Collective decision-making in Pseudomonas aeruginosa involves transient segregation of quorum-sensing activities across cells.

A hallmark of bacterial sociality is that groups can coordinate cooperative actions through a cell-to-cell communication process called quorum sensing (QS). QS regulates key bacterial phenotypes such as virulence in infections and digestion of extracellular compounds in the environment. Although QS responses are typically studied as group-level phenotypes, it is unclear whether individuals coordinate their actions at the single-cell level or whether group phenotypes simply reflect the sum of their noisy members. Here, we studied the behavior of Pseudomonas aeruginosa individuals by tracking their temporal commitments to the two intertwined Las and Rhl-QS systems, from low to high population density. Using chromosomally integrated fluorescent gene reporters, we found that QS gene expression (signal, receptor, and cooperative exoproduct) was noisy with heterogeneity peaking during the build-up phase of QS. Moreover, we observed the formation of discrete subgroups of cells that transiently segregate into two gene expression states: low Las-receptor expressers that instantly activate exoproduct production and high Las-receptor expressers with delayed exoproduct production. Later, gene expression activities converged with all cells fully committing to QS. We developed general mathematical models to show that gene expression segregation can mechanistically be spurred by molecular resource limitations during the initiation phase of regulatory cascades such as QS. Moreover, our models indicate that gene expression segregation across cells can operate as a built-in brake enabling a temporary bet-hedging strategy in unpredictable environments. Altogether, our work reveals that studying the behavior of bacterial individuals is key to understanding emergent collective actions at the group level.

Bacterial Quorum Sensing Allows Graded and Bimodal Cellular Responses to Variations in Population Density.

Quorum sensing (QS) is a mechanism of cell-cell communication that connects gene expression to environmental conditions (e.g., cell density) in many bacterial species, mediated by diffusible signal molecules. Current functional studies focus on qualitatively distinct QS ON/OFF states. In the context of density sensing, this view led to the adoption of a "quorum" analogy in which populations sense when they are above a sufficient density (i.e., "quorate") to efficiently turn on cooperative behaviors. This framework overlooks the potential for intermediate, graded responses to shifts in the environment. In this study, we tracked QS-regulated protease (lasB) expression and showed that Pseudomonas aeruginosa can deliver a graded behavioral response to fine-scale variation in population density, on both the population and single-cell scales. On the population scale, we saw a graded response to variation in population density (controlled by culture carrying capacity). On the single-cell scale, we saw significant bimodality at higher densities, with separate OFF and ON subpopulations that responded differentially to changes in density: a static OFF population of cells and increasing intensity of expression among the ON population of cells. Together, these results indicate that QS can tune gene expression to graded environmental change, with no critical cell mass or "quorum" at which behavioral responses are activated on either the individual-cell or population scale. In an infection context, our results indicate there is not a hard threshold separating a quorate "attack" mode from a subquorate "stealth" mode. IMPORTANCE Bacteria can be highly social, controlling collective behaviors via cell-cell communication mechanisms known as quorum sensing (QS). QS is now a large research field, yet a basic question remains unanswered: what is the environmental resolution of QS? The notion of a threshold, or "quorum," separating coordinated ON and OFF states is a central dogma in QS, but recent studies have shown heterogeneous responses at a single cell scale. Using Pseudomonas aeruginosa, we showed that populations generate graded responses to environmental variation through shifts in the proportion of cells responding and the intensity of responses. In an infection context, our results indicate that there is not a hard threshold separating a quorate "attack" mode and a subquorate "stealth" mode.

Author Correction: In silico bacteria evolve robust cooperation via complex quorum-sensing strategies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In silico bacteria evolve robust cooperaion via complex quorum-sensing strategies.

Many species of bacteria collectively sense and respond to their social and physical environment via 'quorum sensing' (QS), a communication system controlling extracellular cooperative traits. Despite detailed understanding of the mechanisms of signal production and response, there remains considerable debate over the functional role(s) of QS: in short, what is it for? Experimental studies have found support for diverse functional roles: density sensing, mass-transfer sensing, genotype sensing, etc. While consistent with theory, these results cannot separate whether these functions were drivers of QS adaption, or simply artifacts or 'spandrels' of systems shaped by distinct ecological pressures. The challenge of separating spandrels from drivers of adaptation is particularly hard to address using extant bacterial species with poorly understood current ecologies (let alone their ecological histories). To understand the relationship between defined ecological challenges and trajectories of QS evolution, we used an agent-based simulation modeling approach. Given genetic mixing, our simulations produce behaviors that recapitulate features of diverse microbial QS systems, including coercive (high signal/low response) and generalized reciprocity (signal auto-regulation) strategists - that separately and in combination contribute to QS-dependent resilience of QS-controlled cooperation in the face of diverse cheats. We contrast our in silico results given defined ecological challenges with bacterial QS architectures that have evolved under largely unknown ecological contexts, highlighting the critical role of genetic constraints in shaping the shorter term (experimental evolution) dynamics of QS. More broadly, we see experimental evolution of digital organisms as a complementary tool in the search to understand the emergence of complex QS architectures and functions.

Focal urethral stricturing following intraurethral mitomycin-C gel and the use of a penile clamp.

We present a case of a 51-year-old gentleman, previously diagnosed with high-grade superficial transitional cell carcinoma of the bladder and treated with intravesical mitomycin C and BCG, who developed serial recurrences in the prostatic urethra. This was resected and treated further with intraurethral mitomycin-C gel. He subsequently developed an almost impassable distal penile urethral stricture, corresponding to the site of penile clamp application which we hypothesise is secondary to a combination of the mitomycin-C gel and penile clamp pressure.

Oncological outcomes after laparoscopic and open radical nephroureterectomy: results from an international cohort.

OBJECTIVE: • To compare oncological outcomes in patients undergoing open radical nephroureterectomy (ONU) with those in patients undergoing laparoscopic radical nephroureterectomy (LNU). PATIENTS AND METHODS: • A total of 773 patients underwent radical nephroureterectomy at nine centres worldwide; 703 patients underwent ONU and 70 underwent LNU. • Demographic, perioperative and oncological outcome data were collected retrospectively. • Statistical analysis of data was performed using chi-squared, Mann-Whitney U- and log-rank tests, and Cox regression analyses. • The median (interquartile range) follow-up for the cohort was 34 (15-65) months. RESULTS: • The two groups were well matched for tumour stage, presence of lymphovascular invasion (LVI) and concomitant carcinoma in situ (CIS). • There were more high-grade tumours (77.1% vs. 56.3%; P < 0.001) but fewer lymph node positive patients (2.9% vs. 6.8%; P= 0.041) in the LNU group. • Estimated 5-year recurrence-free survival (RFS) was 73.7% and 63.4% for the ONU and LNU groups, respectively (P= 0.124) and estimated 5-year cancer-specific survival (CSS) was 75.4% and 75.2% for the ONU and LNU groups, respectively (P= 0.897). • On multivariable analyses, which included age, gender, race, previous endoscopic treatment for bladder cancer, technique for distal ureter management, tumour location, pathological stage, grade, lymph node status, LVI and concomitant CIS, the procedure type (LNU vs. ONU) was not predictive of RFS (Hazard ratio [HR] 0.80; P= 0.534) or CSS (HR 0.96; P= 0.907). CONCLUSION: • The present study is the second large, independent, multicentre cohort to show oncological equivalence between ONU and LNU for well selected patients with upper urinary tract urothelial cancer, and the first to suggest parity for the techniques in patients with unfavourable disease.