were the dominant strains in the fecal samples colonizing 31 and 35% of the infants, respectively. Those isolates causing bacteremia in two infants clustered in PFGE pattern 3. Conclusion: S. marcescens is a bacterial species closely associated to the NICU environment. It can be frequently isolated from preterm's feces although only some genetic lineages seem to be associated to sepsis. Enteral feeding tubes act as important reservoirs to keep the S. marcescens population in the NICU. Trial registration: The local ethic committee approved this trial with the reference 09/157.

SME-4-producing Serratia marcescens from Argentina belonging to clade 2 of the S. marcescens phylogeny.

BACKGROUND: SME carbapenemases are increasingly reported, especially from North and South America. Here, we describe an SME-4-producing Serratia marcescens (SME-Sm) clinical isolate from Argentina and compare its genome with other SME-Sm and Sm isolates recovered from public databases. METHODS: Sm isolates were characterized by WGS using Illumina technology, susceptibility testing and MIC determination. Carbapenemase activity was revealed by biochemical tests based on imipenem hydrolysis. A whole-genome phylogeny was estimated for all the Sm isolates retrieved from public databases with kSNP3 and a whole-genome phylogenetic analysis based on non-recombinant core SNPs was inferred for Sm complete genomes and for those encoding any blaSME variants. RESULTS: Sm163 was resistant to amoxicillin, temocillin, aztreonam and carbapenems, remaining susceptible to extended-spectrum cephalosporins. WGS analysis of Sm163 revealed a genome of 5139329 bp and a chromosomally encoded blaSME-4 carbapenemase gene located on a genomic island closely related to SmarGI1-1 of Sm N11-02820. Comparison of the Sm genomes revealed that the 14 SME-Sm isolates possess this genomic island inserted at the same loci, that 13/14 belong to clade 1 and that 11/14 form a well-defined subcluster of cluster I of Sm clade 1, while Sm163 belongs to clade 2, suggesting that an SME-encoding genomic island may have been transferred between isolates from different clades. CONCLUSIONS: To the best of our knowledge this is the first report of an SME-4-encoding Sm from Argentina. The blaSME-4 gene is located on a SmarGI1-1-like genomic island. The genome of Sm163 belongs to clade 2, unlike all the other SME-Sm isolates, which belong to clade 1.

Identification and molecular characterization of Serratia marcescens phages vB_SmaA_2050H1 and vB_SmaM_2050HW.

Serratia marcescens is a rod-shaped, Gram-negative bacterium causing nosocomially acquired infections. Bacteriophages are natural opponents of their pathogenic bacterial hosts and could be an alternative to traditional antibiotic treatments. In this study, two S. marcescens-specific bacteriophages, vB_SmaA_2050H1 and vB_SmaM_2050HW, were isolated from two different waste samples in China. Phage plaque assays, transmission electron microscopy, host-range determination, and one-step growth curve analyses were performed for both phages. vB_SmaA_2050H1 was classified as belonging to the family Ackermannviridae, and vB_SmaM_2050HW was classified as belonging to the family Myoviridae. One-step growth curve analysis showed that the latent and rise period of vB_SmaA_2050H1 were 80 min and 50 min, respectively, with a burst size of approximately 103 phage particles per infected cell. For vB_SmaM_2050HW, latent and rise periods of 40 min and 60 min, respectively, were determined, with a burst size of approximately 110 phage particles per infected cell. vB_SmaA_2050H1 infected 10 of the 15 (66.67%) S. marcescens strains tested, while vB_SmaM_2050HW infected 12 (80%) of the strains. Whole-genome sequencing and annotation of each of the phage genomes revealed genome sizes of 159,631 bp and 276,025 bp for vB_SmaA_2050H1 and vB_SmaM_2050HW, respectively, with the respective genomes containing 213 and 363 putative open reading frames. Sequence analysis of the genomes revealed that vB_SmaA_2050H1 is a member of the ViI-like family, while vB_SmaM_2050HW is a novel virulent bacteriophage. These findings provide further insights into the genomic structures of S. marcescens bacteriophages.

Epidemiological Typing of Serratia marcescens Isolates by Whole-Genome Multilocus Sequence Typing.

Serratia marcescens is an opportunistic bacterial pathogen. It is notorious for its increasing antimicrobial resistance and its potential to cause outbreaks of colonization and infections, predominantly in neonatal intensive care units (NICUs). There, its spread requires rapid infection control response. To understand its spread, detailed molecular typing is key. We present a whole-genome multilocus sequence typing (wgMLST) method for S. marcescens Using a set of 299 publicly available whole-genome sequences (WGS), we developed an initial wgMLST system consisting of 9,377 gene loci. This included 1,455 loci occurring in all reference genomes and 7,922 accessory loci. This closed system was validated using three geographically diverse collections of S. marcescens consisting of 111 clinical isolates implicated in nosocomial dissemination events in three hospitals. The validation procedure showed a full match between epidemiological data and the wgMLST analyses. We set the cutoff value for epidemiological (non)relatedness at 20 different alleles, though for the majority of outbreak-clustered isolates, this difference was limited to 4 alleles. This shows that the wgMLST system for S. marcescens provides prospects for successful future monitoring for the epidemiological containment of this opportunistic pathogen.

Use of MALDI-TOF mass spectrometry to detect nosocomial outbreaks of Serratia marcescens and Citrobacter freundii.

MALDI-TOF mass spectrometry (MS) may be used as a rapid typing method for nosocomial pathogens. Here, we evaluated MALDI-TOF MS for discrimination of hospital outbreak-related clusters of Serratia marcescens and carbapenemase-producing Citrobacter freundii. Thirty-three S. marcescens isolates collected from neonatal intensive care unit (NICU) patients, and 23 C. freundii isolates including VIM-positive isolates from a hospital colonization outbreak were measured by Vitek MS. Consensus spectra of each isolate were clustered using SARAMIS software. Genotyping was performed by whole-genome sequencing (WGS). First, a set of 21 S. marcescens isolates from 2014 with seven genotypes including three monoclonal clusters was used for the evaluation of MALDI-TOF typing. MS clustering was largely in agreement with genotyping results when the similarity cut-off for clonal identity was set on 90%. MALDI-TOF cluster analysis was then investigated for the surveillance of S. marcescens in the NICU in 2017 and demonstrated the introduction of new strains into the hospital and nosocomial transmissions. MS analysis of the C. freundii outbreak in 2016 revealed a monoclonal cluster of VIM-positive isolates and the separation of epidemiologically non-related VIM-positive and negative isolates. Two additional VIM-positive Citrobacter isolates from food samples were closely related to the large monoclonal cluster. WGS confirmed the MS results. MALDI-TOF MS may be used as a first-line typing tool for S. marcescens and C. freundii to detect transmission events in the hospital because isolates of an identical WGS type were grouped into the same MS cluster.

Molecular cloning and characterization of a methyl parathion hydrolase from an organophosphorus-degrading bacterium, Serratia marcescens MEW06.

An organophosphorus-degrading bacterium MEW06, which exhibited excellent biodegradation capabilities towards 50 mg/L of methyl parathion (MP), paraoxon and dimethoate, was isolated from Sand Lake (Wuhan, China) and identified as Serratia marcescens subsp. marcescens based on physiological-biochemical characteristics and a 16S rDNA sequence-based phylogenetic tree. MEW06 genome contains a 31.09-kDa methyl parathion hydrolase (MPH) (MPHGM004539) that was 54.9% similar to Pseudomonas sp. WBC-3's MPH. RT-qPCR revealed that mphGM004539 gene expression was significant up-regulated when co-cultured with MP. mphGM004539 without signal peptide (mphGM004539Δsp) was successful cloned and expressed in Escherichia coli BL21 (DE3). Optimized specific enzyme activity of MPHGM004539ΔSP was 5.26 U/mg under 35°C and pH 11.0 conditions when MP as the substrate. Additionally, Co2+, Cd2+and Fe2+ increased the enzyme activity level. MP could be degraded by MPHGM004539ΔSP into p-nitrophenol probably by hydrolyzing the P-O ester bond. Virulence of MP towards Drosophila melanogaster W1118 was reduced by MEW06 or MPHGM004539ΔSP biodegradation. This is the first cloning and characterization of MPH from the organophosphorus-degrading bacterium S. marcescens. MEW06 and its MPH have potential roles in the bioremediation of organophosphorus pesticide-contaminated eco-systems.

Serratia marcescens Outbreak in a Neonatal Intensive Care Unit: New Insights from Next-Generation Sequencing Applications.

Serratia marcescens is an environmental bacterium that is commonly associated with outbreaks in neonatal intensive care units (NICUs). Investigations of S. marcescens outbreaks require efficient recovery and typing of clinical and environmental isolates. In this study, we investigated how the use of next-generation sequencing applications, such as bacterial whole-genome sequencing (WGS) and bacterial community profiling, could improve S. marcescens outbreak investigations. Phylogenomic links and potential antibiotic resistance genes and plasmids in S. marcescens isolates were investigated using WGS, while bacterial communities and relative abundances of Serratia in environmental samples were assessed using sequencing of bacterial phylogenetic marker genes (16S rRNA and gyrB genes). Typing results obtained using WGS for the 10 S. marcescens isolates recovered during a NICU outbreak investigation were highly consistent with those obtained using pulsed-field gel electrophoresis (PFGE), the current standard typing method for this bacterium. WGS also allowed the identification of genes associated with antibiotic resistance in all isolates, while no plasmids were detected. Sequencing of the 16S rRNA and gyrB genes both showed greater relative abundances of Serratia at environmental sampling sites that were in close contact with infected babies. Much lower relative abundances of Serratia were observed following disinfection of a room, indicating that the protocol used was efficient. Variations in the bacterial community composition and structure following room disinfection and among sampling sites were also identified through 16S rRNA gene sequencing. Together, results from this study highlight the potential for next-generation sequencing tools to improve and to facilitate outbreak investigations.