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.

Keywords: Biosensor; Ionic self-assembled multilayer films; Long-period gratings optical fiber; Methicillin-resistant Staphylococcus spp.; Monoclonal antibody; Penicillin-binding protein 2a.