Rapid detection and differentiation of the exfoliative toxin A-producing Staphylococcus aureus strains based on ϕETA prophage polymorphisms Pavla Holochová a , Vladislava Růžičková a, ⁎ , Lucie Dostálová a , Roman Pantůček a , Petr Petráš b , Jiří Doškař a a Faculty of Science, Department of Genetics and Molecular Biology, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic b Reference Laboratory for Staphylococci, National Institute of Public Health, 100 42 Prague, Czech Republic Received 9 June 2009; accepted 7 October 2009 Abstract The exfoliative toxin A (ETA) is encoded by the gene located on Staphylococcus aureus prophages. We have developed a single-reaction multiplex polymerase chain reaction (PCR) assay for rapid and specific detection of various ϕETA prophages of serogroup B responsible for dissemination of eta gene and ETA production in clinical strains. This PCR strategy enabled to classify the ETA-positive strains into 6 groups designated ETA-B1, ETA-B2, ETA-B3, ETA-B4, ETA-B5, and ETA-B6. The method was tested on a diverse set of 101 ETA and/or ETB-positive S. aureus strains isolated in 22 Czech maternity hospitals and 1 Slovak maternity hospital between 1998 and 2009. This novel PCR strategy is reliable in the rapid identification of yet undescribed ETA-converting B prophages and differentiation of the closely related ETA-positive strains, and it is a convenient tool for hospital epidermolytic infection control. © 2010 Elsevier Inc. All rights reserved. Keywords: Staphylococcus aureus; Pemphigus neonatorum; Exfoliative toxin A; Prophages; Multiplex PCR 1. Introduction Exfoliative toxin (ET) produced by Staphylococcus aureus strains is the major causative agent of blistering skin disorders, the most severe of them being staphylococcal scalded skin syndrome (SSSS) that primarily affects neonates and young children (Ladhani et al., 1999; Plano, 2004). Serologically, this toxin isolated from human strains has been divided into 3 isoforms, the most frequently occurring ETA and ETB toxins (Kondo et al., 1975; Růžičková et al., 2003) and much less frequently occurring ETD toxin (Yamaguchi et al., 2002). A fourth ETC was isolated from a horse strain of S. aureus (Sato et al., 1994); however, it has not been associated with human disease. The eta gene encoding ETA is located on a prophage that is integrated into the S. aureus chromosome. To date, a few ETA phages have been induced from bacterial cells and found capable of transferring the eta gene into a prophage- less strain (Endo et al., 2003; Yamaguchi et al., 2000; Yoshizawa et al., 2000). Between 1998 and 1999, the ETA-producing S. aureus strains responsible for 2 outbreaks of pemphigus neona- torum in the Czech maternity hospitals were genotyped and examined for the content of prophages of serogroups A, B, and F. Analysis of the prophage carriage revealed the presence of at least 1 prophage in all strains (Růžičková et al., 2003). Prophages of serogroup B significantly pre- dominated over the other prophages. Identification of several monolysogenic ETA-positive strains harboring single B prophage indicates that the eta gene is carried by a B phage. Because closely related strains can contain different ETA-converting prophages, their detection and characterization is very important for diagnostics of the strains responsible for outbreaks of SSSS or other forms of toxic epidermolytic diseases. There have been several reports describing the use of molecular typing methods for characterization of ET- Available online at www.sciencedirect.com Diagnostic Microbiology and Infectious Disease 66 (2010) 248 – 252 www.elsevier.com/locate/diagmicrobio ⁎ Corresponding author. Tel.: +420-5-49496827; fax: +420-5- 49492570. E-mail address: vladkar@sci.muni.cz (V. Růžičková). 0732-8893/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2009.10.008