Abstract
Klebsiella pneumoniae is a well-known opportunistic pathogen associated with nosocomial infections such as urinary tract infections, pneumonia and septicaemia (Podschun & Ullmann, 1998). In recent years, a high incidence of community-acquired K. pneumoniae pyogenic liver abscess with a high mortality rate has been reported, especially from Taiwan, and more sporadic cases have been reported from other Asian countries, Europe and North America (Wang et al., 1998; Fung et al., 2002; Okano et al., 2002; Rahimian et al., 2004; Fang et al., 2005). Most recently, K. pneumoniae was reported as the most common organism isolated from pyogenic liver abscesses in two independent USA studies (Rahimian et al., 2004; Lederman & Crum, 2005). Although diabetes mellitus is considered an important risk factor, approximately half the cases occur in otherwise healthy people. Moreover, severe metastatic infections including endophthalmitis and meningitis occur more frequently in patients infected with K. pneumoniae than in patients with liver abscess of other aetiological origins (Wang et al., 1998; Yang et al., 2004).
The pathogenic mechanism of this infectious disease is not well understood. K. pneumoniae characteristically produce vast amounts of capsular polysaccharide covering the bacterial surface. Of the 77 capsular (K) serotypes recognized, the majority of liver abscess isolates belong to the K1 and K2 serotypes (Fung et al., 2002). A new virulence gene magA (mucoviscosity-associated gene) was recently identified in pathogenic strains from Taiwan causing liver abscess (Fang et al., 2004). This gene was detected in the vast majority of K. pneumoniae liver abscess isolates and was associated with hypermucoviscosity, and resistance to killing by human serum and phagocytosis as well as high virulence in an animal model. We investigated the prevalence of the magA gene in our collection of K. pneumoniae at the International Escherichia coli and Klebsiella Reference Centre (WHO), Statens Serum Institut (SSI), Denmark, and found that magA was restricted to the capsular gene cluster of serotype K1.
Initially, eight isolates were checked for the presence of magA by PCR analysis. magA-specific primers MagA-F (5'-TAGGACCGTTAATTTGCTTTGT-3') and MagA-R (5'-GAATATTCCCACTCCCTCT CC-3') were designed from the published magA sequence (GenBank accession no. AB085741). The eight strains belonged to the most frequent capsule serotypes associated with liver abscess, K1 and K2, and displayed the hypermucoid phenotype associated with magA (Fang et al., 2004). Of the eight isolates, only the three isolates belonging to the K1 serotype, including the K1 serotype reference strain (A5054), were magA positive.
To further investigate the prevalence of magA and the association between magA and capsular serotype, a collection of 495 international K. pneumoniae isolates from the Reference Centre (WHO) was screened by colony blot hybridization. The strain collection included the reference strains of all 77 K. pneumoniae capsule serotypes. The additional 418 clinical isolates included in the collection were serotyped by counter-current immuno-electrophoresis as previously described (Hansen et al. 1998). From the K1 serotype test strain, a digoxigenin (DIG)-labelled nucleotide probe for magA was prepared by use of the PCR DIG Synthesis Kit (Roche), as described by the manufacturer, using the primer pair MagA-F and MagA-R. Colony hybridizations were performed on Hybond-N+ membranes (Amersham) under stringent conditions according to the manufacturer's directions. The screening revealed 39 magA-positive isolates (7.8 %). None of the 456 non-K1 serotypes in the strain collection contained magA and all 39 magA-positive isolates were of the K1 capsule serotype, indicating a close relationship between magA and the K1 capsule serotype.
The K. pneumoniae capsule gene cluster contains conserved and serotype-specific regions (Rahn et al., 1999; Brisse et al., 2004). So far, only the serotype-specific region of the K2 serotype has been published (Arakawa et al., 1995). By PCR analysis we established the relationship between magA and the K1 serotype. The primer pair CPS-1 (5'-GCTGGTAGCTGT TAAGCCAGGGGCGGTAGCG-3') and rCPS (5'-TATTCATCAGAAGCAGCACG CAGCTGGGAGAAGCC-3') is specific for conserved regions flanking the K. pneumoniae capsule gene cluster (Brisse et al., 2004). By combining the primers CPS-1 and rCPS with the magA-specific primers UmagA (5'-TTTGCGGCGAGAAATGCAT AAACGATAGGA-3') and DmagA (5'-AAG GGGATGT CAAAACTCCTGTAGTAGCG GCAATGTTCAT-3') we identified magA as a gene in the serotype-specific region of the K1 capsule gene cluster (Fig. 1).