Growth Characteristics ofBartonella henselaein a Novel Liquid Medium: Primary Isolation, Growth-Phase-Dependent Phage Induction, and Metabolic Studies

Abstract

Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 ؋ 10 8 CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.Bartonella henselae is a medically important gram-negative, zoonotic pathogen. Cats are the reservoir, and infections are generally asymptomatic. B. henselae causes a diverse and emerging disease spectrum in humans, from self-limiting lymphadenopathy to life-threatening conditions such as bacillary angiomatosis. Cat scratch disease (CSD), a generally benign infection characterized by regional lymphadenopathy and persistent fever, is the most-common manifestation of B. henselae infection in humans. CSD occurs in healthy individuals and affects an estimated 24,000 persons a year in the United States (11). In immunocompromised patients, B. henselae causes more-serious infections, including bacillary angiomatosis and bacillary peliosis, which can be fatal when misdiagnosed and improperly treated. A major contributing factor to misdiagnosis is the inherent difficulty in culturing the bacterium. B. henselae takes an average of 21 days to form colonies during primary isolation on blood agar plates, and no reliable liquid growth medium exists (8). In clinical settings, the protracted growth phase ofte...

Predominant Outer Membrane Antigens of Bartonella henselae

“…The first is that these proteins are expressed only in vitro or in the arthropod vector and therefore are not presented to the feline immune system. However, this seems very unlikely because these proteins are involved in heme uptake and B. henselae appears to utilize heme as its sole iron source (4,33). It is more plausible that these proteins are not immunogenic or are undergoing antigenic variation.…”

“…Virulent Houston-1 B. henselae (32) was grown in BBH-H medium as described previously (4). Cell densities of liquid cultures were determined by plate counts on BBH-H agar.…”

“…To determine which membrane fractions contained LPS, protein from each fraction (10 g/lane) was separated by SDS-PAGE in 15% acrylamide gels and LPS was visualized with a modified silver stain (37). To determine the levels of cytoplasmic proteins present in the membrane fractions, malate dehydrogenase, a soluble enzyme, was assayed at 37°C as described previously (4).…”

Possible role of tick‐borne infection in “cat‐scratch disease”: Comment on the article by Giladi et al

“…Of interest, B henselae utilizes amino acid catabolism rather than glycolysis to derive energy from its host, generating ammonia in the process (16). Ammonia production may contribute to the encephalopathy reported in patients with chronic B henselae infection, and it may also play a role in arthropathy, as noted with other arthritogenic bacteria (17).…”

Bartonellaspecies detection in captive, stranded and free-ranging cetaceans

“…Recently, the development of a more sensitive isolation approach, using Bartonella Alpha-Proteobacteria growth medium (BAPGM) followed by real time PCR has facilitated the molecular detection or isolation of Bartonella spp. from the blood of sick and healthy animals [8,13,29]. These improved techniques led to the first recognition of bloodborne Bartonella spp.…”