Antibiotics modulate attractive interactions in bacterial colonies affecting survivability under combined treatment

Abstract

Biofilm formation protects bacteria from antibiotics. Very little is known about the response of biofilm-dwelling bacteria to antibiotics at the single cell level. Here, we developed a cell-tracking approach to investigate how antibiotics affect structure and dynamics of colonies formed by the human pathogen Neisseria gonorrhoeae. Antibiotics targeting different cellular functions enlarge the cell volumes and modulate within-colony motility. Focusing on azithromycin and ceftriaxone, we identify changes in type 4 pilus (T4P) mediated cell-to-cell attraction as the molecular mechanism for different effects on motility. By using strongly attractive mutant strains, we reveal that the survivability under ceftriaxone treatment depends on motility. Combining our results, we find that sequential treatment with azithromycin and ceftriaxone is synergistic. Taken together, we demonstrate that antibiotics modulate T4P-mediated attractions and hence cell motility and colony fluidity.Author SummaryAggregation into colonies and biofilms can enhance bacterial survivability under antibiotic treatment. Aggregation requires modulation of attractive forces between neighboring bacteria, yet the link between attraction and survivability is poorly characterized. Here, we quantify these attractive interactions and show that different antibiotics enhance or inhibit them. Live-cell tracking of single cells in spherical colonies enables us to correlate attractive interactions with single cell motility and colony fluidity. Even moderate changes in cell-to-cell attraction caused by antibiotics strongly impact on within-colony motility. Vice versa, we reveal that motility correlates with survivability under antibiotic treatment. In summary, we demonstrate a link between cellular attraction, colony fluidity, and survivability with the potential to optimize the treatment strategy of commonly used drug combinations.

Spatiotemporal dynamics of growth and death within spherical bacterial colonies

“…The rapid growth arrest at the centers of the gonococcal colonies suggests that aggregation can rapidly cause tolerance against antibiotics acting on growing bacteria. This is consistent with previous reports showing that gonococcal aggregation enhances their survivability under ceftriaxone treatment with the fraction of dead cells being highest at the colony edge (21,47). Our methods are applicable to all spherical colonies, and it will be interesting to compare the growth profiles between different species.…”

“…We note that type 4 pili generate bacterial movement within colonies as pili belonging to neighboring cells bind to each other and retract (28). The pilus-mediated motility has been described as diffusive (21,26,30). Thus, the mean velocity of pilus-driven movement is 0 and does not contribute to the radial velocity analyzed in this study.…”

“…Therefore, the growth rate could not be determined in this region with the second method. Some external stresses cause swelling of cells (21). Swelling would cause the colony to expand and would introduce errors to the determination of growth rates.…”

“…Within two-dimensional colonies, individual cell division events are detectable by means of bright-field or fluorescence microscopy, and therefore, lineages arising from a single cell can be tracked (17,18). In 3D colonies, confocal microscopy or light sheet microscopy enable single-cell detection within colonies (19)(20)(21). Advanced image analysis techniques have been developed for characterizing local order, gene expression, and stress responses with spatial and temporal resolution (22)(23)(24).…”

Reduced metabolic activity in established Gardnerella spp. biofilms contributes to protection from the bactericidal effects of metronidazole

“…Although no differences were observed between treated and untreated biofilms formed by isolate N170, we observed that the biofilm was largely composed of bacterial cells in compact masses, and extracellular polymeric substance was not a major component of the structure. Although SEM alone cannot determine if any other changes were happening in the biofilms treated with metronidazole (sensors, florescence microscopy, and nanoparticles can provide a better understanding of what changes may occur in a biofilm treated with antibiotics), the fact that biofilm formation was not enhanced by sub-inhibitory concentrations of metronidazole and extracellular polymeric substance was not abundant in Gardnerella biofilm suggest that extracellular polymeric substance does not play a major role in providing protection against metronidazole (46,47).…”