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.

A highly specific Serratia-infecting T7-like phage inhibits biofilm formation in two different genera of the Enterobacteriaceae family.

Due to the emergence of multidrug-resistant bacteria, bacteriophages have become a viable alternative in controlling bacterial growth or biofilm formation. Biofilm is formed by extracellular polymeric substances (EPS) and is one of the factors responsible for increasing bacterial resistance. Bacteriophages have been studied as a bacterial control agent by use of phage enzymes or due to their bactericidal activities. A specific phage against Serratia marcescens was isolated in this work and was evaluated its biological and genomic aspects. The object of this study was UFV01, a bacteriophage belonging to the Podoviridae family, genus Teseptimavirus (group of lytic viruses), specific to the species S. marcescens, which may be related to several amino acid substitutions in the virus tail fibers. Despite this high specificity, the phage reduced the biofilm formation of several Escherichia coli strains without infecting them. UFV01 presents a relationship with phages of the genus Teseptimavirus, although it does not infect any of the E. coli strains evaluated, as these others do. All the characteristics make the phage an interesting alternative in biofilm control in hospital environments since small breaks in the biofilm matrix can lead to a complete collapse.

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.

Complete genome sequence analysis of PS2, a novel T4-like bacteriophage that infects Serratia marcescens clinical isolates.

In this study, we isolated and characterized a lytic phage, named vB_SmaM_PS2 (abbreviated as PS2), which can infect Serratia marcescens clinical isolates. Morphologically, phage PS2 can be classified within the Myoviridae family. The 167,276 bp double-stranded DNA genome of PS2 possesses 41.7% GC content. A total of 276 protein-coding genes and 4 tRNA genes were predicted in the PS2 genome. Of the 276 genes, 131 (47%) encoded T4-like genes, most of which are DNA replication and virion structural genes. Therefore, phage PS2 should be a new member of the T4-like Serratia phage.

Protist predation can select for bacteria with lowered susceptibility to infection by lytic phages.

BACKGROUND: Consumer-resource interactions constitute one of the most common types of interspecific antagonistic interaction. In natural communities, complex species interactions are likely to affect the outcomes of reciprocal co-evolution between consumers and their resource species. Individuals face multiple enemies simultaneously, and consequently they need to adapt to several different types of enemy pressures. In this study, we assessed how protist predation affects the susceptibility of bacterial populations to infection by viral parasites, and whether there is an associated cost of defence on the competitive ability of the bacteria. As a study system we used Serratia marcescens and its lytic bacteriophage, along with two bacteriovorous protists with distinct feeding modes: Tetrahymena thermophila (particle feeder) and Acanthamoeba castellanii (surface feeder). The results were further confirmed with another study system with Pseudomonas and Tetrahymena thermophila. RESULTS: We found that selection by protist predators lowered the susceptibility to infections by lytic phages in Serratia and Pseudomonas. In Serratia, concurrent selection by phages and protists led to lowered susceptibility to phage infections and this effect was independent from whether the bacteria shared a co-evolutionary history with the phage population or not. Bacteria that had evolved with phages were overall more susceptible to phage infection (compared to bacteria with history with multiple enemies) but they were less vulnerable to the phages they had co-evolved with than ancestral phages. Selection by bacterial enemies was costly in general and was seen as a lowered fitness in absence of phages, measured as a biomass yield. CONCLUSIONS: Our results show the significance of multiple species interactions on pairwise consumer-resource interaction, and suggest potential overlap in defending against predatory and parasitic enemies in microbial consumer-resource communities. Ultimately, our results could have larger scale effects on eco-evolutionary community dynamics.

The genome and proteome of Serratia bacteriophage η which forms unstable lysogens.

BACKGROUND: Serratia marcescens phage η is a temperate unclassified member of the Siphoviridae which had been reported as containing hypermodified guanine residues. METHODS: The DNA was characterized by enzymatic digestion followed by HPLC analysis of the nucleoside composition, and by DNA sequencing and proteomic analysis. Its ability to form stable lysogens and integrate was also investigated. RESULTS: Enzymatic digestion and HPLC analysis revealed phage η DNA did not contain modified bases. The genome sequence of this virus, determined using pyrosequencing, is 42,724 nucleotides in length with a mol% GC of 49.9 and is circularly permuted. Sixty-nine putative CDSs were identified of which 19 encode novel proteins. While seven close genetic relatives were identified, they shared sequence similarity with only genes 40 to 69 of the phage η genome, while gp1 to gp39 shared no conserved relationship. The structural proteome, determined by SDS-PAGE and mass spectrometry, revealed seven unique proteins. This phage forms very unstable lysogens with its host S. marcescens.

High temperature and bacteriophages can indirectly select for bacterial pathogenicity in environmental reservoirs.

The coincidental evolution hypothesis predicts that traits connected to bacterial pathogenicity could be indirectly selected outside the host as a correlated response to abiotic environmental conditions or different biotic species interactions. To investigate this, an opportunistic bacterial pathogen, Serratia marcescens, was cultured in the absence and presence of the lytic bacteriophage PPV (Podoviridae) at 25°C and 37°C for four weeks (N = 5). At the end, we measured changes in bacterial phage-resistance and potential virulence traits, and determined the pathogenicity of all bacterial selection lines in the Parasemia plantaginis insect model in vivo. Selection at 37°C increased bacterial motility and pathogenicity but only in the absence of phages. Exposure to phages increased the phage-resistance of bacteria, and this was costly in terms of decreased maximum population size in the absence of phages. However, this small-magnitude growth cost was not greater with bacteria that had evolved in high temperature regime, and no trade-off was found between phage-resistance and growth rate. As a result, phages constrained the evolution of a temperature-mediated increase in bacterial pathogenicity presumably by preferably infecting the highly motile and virulent bacteria. In more general perspective, our results suggest that the traits connected to bacterial pathogenicity could be indirectly selected as a correlated response by abiotic and biotic factors in environmental reservoirs.

Morphological and genetic analysis of three bacteriophages of Serratia marcescens isolated from environmental water.

Increases in multidrug-resistant strains of Serratia marcescens are of great concern in pediatrics, especially in neonatal intensive care units. In the search for bacteriophages to control infectious diseases caused by multidrug-resistant S. marcescens, three phages (KSP20, KSP90, and KSP100) were isolated from environmental water and were characterized morphologically and genetically. KSP20 and KSP90 belonged to morphotype A1 of the family Myoviridae, and KSP100 belonged to morphotype C3 of the family Podoviridae. Analysis of the DNA region coding virion proteins, together with their morphological features, indicated that KSP20, KSP90, and KSP100 were related to the P2-like phage (temperate), T4-type phage (virulent), and phiEco32 phage (virulent), respectively. Based on amino acid sequences of the major capsid protein, KSP90 formed a new branch with a Stenotrophomonas maltophilia phage, Smp14, in the T4-type phage phylogeny. Both Smp14 and phiEco32 have been reported as potential therapeutic phages. These results suggest that KSP90 and KSP100 may be candidate therapeutic phages to control S. marcescens infection.

[Isolation and characterization of Serratia marcescens phage].

OBJECTIVE: To isolate the bacteriophage of Serratia marcescens (8039) from sewage, and to study on its biological characteristics. METHODS: We used Serratia marcescens (8039) as the host to isolate phage from raw sewage from the Sewage Treatment Center of Hospital. Phage SM701 was examined in electron microscope. The genome of SM701 was extracted and the size of its nucleic acid was identified with restriction enzyme analysis. Phage isolated was subjected to plaque forming unit (PFU) assay using double layer agar medium plate method and morphological properties of plaque was observed, and finally optimal MOI multiplicity of infection and one-step growth experiments were carried out. RESULTS: Phage SM701 specific to Serratia marcescens (8039) was isolated successfully from the raw sewage. SM701 had an isometric polyhedral head (about 64 nm in diameter) and a long noncontractile tail (about 143 nm long). The nucleic acid could be cut off by dsDNA restriction enzyme BamH I or HindIII and its complete size was about 57kb. The plaque of SM701 was transparent about 1mm in diameter at the 12th hour. When MOI equaled 10, the number of phage offspring was higher. One-stepgrowth kinetics was determined according to the results of one-step growth experiment, which showed that the latent period was about 30 min, the rise period was about 100 min, and the average bust size was about 63 pfu/cell. CONCLUSION: Phage SM701 belonged to tailed family: siphoviridae and lytic bacteriophage. It was quite easy to observe and count the plaques due to existing color differences between lytic and nonlytic zone.

A generalized transducing phage (phiIF3) for the genomically sequenced Serratia marcescens strain Db11: a tool for functional genomics of an opportunistic human pathogen.

A bacteriophage (phiIF3) capable of mediating generalized transduction in Serratia marcescens strain Db11 has been isolated and characterized. The genome of this Serratia strain has recently been sequenced and is likely to become the reference strain for S. marcescens researchers. phiIF3 is most likely a virulent phage, which can transduce markers at frequencies of 10(-6) transductants per p.f.u. It has a lipopolysaccharide receptor and was determined to have a latent period of 50 min and a burst size of approximately 100 phages. The phage DNA was resistant to digestion with restriction enzymes. Electron microscopy showed phiIF3 to be a member of the family Myoviridae. This is the first report of a generalized transducing phage able to infect Db11 and this phage will be a valuable tool for functional genomic analysis of the pathogen host.

Two genes in prophage y of Serratia marcescens with functional counterparts in prophage Psi tested by Mu insertions as to order and homology.

Two special genes carried in pairs by the native prophages y and psi of Serratia marcescens HY, with functionally rather similar counterparts each, were assigned to restriction fragments and tested for homology. The y genes any and sky, as well as the psi genes anp and skp, are specifically activated by infection of HY cells with kappa phage. The kappa genes exerting this effect are tay for y and tap for psi. By means of tay and tap mutants, insertions of phage Mu DNA in the relevant parts of y and psi prophage, respectively, could be discovered. These insertions and subsequent deletions of Mu were the basis of our studies. The use of Mu was made possible by the isolation of an HY variant giving appropriate plaques with Mu particles of the G(-) type. In the case of another HY variant, Mu plaque formation depended on the presence of a host range mutation isolated by its ability to allow plaque formation on E. coli C, an indicator only for G(-) particles. Unexpectedly, this HY strain was an indicator of both G(-) and G(+) particles, but unfortunately had become adsorption resistant to y and psi. As an accessory result, it provided evidence for a second restriction/modification system. In both cases the O-specific polysaccharides were reduced. The main results of our paper concerning the two pairs of y and psi genes are as follows: the orders any-sky and anp-skp correspond with each other in y and psi; the sky gene, just as the skp gene, lies near one prophage end. However, despite the similarity in function and order, these genes are not homologous, in contrast to any and anp, which are at least partially homologous. The homologous regions of y and psi amount to only about 0.5 kbp. Another observation was that bacteria with a Mu insertion near the skp end of psi prophage were no longer cured of psi when infected with kappa tay-l, in contrast to the efficient curing observed with an ordinary psi prophage.

Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase.

Biofilm bacteria Enterobacter agglomerans 53b and Serratia marcescens Serr were isolated from a food processing factory. A bacteriophage (SF153b), which could infect and lyse strain 53b, was isolated from sewage. This has been shown to possess a polysaccharide depolymerase enzyme specific for the exopolysaccharide (EPS) of strain 53b. Using batch culture and chemostat-linked Modified Robbins Device systems it was observed that SF153b could degrade the EPS of a mono-species biofilm (strain 53b) and infect the cells. The disruption of the biofilm by phage was a combination of EPS degradation by the depolymerase and infection and subsequent cell lysis by the phage. Strain Serr biofilms were not susceptible to the phage and the biofilm EPS was not degraded by the phage glycanase, with the result that the biofilm was unaffected by the addition of SF153b phage. Scanning electron microscopy confirmed that specific phage could extensively degrade susceptible biofilms and continue to infect biofilm bacteria whilst EPS degradation was occurring.

Different restriction profiles of kappa prophage in Serratia marcescens K and HY.

Temperate phage kappa originated from the defectively lysogenic Serratia marcescens strain K, from where it was liberated after uv irradiation with low efficiency. The phage is usually indicated on strain HY that can be easily lysogenized by it and rather efficiently uv-induced. Comparing the Eco RI restriction profiles of the kappa prophage in HY revealed a DNA rearrangement, by which the precursor structure in K is converted into the non-defective form. Apparently the fragment containing the pac sequence is concerned since a phage DNA probe prepared from the assumed initiation fragments of the first particles of the packaging series gave two signals instead of one with genomic K DNA. Since several independent new kappa isolates showed the same Eco RI restriction pattern as the original phage of ELLMAUER and KAPLAN (1959), the generation of kappa is a reproducible event.