Microbiological and molecular assessment of bacteriophage ISP for the control of Staphylococcus aureus - PubMed (original) (raw)

Microbiological and molecular assessment of bacteriophage ISP for the control of Staphylococcus aureus

Katrien Vandersteegen et al. PLoS One. 2011.

Abstract

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the 'Twort-like viruses'. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Dendrogram of the 22 identified S. aureus and S. haemolyticus genotypes.

The dendrogram was generated based on rep-PCR patterns using a 98% similarity level. For each genotype, the representative strain and its fingerprint, the constituting isolates and the ISP sensitivity are indicated.

Figure 2

Figure 2. Infection curves of Staphylococcus phage ISP.

Three independent experiments of a non-infected culture and cultures infected with MOI 0.1, 1 and 10 of S. aureus phage propagation strain 47 (A) and S. aureus subsp. aureus Rosenbach ATCC 6538 (B) are shown. Standard deviations are indicated.

Figure 3

Figure 3. Summary of the bio-informatical analysis of the phage ISP genome.

(A) Representation of the genome of phage ISP. Predicted proteins are indicated as bars, putative promoters are depicted as arrows and putative terminators as hair pins and the 4 tRNA's are indicated in light blue. The DNA polymerase and the endolysin, both interrupted by endonucleases, are colored red and orange respectively. Genes which were not annotated in phage G1 are colored purple and genes mistakenly annotated in phage G1 in dark blue, additional genes in phage ISP are framed in red. (B, C and D) Pairwise DNA homology of ISP and G1, ISP and K and ISP and Twort, compared using a sliding window of 50 bp.

Figure 4

Figure 4. Coomassie G-250 stained SDS-PAGE gel of the structural proteome of phage ISP.

On the left a low molecular weight marker is shown. The numbers on the right correspond to the analyzed gel pieces of which the identified proteins are given in Table 1. Clearly visible protein bands are indicated with capital letters.

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