Genomic characterization of two Staphylococcus epidermidis bacteriophages with anti-biofilm potential - PubMed (original) (raw)

Genomic characterization of two Staphylococcus epidermidis bacteriophages with anti-biofilm potential

Diana Gutiérrez et al. BMC Genomics. 2012.

Abstract

Background: Staphylococcus epidermidis is a commensal bacterium but can colonize the hospital environment due to its ability to form biofilms favouring adhesion to host tissues, medical devices and increasing resistance to antibiotics. In this context, the use of phages to destroy biofilms is an interesting alternative.

Results: The complete genomes of two Staphylococcus epidermidis bacteriophages, vB_SepiS-phiIPLA5 and vB_SepiS-phiIPLA7, have been analyzed. Their genomes are 43,581 bp and 42,123 bp, and contain 67 and 59 orfs. Bioinformatic analyses enabled the assignment of putative functions to 36 and 29 gene products, respectively, including DNA packaging and morphogenetic proteins, lysis components, and proteins necessary for DNA recombination, regulation, modification and replication. A point mutation in vB_SepiS-phiIPLA5 lysogeny control-associated genes explained its strictly lytic behaviour. Comparative analysis of phi-IPLA5 and phi-IPLA7 genome structure resembled those of S. epidermidis ϕPH15 and ϕCNPH82 phages. A mosaic structure of S. epidermidis prophage genomes was revealed by PCR analysis of three marker genes (integrase, major head protein and holin). Using these genes, high prevalence (73%) of phage DNA in a representative S. epidermidis strain collection consisting of 60 isolates from women with mastitis and healthy women was determined. Putative pectin lyase-like domains detected in virion-associated proteins of both phages could be involved in exopolysaccharide (EPS) depolymerization, as evidenced by both the presence of a clear halo surrounding the phage lysis zone and the phage-mediated biofilm degradation.

Conclusions: Staphylococcus epidermidis bacteriophages, vB_SepiS-phiIPLA5 and vB_SepiS-phiIPLA7, have a mosaic structure similar to other widespread S. epidermidis prophages. Virions of these phages are provided of pectin lyase-like domains, which may be regarded as promising anti-biofilm tools.

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Figures

Figure 1

Figure 1

Physical and genetic map of phages phi-IPLA5 (A) and phi-IPLA7 (B). The ORFs are sequentially numbered, indicated by arrows proportional to their lengths and pointing toward their direction of transcription. Some ORFs have been placed below for clarity. The functional modules are indicated on top of the scheme, and the names of several putatively or experimentally identified genes are shown. Putative promoter (P) and terminator (T) sequences are also indicated.

Figure 2

Figure 2

Alignment of the genome of the two S. epidermidis phages phi-IPLA5 and phi-IPLA7 with those of other Staphylococcus phages using the Mauve software. Each block represents a region of the genome sequence that aligned and is homologous to part of another genome. Regions outside blocks lack detectable homology among the input genomes. Inside each block nucleotide sequence similarity is indicated by the height of the colored bars, while regions that are dissimilar are in white. Lines connecting blocks are indicative of homologous regions.

Figure 3

Figure 3

Aminoacid sequence analysis of the virion-associated protein gp18 from phi-IPLA7. A) ClustalW alignment of the predicted binding site. Positions with a single, fully conserved residue are marked with an asterisk; the colon marks the conserved residues between groups of strongly similar properties, and the period marks the residues weakly conserved between groups based on the Gonnet PAM 250 matrix, score <0,5. Highlighted are those residues involved in ligand association. B) Predicted 3D structure of phi-IPLA7 gp18. C) Predicted 3D structure of the domain pectin lyase like (D1) (amino acids 316- 374).

Figure 4

Figure 4

Effect of phages A) phi-IPLA5 and B) phi-IPLA7 on a lawn of S. epidermidis . I) Morphology of phage lytic zone when dropped on different S. epidermidis strains. (S) Sensitive strain; (R) resistant strain; (DS) drop-sensitive strain. II) Bacteria viable number in drop-zone (white), bacteria lawn (black) and phage titre (grey) of phages. Each value correspond with the mean of five different experiments, the standard error is represented by bars.

Figure 5

Figure 5

Killing of S. epidermidis cells forming a biofilm on microtiter wells by phage phi-IPLA7 after incubation for 1 h (A) and 3 h (B). Results are depicted as the percentage of attached cells (dark gray square) and planktonic cells (light gray square) detected in control biofilms treated with SM buffer (C) and in biofilms treated with phage phi-IPLA7 (PT). Each value corresponds with the mean of five different experiments and the standard error is represented by bars

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