According to WHO, gonorrhea is currently the second most common STI worldwide. Vaccination is not available and the probability of failure of treatment with antibiotics is rising rapidly. Some aspects of (multi-) drug resistance are understood at the genetic level, but the effect of biofilm formation and its structure on efficiency of treatment remains poorly understood. This project aims at elucidating potential mechanisms of antibiotic tolerance in gonococcal biofilms.
Communities of bacterial cells can live together as a biofilm. This allows the bacteria to hide from external stresses including antibiotic treatment. The causative agent of gonorrhea, Neisseria gonorrhoeae, forms biofilms, but whether and how biofilm formation favors antibiotic tolerance remains largely unknown. We hypothesize that heterogeneous environments within biofilms induce different physiological states that cause antibiotic tolerance. In this project, we aim at deciphering molecular mechanisms causing tolerance by correlating structure of early biofilms with putative factors enabling tolerance. 

1. We will develop time-resolved single cell methods for measuring growth and death rates and for reporting cellular state. Combining confocal microscopy with advanced image analysis, we aim at establishing lineage tracking, and characterization of cellular physiology. 
2. We aim at defining factors that confer antibiotic tolerance. Antibiotic tolerance is usually associated with changes in cellular physiology. We will correlate death dynamics during drug treatment with these factors. 
3. We will link cellular state and antibiotic tolerance to biofilm structure. We will build on previous biophysical projects and on a collection of clinical isolates with different biofilm structures to address the correlation between biofilm structure and antibiotic tolerance.

The major goal of the project will be to find out whether and how antibiotic tolerance develops in gonococcal biofilms. Specifically, we will;

1. develop methods for lineage tracking and reporting cellular state at the single cell level with temporal resolution
2. correlate death dynamics during antibiotic treatment with cellular physiology
3. characterize variability of biofilm structure across isolates and its relation to antibiotic tolerance

Bacterial type 4 pili govern colony structure, dynamics, and antibiotic tolerance

The bacterial type 4 pilus performs so many different functions that it has been termed the bacterial "Swiss army knife". (Craig et al, 2019, Maier & Wong, 2015). It is an extracellular polymer involved in adhesion to host cells and other surfaces, motility and force generation, colony formation, transformation and electron transport. We showed that the pilus may be used as a tool for controlling structure, dynamics, and materials properties of gonococcal colonies (Oldewurtel et, 2015, Welker et al 2018, Maier, 2021, Hennes et al, 2022). We developed tools that allow linking the attractive force generated by pili with the physical properties of the colonies and antibiotic tolerance (Fig. 1) (Cronenberg, 2021).

Using these tools, we demonstrate a link between cellular attraction, colony fluidity, and survivability with the potential to optimize the treatment strategy of commonly used drug combinations. Combining laser tweezers, single pilus fluorescence, and transcriptomics, we quantify the effect of antibiotic treatment on pilus-mediated cellular attraction (Cronenberg et al, 2021, Kraus-Römer et al, 2022). We find that even moderate changes of the attractive force caused by antibiotics strongly impact on the physical properties of the colony, in particular its fluidity. Vice versa, we show that fluidity correlates with survivability under antibiotic treatment (Fig. 1c).
In addition to antibiotic treatment, we are currently testing the hypothesis that antigentic variation of the type 4 pilus structure alters the local physical interactions within biofilms. This variation is an important and possibly common contributor to the dynamics of biofilm growth, dispersion and adaptation. This hypothesis will be addressed using a collection of clinical isolates. The isolates were collected from patients with symptoms of urethritis between November 2015 and May 2019 (Fig. 2). All isolates have had their susceptibility to penicillin, ciprofloxacin, tetracycline, azithromycin and ceftriaxone determined at the Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne. All isolates have had their genomes sequenced, resistome identified, and been genotyped to determine their molecular epidemiology.