Advances in Biosensors and Bioelectronics (ABB) Volume 2 Issue 1, March 2013 www.seipub.org/abb/ 1 MRSA Biosensor Based on CCD Detection Rajesh Guntupalli 1 , Iryna Sorokulova 1 , Eric Olsen 2 , and Vitaly Vodyanoy 1* 1 College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA, 2 Clinical Research Laboratory, 81st Medical Group Keesler AFB MS 39534, USA 1 guntura@auburn.edu; 2 sorokib@auburn.edu; 3 eric.olsen@us.af.mil; * 4 vodyavi@auburn.edu Abstract A method for the detection of methicillin resistant Staphylococcus aureus (MRSA) using Charge Coupled Device (CCD) detector is described. Monolayers of bacteriophage were formed at an air–water interface and transferred onto silica substrates by Langmuir-Blodgett (LB) method. Firstly, the interactions of a wide host range of Staphylococcus aureus lytic bacteriophage and S. aureus were characterized on silica substrates by CCD detector. Experiment results indicated that this biosensor system has a detection limit of 10 5 cfu/ml. To distinguish MRSA and methicillin sensitive (MSSA) strains, a penicillin-binding protein (PBP 2a) specific antibody was used as a secondary probe. A simple agglutination test was carried out using a latex reagent sensitized with monoclonal antibody against PBP 2a. Agglutination indicated the presence of PBP 2a in MRSA strains. Keywords Lytic phage; Biosensosr; CCD; Phage Monolayer Introduction Methicillin resistant strains of MRSA are associated with significant infections and nosocomial outbreaks (Byun et al.; Giamarellou et al.; Knopf). MRSA shows resistance to a wide range of antibiotics thus limiting the treatment options (Broughan et al.; Durgaryan et al.). Recent outbreaks of MRSA in Europe and USA caused panic. They demonstrated lack of approaches to timely recognize dangerous infection and properly handle curing of that disease (Shorr et al.). Currently existing methods of MRSA detection have some limitations and lack sensitivity or specificity. Usually these tests can be used just for the confirmation of methicillin resistance after the specific identification of S. aureus, which require at least several hours (Diekema). Therefore, rapid detection of MRSA is of critical importance in prevention and prognosis of infections due to antibiotic resistant S. aureus. In this study, biosensor based on CCD detector was employed for the detection of MRSA using lytic phage probes. Optical biosensors based on CCD detectors provide an expedient method for quantifying bioprobe and analyte interactions on the biosensor surface. CCD based biosensors have proven to be sensitive and specific for detecting various analytes of interest. Usage of antibodies as molecular probes and biosensors based on CCD were described for detecting avian influenza virus (Qi et al.), filamentous phage M13KO7 (Qi et al.), hepatitis virus (Huang et al.), and Salmonella typhimurium (Bae et al.). In the present work, we use lytic phages as probes for recognition of MRSA and CCD to detect bacterial binding. It was previously demonstrated that bacteriophage 12600 can be used as a recognition probe for Staphylococcus aureus strains including those having methicillin-resistance (Balasubramanian et al.; Guntupalli et al.). Recently we demonstrated discrimination of MRSA and MSSA by using specially modified lytic bacteriophage (spheroids) and a penicillin-binding protein (PBP 2a) specific antibody using quartz crystal microbalance (Guntupalli et al.). In present work, we proposed a different approach in specific recognition and detection of MRSA, including identification of bacteria together with the conformation of MRSA in real time. For this purpose, we use an intact unmodified S. aureus bacteriophage along with monoclonal antibody against protein (PBP 2a). PBP 2a creates a bacterial cell wall structure and it is responsible for antibiotic resistively of MRSA. PBP 2a antibody may not recognize MRSA alone as the interaction between PBP 2a protein and antibodies is not specific for Staphylococcus aureus and other bacteria have antibiotic binding proteins with large sequence similarity to PBP 2a (Wei et al.). In order to build a biosensor to specifically detect and identify MRSA,we will employ a device with a two-step action. The first step will use an S. aureus bacteriophage monolayer as a sensor probe, while the second step will utilize an agglutination test using PBP 2a specific antibodies. Consequently, the step one will identify S. aureus bacteria; step two will confirm the presence/absence of antibiotic resistance. It is believed that simultaneous recognition of S. aureus bacteria and PBP 2a protein enhances specificity and consistency of MRSA sensing.