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DdeI RFLP for 16S rDNA typing in Bartonella henselae

Centre for Infectious Diseases and Microbiology, University of Sydney, Westmead Hospital, NSW 2145, Australia

Correspondence: J. Iredell (joni{at}icpmr.wsahs.nsw.gov.au)

Bartonella henselae is a fastidious Gram-negative bacterial pathogen of cats that may cause lethal human infection (Regnery et al., 1992; Welch et al., 1992), and is the only microbial pathogen shown to directly mediate reversible angiogenesis in human cells (Kempf et al., 2001).

The epidemiology is complex and while it may cause severe illness in the immune competent host, some infections appear to be relatively benign. 16S rDNA typing suggests two distinct lineages corresponding to serotype. The Marseille serotype corresponds to 16S type II (Drancourt et al., 1996; La Scola et al., 2002), and may also be distinguished from the type strain (Houston serotype) by protein profile (Iredell et al., 2002; La Scola et al., 2002), and by single- and multilocus DNA typing schemes (Birtles & Raoult, 1996; Iredell et al., 2003; Zeaiter et al., 2002a, b).

Sequencing and RFLP within the 16S–23S region is useful for interspecies but not intraspecies distinctions (Dillon et al., 2002). Multilocus sequence (MLS) analysis suggests a tightly clustered Marseille lineage, although the 16S rDNA type II was also found outside the Marseille lineage and there is some support for horizontal gene transfer within the species (Iredell et al., 2003). 16S rDNA typing nevertheless remains the most accepted and widely used method for Bartonella subtyping, and there is relative over-representation of 16S rDNA type I strains in human compared with feline isolates (Bergmans et al., 1996; Dillon et al., 2002; Ehrenborg et al., 2000). In addition, a recent epidemiological study suggests that clinical outcomes may vary with 16S type (Bergmans et al., 1996; Chang et al., 2002).

In the original description of PCR-based 16S subtyping of B. henselae, some were described as untypable, and some gave positive reactions for both type I and type II (Bergmans et al., 1996). Using exactly the same methods in a larger study (Dillon et al., 2002), both type I and type II 16S product was obtained in 11 of 59 isolates. Raising the annealing temperature resolved this apparent lack of PCR specificity in five, but the remainder required sequencing as further increases in temperature caused reaction failure (Dillon et al., 2002). 16S rDNA sequences were unambiguous, arguing against multicopy 16S rDNA genes. In addition, three strains with reproducibly positive and specific reactions were thus shown to have been incorrectly assigned by the usual method (Iredell et al., 2003).

This pathogen may be difficult to culture (La Scola & Raoult, 1999), and these difficulties may relate to genetic variation (Kyme et al., 2003). However, since 16S rDNA is readily obtained directly from clinical samples (Rodrick et al., 2004), we developed a simple 16S RFLP, and describe results in illustrative strains from a published collection (Dillon et al., 2002; Iredell et al., 2003).

Boiled lysates (Dillon et al., 2002) provided template for the original PCR primers (Tables 1 and 2; Fig. 1) and methods were as described by Bergmans et al. (1996). DdeI is predicted to yield 228, 146, 95, 27 and 16 bp fragments (total size 512 bp for Houston-1; M73229), or 242, 228, 27 and 16 bp fragments (total size 513 bp for Marseille strains; GenBank accession no. AJ223779). Five microlitres of product was restricted without purification in 20 µl according to the manufacturer's instructions (New England Biolabs). Excellent resolution is obtained by PAGE (not shown), as described for infrequent restriction site typing (Dillon et al., 2002), but even slightly overloaded or indistinct tracks on 2 % agarose give clear results (Fig. 2, lanes 3, 6 and 9). Thus while MLS typing is expensive and published 16S subtyping tools may be misleading, this simple rapid method provides an unambiguous and accurate 16S type. Its broad application, even in culture-negative infected material (Rodrick et al., 2004), can be expected to add significantly to our understanding of the epidemiology of this important human pathogen.

View larger version (34K): [in this window] [in a new window]   Fig. 1. PCR subtyping of 16S rDNA (16SF/BH1 or /BH2) at Tm 56 °C. Lanes: 1 and 9, HC71; 2 and 10, R987; 3 and 11, NU4423; 4 and 12, RMC10; 5 and 13, HC48; 6 and 14, ATCC 49882 (Houston-1); 7 and 15, URLLY-8 (Marseille); 8, molecular size marker (Invitrogen): sizes indicated on the right in bp. All strains as previously described in detail (Dillon et al., 2002; Iredell et al., 2003). Products were electrophoresed in 2 % (w/v) agarose and visualized with ethidium bromide on a UV transilluminator.

View larger version (45K): [in this window] [in a new window]   Fig. 2. DdeI RFLP of partial 16S rDNA (16SF/16SR product). Lanes: 1, Houston-1/ATCC 49882 (type I); 2, HC62 (type I); 3, BH3 (type I); 4, JR2 (type I); 5, BH2 (type I); 6, HC48 (type II); 7, molecular size marker (Invitrogen): sizes indicated on the right in bp; 8, RMC10 (type I); 9, HC60 (type II); 10, NU4423 (type II); 11, Marseille URLLY-8 (type II). These strains have been previously described in detail and the 16S rDNA fully sequenced (Dillon et al., 2002; Iredell et al., 2003).

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