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Applied and Environmental Microbiology, July 2009, p. 4483-4490, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.02641-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Isolation from Sewage Influent and Characterization of Novel Staphylococcus aureus Bacteriophages with Wide Host Ranges and Potent Lytic Capabilities{triangledown}

Aidan J. Synnott,1 Ying Kuang,1 Miki Kurimoto,1 Keiko Yamamichi,1 Hidetomo Iwano,2 and Yasunori Tanji1*

Department of Bioengineering, Tokyo Institute of Technology, 4259 J2-15 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan,1 Department of Bioscience, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japan2

Received 18 November 2008/ Accepted 3 April 2009

Staphylococcus aureus is a gram-positive pathogen that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to a need for alternative treatments, such as bacteriophage therapy. Forty-nine S. aureus isolates were obtained from the milk of mastitic cows for use in screening of staphylococcal phages. Fifteen isolates which were positive for both coagulase and hemolysin were assayed by PCR for variation in the X region and the immunoglobulin G-binding region of the protein A gene (spa) and in the carboxy terminus of the coagulase gene (coa) and for the presence of enterotoxin C, G, H, and I genes. The host ranges of 52 phages isolated from sewage influent were determined by performing spot tests with the 15 S. aureus isolates, and two phages were subsequently chosen for further analysis. {Phi}SA039 had the widest host range, producing clear plaques on 13 of the 15 isolates (87%), while {Phi}SA012 produced clear plaques on 8 isolates (53%) and was the only phage that produced a clear plaque on a nonmastitic S. aureus strain. Transmission electron microscopy revealed that the phages were similar sizes and belonged to the Myoviridae family. Measurement of optical densities during coculture with S. aureus isolates confirmed the breadth of the {Phi}SA039 host range and showed that {Phi}SA012 had potent lytic capability. {Phi}SA012-resistant bacteria did not appear for three of seven isolates tested (43%) after 65 h of incubation. These two phages are proposed as candidates for phage therapy of bovine mastitis.

* Corresponding author. Mailing address: Department of Bioengineering, Tokyo Institute of Technology, 4259 J2-15 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Phone: 81-45-924-5763. Fax: 81-45-924-5818. E-mail: ytanji{at}bio.titech.ac.jp

{triangledown} Published ahead of print on 1 May 2009.

Applied and Environmental Microbiology, July 2009, p. 4483-4490, Vol. 75, No. 13
0099-2240/09/$08.00+0     doi:10.1128/AEM.02641-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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