Klebsiella pneumoniae type 3 fimbriae agglutinate yeast in a mannose-resistant manner Steen G. Stahlhut, Carsten Struve and Karen A. Krogfelt Correspondence Carsten Struve cas@ssi.dk Received 14 July 2011 Accepted 15 October 2011 Department of Microbiological Surveillance and Research, Statens Serum Institut, 2300 Copenhagen S, Denmark The ability of bacterial pathogens to express different fimbrial adhesins plays a significant role in virulence. Thus, specific detection of fimbrial expression is an important task in virulence characterization and epidemiological studies. Most clinical Klebsiella pneumoniae isolates express type 1 and type 3 fimbriae, which are characterized by mediation of mannose-sensitive agglutination of yeast cells and agglutination of tannic acid-treated ox red blood cells (RBCs), respectively. It has been observed that K. pneumoniae isolates agglutinate yeast cells and commercially available sheep RBCs in a mannose-resistant manner. Thus, this study was initiated to identify the adhesin involved. Screening of a mutant library surprisingly revealed that the mannose-resistant agglutination of yeast and sheep RBCs was mediated by type 3 fimbriae. Specific detection of type 1 fimbriae expression in K. pneumoniae was feasible only by the use of guinea pig RBCs. This was further verified by the use of isogenic fimbriae mutants and by cloning and expressing K. pneumoniae fimbrial gene clusters in Escherichia coli. Yeast agglutination assays are commonly used to detect type 1 fimbriae expression but should not be used for bacterial species able to express type 3 fimbriae. For these species, the use of guinea pig blood for specific type 1 fimbriae detection is essential. The use of commercially available sheep RBCs or yeast is an easy alternative to traditional methods to detect type 3 fimbriae expression. Easy and specific detection of expression of type 1 and type 3 fimbriae is essential in the continuous characterization of these important adhesive virulence factors present in members of the Enterobacteriaceae. INTRODUCTION Klebsiella pneumoniae is recognized as an important oppor- tunistic pathogen and is a common cause of urinary tract infections, respiratory tract infections and sep- ticaemia, especially in immunocompromised individuals (Podschun & Ullmann, 1998). In recent years, an emerging syndrome of community-acquired pyogenic liver abscess caused by highly virulent K. pneumoniae strains has occurred (Keynan et al., 2007; Ko et al., 2002; Lederman & Crum, 2005; Sobirk et al., 2010; Wang et al., 1998). These severe infections often occur in otherwise healthy individuals and are frequently complicated by devastating dissemination of the infection to the eyes and central nervous system. The rising incidence of serious K. pneumoniae infections stresses the need to elucidate the pathogenic mechanisms that are responsible for transform- ing this otherwise opportunistic pathogen into a highly virulent pathogen. Adherence to tissue surfaces as well as artificial surfaces is an important first step for bacteria in the development of infections. Using PCR and anti-fimbrial serum, it has been established that .80 % of K. pneumoniae isolates are able to express the two fimbrial adhesins type 1 and type 3 fimbriae (Schurtz et al., 1994; Stahlhut et al., 2009; Tarkkanen et al., 1992). Type 1 fimbriae, originally described in the late 1950s, are present in the majority of enterobacterial species (Duguid et al., 1955). They belong to the chaperone/usher class fimbriae family and are encoded by fimABCDEFGHIK, with fimA being the major structural subunit whilst fimH encodes the adhesive subunit. FimH has been shown to mediate adhesion to mannose-containing structures present on host tissue surfaces and extracellular matrix (Klemm et al., 1990; Krogfelt et al., 1990; Madison et al., 1994). We recently established that type 1 fimbriae are essential for the ability of K. pneumoniae to cause urinary tract infections (Struve et al., 2008). Like type 1 fimbriae, type 3 fimbriae belong to the chaperone/usher class fimbriae. Type 3 fimbriae were first described in Klebsiella species exhibiting mannose- resistant haemagglutination of tannic acid-treated ox erythrocytes (Duguid, 1959; Thornley & Horne, 1962). In addition to Klebsiella species, type 3 fimbriae are common in Enterobacter, Serratia, Proteus and Providencia isolates (Clegg et al., 1994). The fimbriae are encoded by the mrkABCDF gene cluster, where the mrkD gene encodes the Abbreviation: RBC, red blood cells. Journal of Medical Microbiology (2012), 61, 317–322 DOI 10.1099/jmm.0.036350-0 036350 G 2012 SGM Printed in Great Britain 317