Identification of Burkholderia cepacia complex bacteria with a lipopolysaccharide-specific monoclonal antibody

Abstract

The genus Burkholderia includes many bacteria that cause serious human infections. As is the case with other Gram-negative bacteria, Burkholderia species produce LPS, which is an abundant component of the bacterial cell surface. Burkholderia cepacia complex (Bcc) bacteria (which include at least 17 separate species) produce LPS structures that are quite different. In an attempt to determine the degree of LPS epitope variation among Bcc species, a mAb was produced, designated 5D8, specific for the LPS of B. cepacia. Western blot analysis determined that mAb 5D8 was able to produce the classic 'ladder pattern' when used to probe B. cepacia and Burkholderia anthina lysates, although 5D8 did not produce this pattern with the other seven Bcc species tested. mAb 5D8 reacted with varying intensity to most but not all of the additional B. cepacia and B. anthina strains tested. Therefore, there seems to be significant epitope variation among Bcc LPS both between and within species. Additionally, mAb 5D8 reacted with a proteinase-K-sensitive 22 kDa antigen in all Bcc strains and also in a strain of Burkholderia pseudomallei. INTRODUCTIONBacteria in the genus Burkholderia are Gram-negative bacilli that include species with the potential to cause lifethreatening human infections. People with cystic fibrosis (CF) are susceptible to infection by several Burkholderia species comprising the Burkholderia cepacia complex (Bcc) (LiPuma, 2005). 'Cepacia syndrome', which is characterized by necrotizing pneumonia and septicaemia resulting in rapid clinical deterioration, can develop in a small percentage of infected CF patients (Mahenthiralingam et al., 2005).Although much remains unknown about the pathogenic mechanisms of Burkholderia, these species produce a number of putative virulence determinants. One such determinant is LPS, which is present in abundance on the outer membrane of Gram-negative bacteria. Bcc LPS has potent endotoxic activity and elicits significantly higher levels of pro-inflammatory cytokines such as tumour necrosis factor, interleukin-6 and interleukin-8 than LPS from Pseudomonas aeruginosa, another common CF pathogen (Shaw et al., 1995;Vinion-Dubiel & Goldberg, 2003).The O antigen or O-specific polysaccharide, along with an oligosaccharide core and a lipid A molecule, are components of LPS (De Soyza et al., 2008). Bcc species produce many structurally distinct LPS O antigens (Burtnick et al., 2002;Vinion-Dubiel & Goldberg, 2003). In fact, 16 Oantigen serotypes have been identified among Bcc isolates recovered from CF patients (Nakamura et al., 1986;Soldatkina et al., 1989;Vinion-Dubiel & Goldberg, 2003). These serotypes do not correlate with Bcc species (Kenna et al., 2003;Rabkin et al., 1989). As a consequence, Oantigen serotype cannot be used to determine the specific Bcc species causing an infection. The O-antigen biosynthetic genes of Burkholderia stabilis (a member of the Bcc) have been mapped based on homology to other polysaccharide biosynthetic genes (Vinion-Dubiel & Goldberg, 2003). One gene, wbiI, wa...

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