Culture and antibiotic susceptibility of Bartonella quintana in human erythrocytes

Abstract

Bartonella quintana, the agent of trench fever, has recently been implicated in various diseases, in particular, bacteremia and endocarditis in homeless people. The host cell of Bartonella spp. is believed to be the erythrocyte, and in the present study we demonstrate that B. quintana can be cultured in vitro in human erythrocytes. The bacteria were found to be intraerythrocytic by laser confocal microscopy with Bartonella species-specific monoclonal antibodies. Infections with B. quintana decreased the life span of erythrocytes in culture from 8.6 to 4.8 days. In the culture system we found that most of the antibiotics that we tested (doxycycline, fluoroquinolone compounds, and beta-lactams) were not bactericidal. Gentamicin was bactericidal at 4 micro g/ml, as was rifampin, but to a lesser extent. At this concentration, gentamicin has been shown to enter erythrocytes slowly and to reach a peak level of 0.26 micro g/ml after 24 h. At 0.26 micro g/ml, however, we found that gentamicin was not able to kill extracellular B. quintana, even after 96 h of incubation. We hypothesize that erythrocytes may be a reservoir for B. quintana and that the bactericidal activity of gentamicin that we observed occurs mainly when the bacteria emerge from the erythrocytes and are found extracellularly. It would appear that gentamicin should be administered for at least 5 days to cure patients infected with B. quintana.

FIG. 1.

Kinetics of growth of B. quintana in human erythrocytes as determined by enumeration of colonies after plating onto blood agar. Nb, number.

FIG. 2.

Laser scanning confocal microscopy of digital sections of cultured human erythrocytes infected with B. quintana. Sections were taken in 0.5-μm increments from top to bottom. B. quintana was revealed with a specific monoclonal antibody.

FIG. 3.

Bactericidal effects of rifampin at 1 μg/ml (▵) and 4 μg/ml (▴) and gentamicin at 1 μg/ml (□) and 4 μg/ml (▪) on B. quintana in human erythrocytes. •, growth control; Nb, number.

FIG. 4.

Kinetics of penetration of gentamicin into human erythrocytes determined by HPLC. Gentamicin was used at 3 μg/ml in medium that was changed each day.