Recognition of Staphylococcus aureus by lytic phage monolayers Phage immobilized glass substrates were exposed to 1 ml of bacterial (MRSA) solutions for 15 minutes. Then these glass slides were gently rinsed in deionized water for 10-15 seconds and air dried. Five different regions of each sensor surface were examined and photographed using a optical microscope (CitoViva Technologies Inc. and Olympus, BH-2 microscope). (Figure 1) The number of cells (N) bound to the substrate surface was measured using Image pro-plus 2.0 software. OBJECTIVE To detect Staphylococcus aureus (including methicillin resistant bacteria) by lytic phage monolayers. Rajesh Guntupalli, Iryna Sorokulova, April Krumnow, Oleg Putsovyy, Eric Olsen, and Vitaly Vodyanoy INTRODUCTION Target pathogen: Staphylococcus aureus Staphylococcus aureus is one of the most common causes of nosocomial or community-based infections, leading to serious illnesses with high rates of morbidity and mortality. In recent years, the increase in the number of bacterial strains that show resistance to methicillin (MRSA) has become a serious clinical and epidemiological problem Bioprobe: Lytic Phage We isolated new bacteriophage S. aureus 12600 with lytic activity against various S. aureus strains. Different S. aureus strains were used for testing isolated bacteriophage: S. aureus ATCC 12600 and methicillin-resistant (MRSA) strains. Immobilization of phage monolayers: LB method The monolayers made of lytic phage were transferred onto the solid substrate at air-water interface by the Langmuir-Blodgett (LB) method (Figure 1). The monolayer was formed on the air-liquid interface by allowing the spreading solution to run down a partially submerged wetted glass rod. Multilayers were obtained by successive dipping of the glass substrate through the monomolecular film deposited at a water-air interface RESULTS (a) Solid Substrate Monolayer on liquid surface Teflon Trough Variable speed Teflon barrier Deposition of monolayer onto the sensor Sub phase Compression of film sub phase Selectivity Test (c) Bacteria bound to Phage No phage (b) (a) (b) Figure 6: (a)–(c) Typical Microscopic images of phage immobilized glass surface after phage-bacterial interaction with different bacterial species at a concentration of 109 cfu/ml. (a) MRSA (b) Bacillus subtilis, (c) Salmonella typhimurium. RESULTS AND DISCUSSION Figure 3: Characteristics of lytic phage monolayers at 20 °C. (a) Surface pressure-area isotherm and (b) Elasticity of monolayers as a function of surface pressure Figure 2. Formation and deposition of lytic phage monolayers using Langmuir-Blodgett (LB) technique. This work has demonstrated the use of lytic bacteriophage as a probe for the detection of S. aureus including MRSA. Lytic phage monolayers were stable and were characterized by high levels of monolayer elasticity. A bacterial coverage of 33±5% was obtained for substrates tested with bacterial suspension of 109 cfu/ml, as compared to 10±1%, 1.1±0.1%, 0.09±0.01 % for the substrates when concentration of bacteria in tested suspensions was 108, 107 and 106 cfu/ml accordingly. Phage was active only against S. aureus strains and did not affect other bacteria (Salmonella typhimurium, Bacillus subtilis). We hypothesize that simultaneous recognition of S. aureus and PBP 2a protein increases specificity and reliability of MRSA detection. High resolution optical microscope for detection of bacteria a b d c Light Microscope Image Acquisition Camera Camera control unit Digital image processing Image storage ACKNOWLEDGMENTS The work was supported under USAF SG research investigation FDG20060049N and CRADA #07-277-60MDG-01. The views expressed in this article are those of the authors, and do not reflect the official policy or position of the United States Air Force, Department of Defense, or the U.S. Government. Figure 4: (a)–(d) Typical Microscopic images of MRSA bound to Phage immobilized glass substrate. Representative images of substrates exposed to solutions containing varying concentrations of bacteria (a) 109 cfu/ml, (b) 108 cfu/ml, (c) 107 cfu/ml and (d) 106 cfu/ml. Figure 5: Percentage of area covered by bacterial cells on a Phage immobilized substrate. Figure 1: Schematic of computer aided high resolution light microscope. AUBURN UNIVERSITY PROPRIETARY AND CONFIDENTIAL INFORMATION Rajesh Guntupalli, Post Doctoral fellow, Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University. Iryna Sorokulova, Research Professor, Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University. April Krumnow, Research Assistant II, Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University. Oleg Putsovyy, Research Assistant III, Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University Eric Olsen, Major, Deputy Director, Clinical Investigation Facility, David Grant USAF Medical Center Vitaly Vodyanoy, Alumni Professor, Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University