title: Detection of methicillin-resistant staphylococci by biosensor assay consisting of nanoscale films on optical fiber long-period gratings.
abstract: Methicillin-resistance among Staphylococcus species is a major health problem in hospitals, communities, and animals. There is a need for culture-free diagnostic assays that can be carried out rapidly, and maintain a high degree of sensitivity and specificity. To address this need an ionic self-assembled multilayer (ISAM) film was deposited on the surface of a long-period grating (LPG) optical fiber by immersion alternately in poly-allylamine hydrochloride and in poly-1-[p-(3'-carboxy-4'-hydroxyphenylazo) benzenesulfonamido]-1,2-ethandiyl (PCBS), resulting in terminal carboxyl groups on the LPG-ISAM. The terminal carboxyl groups were covalently conjugated to monoclonal antibodies (MAb) specific to penicillin-binding-protein 2a of methicillin resistant (MR) staphylococci. After exposure of the LPG-ISAM to 10(2) colony forming units (CFU)/ml of MR S. aureus (MRSA) for 50 min., light transmission was reduced by 19.7%. In contrast, after exposure to 10(6) CFU/ml of methicillin-sensitive S. aureus (MSSA) attenuation of light transmission was less than 1.8%. Exposure of the LPG-ISAM to extracts of liver, lungs, or spleen from mice infected with MRSA attenuated light transmission by 11.7-73.5%. In contrast, exposure of the biosensor to extracts from MSSA-infected mice resulted in 5.6% or less attenuation of light transmission. When the sensor was tested with 36 strains of MR staphylococci, 15 strains of methicillin-sensitive staphylococci, 10 strains of heterologous genera (all at 10(4) CFU/ml), or tissue samples from mice infected with MRSA, there was complete agreement between MR and non-MR bacteria determined by antibiotic susceptibility testing and the biosensor assay when the cutoff value for attenuation of light transmission was 6.3%. Thus, the biosensor described has the potential to detect MR staphylococci in clinical samples with a high degree of sensitivity and specificity.
authors.affiliation: Department of Biomedical Sciences and Pathobiology, Life Sciences 1, 970 Washington Street, SW, Virginia Tech, Blacksburg, VA 24061, USA. Electronic address: abandara@vt.edu.
authors.forename: Aloka B
authors.initials: AB
authors.lastname: Bandara
authors.affiliation: Department of Physics, 850 West Campus Drive, Virginia Tech, Blacksburg, VA 24061, USA. Electronic address: zuo@vt.edu.
authors.forename: Ziwei
authors.initials: Z
authors.lastname: Zuo
authors.affiliation: Department of Electrical and Computer Engineering, 8 Saint Mary's Street, Boston University, Boston, MA 02215, USA. Electronic address: sidr@bu.edu.
authors.forename: Siddharth
authors.initials: S
authors.lastname: Ramachandran
authors.affiliation: Virginia nanoTech, LLC, 2200 Kraft Drive, Blacksburg, VA 24060, USA. Electronic address: rittera@vnanot.com.
authors.forename: Alfred
authors.initials: A
authors.lastname: Ritter
authors.affiliation: Department of Physics, 850 West Campus Drive, Virginia Tech, Blacksburg, VA 24061, USA; Virginia nanoTech, LLC, 2200 Kraft Drive, Blacksburg, VA 24060, USA. Electronic address: rheflin@vt.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Heflin
authors.affiliation: Department of Biomedical Sciences and Pathobiology, Life Sciences 1, 970 Washington Street, SW, Virginia Tech, Blacksburg, VA 24061, USA; Virginia Tech Carilion School of Medicine, 2 Riverside Circle, Roanoke, VA 24016, USA. Electronic address: tinzana@vt.edu.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Inzana
mesh_terms: D058491:Bacterial Load / Q000295:instrumentation*; D015374:Biosensing Techniques / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005336:Fiber Optic Technology / Q000295:instrumentation*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000302:isolation & purification*; D046228:Microarray Analysis / Q000295:instrumentation*; D012031:Refractometry / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
keywords: Biosensor; Ionic self-assembled multilayer films; Long-period gratings optical fiber; Methicillin-resistant Staphylococcus spp.; Monoclonal antibody; Penicillin-binding protein 2a