Discrimination of methicillin-resistant Staphylococcus aureus from methicillin-susceptible Staphylococcus aureus or coagulase-negative staphylococci by detection of penicillin-binding protein 2 and penicillin-binding protein 2′ using a bioluminescent enzyme immunoassay

• Published in: Journal of immunological methods Vol. 388; no. 1-2; pp. 40 - 45
• Main Authors: Shiga, Kazuki, Gomi, Keiko, Nishimura, Motoi, Watanabe, Masaharu, Nomura, Fumio, Kajiyama, Naoki
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 28.02.2013
• Subjects: antibiotic resistance; Antibiotics; bioluminescence; Central nervous system; Clinical isolates; Coagulase - deficiency; Coagulase - metabolism; Coagulase-negative staphylococci; Colonies; Detergents; diagnostic techniques; Drug resistance; Enzyme immunoassay; enzyme immunoassays; Humans; Immunoenzyme Techniques - methods; luciferase; Luciferases - chemistry; Luminescent Measurements - methods; methicillin; Methicillin-Resistant Staphylococcus aureus - chemistry; Methicillin-Resistant Staphylococcus aureus - drug effects; Methicillin-Resistant Staphylococcus aureus - enzymology; Methicillin-Resistant Staphylococcus aureus - isolation & purification; MRSA; PBP2; penicillin-binding protein 2; Penicillin-Binding Proteins - analysis; Staphylococcal Infections - microbiology; Staphylococcus aureus; Staphylococcus aureus - drug effects; Staphylococcus aureus - enzymology; Staphylococcus aureus - isolation & purification; MSSA; PBP2; CFU; CV; S; TBS; BLEIA; MIC; MRSA; Coagulase-negative staphylococci; CNS; PCR
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2012.11.012

For the discrimination of methicillin-resistant S. aureus (MRSA) from methicillin-susceptible S. aureus (MSSA) or coagulase-negative staphylococci (CNS), we developed a bioluminescent enzyme immunoassay (BLEIA) for detecting penicillin-binding protein 2 (PBP2) and penicillin-binding protein 2′ (PBP2′) using biotinylated firefly luciferase. The BLEIA was able to detect recombinant PBP2 at 50pg/ml and recombinant PBP2′ at 500pg/ml. PBP2 and PBP2′ present in the membranes of S. aureus were extracted by acid and detergent treatment. The method was able to detect PBP2 or PBP2′ extracted from 106 colony forming units of S. aureus because of efficient extraction and the high sensitivity of luciferase. In a study of clinical isolates previously characterized as either MRSA or MSSA by antibiotic susceptibility testing, all 34 specimens identified as MRSA were both PBP2 and PBP2′ positive. The 34 MSSA specimens were PBP2 positive and PBP2′ negative. Moreover, the BLEIA could detect PBP2′ extracted from four species of methicillin-resistant CNS, but not PBP2 extracted from four species of methicillin-resistant and methicillin-susceptible CNS. This result suggested that PBP2 could be a unique marker for discrimination of S. aureus from CNS. A BLEIA that is able to detect PBP2 and PBP2′ may be useful in clinical diagnostics. ► We developed a BLEIA for detecting PBP2 and PBP2′ using firefly luciferase. ► The BLEIA could detect PBP2 and PBP2′ from all 34 specimens identified as MRSA. ► Detection of PBP2 and PBP2′ made possible to discriminate MRSA from MSSA or CNS.