Combined MLST and AFLP typing of Bartonella henselae isolated from cats reveals new sequence types and suggests clonal evolution

Abstract

Bartonella species are Gram-negative, fastidious bacteria. Bartonella henselae is found in cats and transmitted to humans via cat scratches or bites causing cat-scratch disease, characterized by clinical symptoms with varying severity. The prevalence of bartonellosis among humans in Germany appears to be high, and severe clinical cases have been described. However, epidemiological data of B. henselae in cats are rare. In this study we determined the detection rates of Bartonella ssp. in cats by culture and real-time PCR. Furthermore, B. henselae isolates were genetically characterized by highly discriminatory amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Bartonella spp. were isolated by culture from 11 (2.2%) of 507 blood samples. Out of 169 blood samples additionally analyzed by PCR, 28 (16.6%) were found positive for Bartonella spp., illustrating the advantage of PCR in Bartonella spp. detection. PCR-REA identified B. henselae in 27 cats and Bartonella clarridgeiae in one cat. B. henselae isolates from different geographical regions in Germany were genetically characterized by AFLP and MLST. Both methods confirmed genetic diversity of B. henselae on the strain level. MLST identified 11 new sequence types, all of them assigned to three clonal complexes as determined by eBURST. AFLP typing revealed genetic relation among the B. henselae isolates from the same geographical region. Combining AFLP typing and MLST/eBURST analyses revealed that B. henselae of the same AFLP subcluster belonged to the same clonal complex. Altogether these results indicate that B. henselae may evolve clonally.

Introduction

Members of the genus Bartonella are small, fastidious Gram-negative rod-shaped bacteria. They infect and persist in mammalian erythrocytes and endothelial cells and are found in a wide range of wild and domesticated mammals throughout the world. Many Bartonella species are zoonotic (Dehio, 2004).

The most frequently encountered Bartonella species is Bartonella henselae, the zoonotic agent of cat-scratch disease in humans, an often self-limiting infection characterized by lymphadenopathy. Moreover, in immunocompromized patients B. henselae can cause severe infections such as bacillary angiomatosis, bacillary peliosis, endocarditis, osteomyelitis and neuroretinitis (Boulouis et al., 2005).

Cats represent the natural host and main reservoir for B. henselae. Prevalence of infection in domestic cats varies from 0% in Norway (Bergh et al., 2002) to 61% in the Philippines (Chomel et al., 1999). Despite its long lasting relapsing bacteraemia, the infection in cats is mostly asymptomatic and self-limiting after several months of duration (Guptill et al., 1997). Infection is transmitted from one cat to another by cat flea (Chomel et al., 1996).

The bacterial isolation from clinical samples is often hampered by difficulties in culturing the organisms. Therefore, the currently preferred mode of detection is the polymerase chain reaction (PCR) based on the amplification of Bartonella specific DNA sequences (Matar et al., 1993, Norman et al., 1995, Raoult et al., 1996, Renesto et al., 2001, Johnson et al., 2003).

Epidemiological studies have been performed to genetically characterize B. henselae. DNA fingerprinting methods as well as sequence based typing methods provided evidence that B. henselae strains are genetically heterogeneous (Rodriguezbarradas et al., 1995, Sander et al., 1998, Ehrenborg et al., 2000, Houpikian and Raoult, 2001, Zeaiter et al., 2002, La Scola et al., 2002, Dillon et al., 2002, Li et al., 2006).

To date, multilocus sequence typing (MLST) of B. henselae identified 15 different sequence types (ST) (Iredell et al., 2003, Arvand et al., 2007, Yanagihara et al., 2010). These studies provided evidence that human isolates assigned to 4 different ST, while feline isolates homogeneously assigned to all ST. Relating the ST to their worldwide distribution revealed conserved ST in different geographical regions of the world.

In this study we investigated the prevalence of Bartonella spp. in cats in the northern and western part of Germany. Since epidemiological studies of Bartonella isolates from different geographically regions of one country have not yet been performed, B. henselae isolates from different regions of Germany were related by combining AFLP and MLST typing.