Assessing intracellular persister formation and antibiotics susceptibility in "Brucella abortus"

Human brucellosis is one of the most common zoonotic disease worldwide with more than 500,000 new cases annually. Brucella melitensis remains the major cause of human infection, followed by B. abortus and B. suis. Human brucellosis mainly causes intermittent bouts of fever, malaise, arthralgia and can evolve into a chronic form leading to serious complications. The treatment currently recommended by the World Health Organization is a combination of doxycycline for 6 weeks and either streptomycin for 3 weeks or rifampicin for 6 weeks. Despite this harsh therapy, a high rate of relapse is observed, ranging from 5% to 15%, representing a significant morbidity factor. The underlying reasons for these relapses are currently unknown.
In this study, we aimed at investigating the occurrence of persisters in Brucella, using B. abortus as a model. Indeed, this sub-population of transiently drug-tolerant bacteria has been recently recognized as a possible underlying cause for many chronic bacterial infections. Furthermore, we investigated the intracellular susceptibility of B. abortus to the drugs used in the treatment of human brucellosis.
Here we were able to demonstrate for the first time the presence of persisters in B. abortus and to show that their formation in broth is growth-phase dependent. Importantly, we discovered that persisters are also formed during macrophage infection and that their proportion varies according to the stage of infection. Moreover, to better understand the state of B. abortus during infection, we developed a pipeline allowing the identification of living intracellular bacteria based on their ability to respond to an inducer. Using this pipeline, we could visualize the presence of viable intracellular B. abortus after antibiotic treatment confirming the ability of a small-population to survive antimicrobials inside the cells. Interestingly, our data suggest that the majority of these viable intracellular bacteria are actually non-cultivable, which is also what we observed in broth. Using the same approach, we measured the susceptibility of intracellular B. abortus against antimicrobials used in the clinic and showed that doxycycline, rifampicin, ciprofloxacin, and tetracycline successfully prevent its intracellular replication. Curiously, whereas streptomycin alone failed to inhibit Brucella growth in macrophages, the combination of streptomycin with doxycycline or tetracycline displayed a synergic antimicrobial effect in preventing the intracellular replication of B. abortus. In addition, we demonstrated that the minimum concentration of rifampicin or tetracycline required to abrogate intracellular transcriptional and translational response of the bacteria is higher than the one required to prevent intracellular growth. Furthermore, we found that from the tested antibiotics only tetracycline and rifampicin showed a response under the concentrations reported in human serum supporting their use over ciprofloxacin in the clinic.
Together, our results reveal the occurrence of persisters during Brucella infection. Although further investigations will be required to define the specific role of these bacteria during human brucellosis, these results open a straightforward path for new strategies to circumvent the establishment of chronic brucellosis in human patients. Moreover, our data highlight the importance of the antibiotic regimen chosen for brucellosis treatment especially in regards to persister cells. We anticipate our method to be a starting point for a larger screen to develop new combinations of antimicrobials targeting intracellular persisters.