Reactivation of hidden-latent Brucella infection after doxycycline and streptomycin treatment in mice

Abstract

Brucellosis has therapeutic challenges due to 3%-15% relapses/therapeutic failures (R/TF) after antibiotic treatment. Therefore, determining the antibiotic concentration in tissues, the physiopathological parameters, and the R/TF after treatment is relevant. After exploring different antibiotic quantities, we found that a combined dose of 100 µg/g of doxycycline (for 45 days) and 7.5 µg/g of streptomycin (for 14 days), respectively, achieved therapeutic levels of more than fourfold minimum inhibitory concentrations (MICs) against Brucella abortus in the spleen, liver, bone marrow, and plasma of mice, causing minimal pathophysiological effects. After 30 days of infection, mice received antibiotics, and hematological, histopathological, biochemical, and immunological analyses were performed. After antibiotic therapy, the pathological, hematological, immunological, and physiological profiles paralleled those described in human brucellosis. Treatment lowered antibody titers, reduced proinflammatory cytokines, and reduced inflammation in the target organs for a protracted period. No bacteria were detected in tissues 8 weeks after treatment, suggesting complete recovery. However, despite high doxycycline and streptomycin concentrations in tissues, relapses appeared in 100% of the animals after 182 days post-infection, estimated by the bacterial counts and PCR from organs. This proportion contrasts with the 15% R/TF observed in humans after antibiotic treatments. None of the B. abortus isolated from relapses showed augmented MICs or mutations coding for antibiotic resistance in chromosomal-relevant regions. We discuss whether our findings constitute a general phenomenon or differences in the exhaustive screening method for bacteria detection related to the murine model. Along these lines, we envision likely mechanisms of bacterial persistence in tissues after antibiotic treatment.

Keywords: INF-γ; antibiotic resistance; antibiotic treatment; brucellosis; cytokines; doxycycline; relapses; streptomycin.

Fig 1

Workflow timeline. Mice were infected with 10⁴ CFU of B. abortus 2308. After 30 days post-infection, analyses (blue color) were performed before the antibiotic. At day 31 post-infection, mice were treated with STR (7.5 µg g⁻¹) for 15 days and DOX (100 µg g⁻¹) for 45 days until day 75 post-infection (red color). At 78, 130, and 183 days post-infection, additional analyses (blue color) were performed.

Fig 2

Antibiotic quantification in tissues. Mice were treated intraperitoneally every 12 h with 50, 100, or 200 µg g⁻¹ of DOX or 7.5, 15, or 30 µg g⁻¹ of STR for 1 week. After 12 h of the last treatment, the antibiotics were extracted and quantified in the liver, spleen, bone marrow, and plasma as described. Extracted samples were analyzed by chromatographic analysis. Dashed lines correspond to each antibiotic’s MIC calculated in vitro.

Fig 3

Cytokine quantification and histopathological score of B. abortus infected mice. Mice were infected with 10⁴ CFU of B. abortus 2308. After 30 days, (A) the levels of INF-γ, TNF-α, MCP-1, IL-6, IL-12, and IL-10 were quantified in plasma, and (B) the histopathological score was evaluated in hematoxylin eosin-stained spleen and liver sections. The P-value of <0.001 (**) is indicated.

Fig 4

Histopathological analysis of B. abortus infected-treated mice. Mice were infected with 10⁴ CFU of B. abortus 2308. After 30 days post-infection, mice were either untreated with antibiotics (infected-nontreated), treated with STR (7.5 µg g⁻¹) for 15 days and DOX (100 µg g⁻¹) for 45 days (infected-treated). At 78 days post-infection, histopathological analysis was evaluated in hematoxylin eosin-stained spleen and liver sections (A–F). Arrows indicate the presence of granulomas. Immunoperoxidase detection of Brucella organisms (anti-lipopolysaccharide) in the red pulp (G) and white pulp around the central arteriole and marginal zone (H) of the spleen of mice at 30 days post-infection. Counterstaining was performed with Harris-hematoxylin. The arrows in G and H indicate the sites where the immunoperoxidase-staining bacteria are localized. The inserts correspond to the amplification of the immunostaining cells pointed by the upper arrow in the G and H panels.

Fig 5

Histopathological score post-treatment. Mice were infected with 10⁴ CFU of B. abortus 2308. After 30 days post-infection, mice were treated with STR (7.5 µg g⁻¹) for 15 days and DOX (100 µg g⁻¹) for 45 days. At 78 days post-infection, the histopathological score was evaluated in eosin-stained spleen and liver sections. The P-value of <0.01 (*) is indicated.

Fig 6

Spleen weights and fluorescent microscopy. Mice were infected with 10⁴ CFU of B. abortus 2308. After 30 days post-infection, mice were treated with STR (7.5 µg g⁻¹) for 15 days and DOX (100 µg g⁻¹) for 45 days. At 30, 78, 130, and 183 days post-infection, (A) spleen weight and (B) fluorescent microscopy in bone marrow cells were performed. Microscope images are at 400× magnification. Representative cells with DAPI-stained nuclei (blue) and intracellular fluorescent B. abortus RFP (red) were photographed under the microscope using the appropriate color filter channel. Images were cut from the microscope field, contrasted, saturated, and merged using Photopea Online Photo Editor. The P-value of <0.01 (*) is indicated.

Fig 7

Anti-Brucella antibody titers. Mice were infected with 10⁴ CFU of B. abortus 2308. After 30 days post-infection, mice were treated with STR (7.5 µg g⁻¹) for 15 days and DOX (100 µg g⁻¹) for 45 days. At 30, 78, 130, and 183 days post-infection, the Brucella antibody titers were quantified by microagglutination in a 96-well plate. The P-value of <0.01 (*) is indicated.

Fig 8

Cytokine quantification in infected-treated mice. Mice were infected with 10⁴ CFU of B. abortus 2308. After 30 days post-infection, mice were treated with STR (7.5 µg g⁻¹) for 15 days and DOX (100 µg g⁻¹) for 45 days. At 78, 130, and 183 days post-infection, the levels of INF-γ, TNF-α, MCP-1, IL-6, IL-12, and IL-10 were quantified in serum. The P-values of <0.001 (**) and <0.0001 (***) are indicated.