Antibiotic persistence of intracellular Brucella abortus

Abstract

Background Human Brucellosis caused by the facultative intracellular pathogen Brucella spp. is an endemic bacterial zoonosis manifesting as acute or chronic infections with high morbidity. Treatment typically involves a combination therapy of two antibiotics for several weeks to month, but despite this harsh treatment relapses occur at a rate of 5–15%. Although poor compliance and reinfection may account for a fraction of the observed relapse cases, it is apparent that the properties of the infectious agent itself may play a decisive role in this phenomenon. Methodology/Principal findings We used B. abortus carrying a dual reporter in a macrophage infection model to gain a better understanding of the efficacy of recommended therapies in cellulo. For this we used automated fluorescent microscopy as a prime read-out and developed specific CellProfiler pipelines to score infected macrophages at the population and the single cell level. Combining microscopy of constitutive and induced reporters with classical CFU determination, we quantified the protective nature of the Brucella intracellular lifestyle to various antibiotics and the ability of B. abortus to persist in cellulo despite harsh antibiotic treatments. Conclusion/Significance We demonstrate that treatment of infected macrophages with antibiotics at recommended concentrations fails to fully prevent growth and persistence of B. abortus in cellulo, which may be explained by a protective nature of the intracellular niche(s). Moreover, we show the presence of bona fide intracellular persisters upon antibiotic treatment, which are metabolically active and retain the full infectious potential, therefore constituting a plausible reservoir for reinfection and relapse. In conclusion, our results highlight the need to extend the spectrum of models to test new antimicrobial therapies for Brucellosis to better reflect the in vivo infection environment, and to develop therapeutic approaches targeting the persister subpopulation.

The putative Type 4 secretion system effector BspD is involved in maintaining envelope integrity of the pathogenBrucella

“…4A and C). To this end we established a CellProfiler pipeline as previously described (32) and detailed in the method section. In brief, thresholds for identifying microcolonies were determined by comparing the size of infection sites of the replication-deficient Δ virB9 strain with microcolonies formed by wild-type B. abortus in RAW264.7 macrophages after 27h of infection (Fig.…”

“…Stained samples were washed three times with PBS prior to imaging. Microscopy was performed with Molecular Devices ImageXpress automated microscopes as described in (32). Nuclei were imaged using DAPI, Bacteria were identified by dsRed.…”

“…Images were analyzed using CellProfiler (version 2.2.0) (41) as described in (32) with some adjustments. In brief, illumination was corrected using an illumination function based on the background method to adjust uneven illumination.…”

Intoxication of antibiotic persisters by host RNS inactivates their efflux machinery during infection

“…For example, bacterial populations treated with chloramphenicol, a translation inhibitor, enter a non-growing state which protects them against the action of β-lactams [11]. As opposed to these inactive cells equipped to survive laboratory conditions, recalcitrant bacteria formed in host cells often retain translational activity despite their growth arrest [12][13][14][15][16][17], indicating that active processes support antibiotic persistence during infection.…”

Environmental, mechanistic and evolutionary landscape of antibiotic persistence

“…For example, there is not always a correlation between growth rate and cell survival during antibiotic treatment (Orman & Brynildsen, 2013; Wakamoto et al , 2013). Furthermore, some persister cells, such as intracellular persisters, still show metabolic activity and synthesize specific proteins that are important for their survival within the host cell (Orman & Brynildsen, 2013; Manina et al , 2015; Stapels et al , 2018; Wilmaerts et al , 2018; Peyrusson et al , 2020; Sulaiman & Lam, 2020; Semanjski et al , 2021; Mode et al , 2022; Ronneau et al , 2023). Additionally, while dormant persister cells are protected from some types of harm, they are not fully shielded from DNA damage (Völzing & Brynildsen, 2015; Wilmaerts et al , 2022).…”