Hepatic HIF2 is a key determinant of manganese excess and polycythemia in SLC30A10 deficiency.

Manganese is an essential yet potentially toxic metal. Initially reported in 2012, mutations in SLC30A10 are the first known inherited cause of manganese excess. SLC30A10 is an apical membrane protein that exports manganese from hepatocytes into bile and from enterocytes into the lumen of the gastrointestinal tract. SLC30A10 deficiency results in impaired gastrointestinal manganese excretion, leading to manganese excess, neurologic deficits, liver cirrhosis, polycythemia, and erythropoietin excess. Neurologic and liver disease are attributed to manganese toxicity. Polycythemia is attributed to erythropoietin excess. The goal of this study was to determine the basis of erythropoietin excess in SLC30A10 deficiency. Here, we demonstrate that transcription factors hypoxia-inducible factor 1a (Hif1a) and 2a (Hif2a), key mediators of the cellular response to hypoxia, are both upregulated in livers of Slc30a10-deficient mice. Hepatic Hif2a deficiency corrected erythropoietin expression and polycythemia and attenuated aberrant hepatic gene expression in Slc30a10-deficient mice, while hepatic Hif1a deficiency had no discernible impact. Hepatic Hif2a deficiency also attenuated manganese excess, though the underlying cause of this is not clear at this time. Overall, our results indicate that hepatic HIF2 is a key determinant of pathophysiology in SLC30A10 deficiency and expand our understanding of the contribution of HIFs to human disease.

AAV-mediated hepatic expression of SLC30A10 and the Thr95Ile variant attenuates manganese excess and other phenotypes in Slc30a10-deficient mice.

The manganese (Mn) export protein SLC30A10 is essential for Mn excretion via the liver and intestines. Patients with SLC30A10 deficiency develop Mn excess, dystonia, liver disease, and polycythemia. Recent genome-wide association studies revealed a link between the SLC30A10 variant T95I and markers of liver disease. The in vivo relevance of this variant has yet to be investigated. Using in vitro and in vivo models, we explore the impact of the T95I variant on SLC30A10 function. While SLC30A10 I95 expressed at lower levels than T95 in transfected cell lines, both T95 and I95 variants protected cells similarly from Mn-induced toxicity. Adeno-associated virus 8-mediated expression of T95 or I95 SLC30A10 using the liver-specific thyroxine binding globulin promoter normalized liver Mn levels in mice with hepatocyte Slc30a10 deficiency. Furthermore, Adeno-associated virus-mediated expression of T95 or I95 SLC30A10 normalized red blood cell parameters and body weights and attenuated Mn levels and differential gene expression in livers and brains of mice with whole body Slc30a10 deficiency. While our in vivo data do not indicate that the T95I variant significantly compromises SLC30A10 function, it does reinforce the notion that the liver is a key site of SLC30A10 function. It also supports the idea that restoration of hepatic SLC30A10 expression is sufficient to attenuate phenotypes in SLC30A10 deficiency.

Delphi Process for Validation of Fluid Treatment Algorithm for Critically Ill Pediatric Trauma Patients.

INTRODUCTION: While intravenous fluid therapy is essential to re-establishing volume status in children who have experienced trauma, aggressive resuscitation can lead to various complications. There remains a lack of consensus on whether pediatric trauma patients will benefit from a liberal or restrictive crystalloid resuscitation approach and how to optimally identify and transition between fluid phases. METHODS: A panel was comprised of physicians with expertise in pediatric trauma, critical care, and emergency medicine. A three-round Delphi process was conducted via an online survey, with each round being followed by a live video conference. Experts agreed or disagreed with each aspect of the proposed fluid management algorithm on a five-level Likert scale. The group opinion level defined an algorithm parameter's acceptance or rejection with greater than 75% agreement resulting in acceptance and greater than 50% disagreement resulting in rejection. The remaining were discussed and re-presented in the next round. RESULTS: Fourteen experts from five Level 1 pediatric trauma centers representing three subspecialties were included. Responses were received from 13/14 participants (93%). In round 1, 64% of the parameters were accepted, while the remaining 36% were discussed and re-presented. In round 2, 90% of the parameters were accepted. Following round 3, there was 100% acceptance by all the experts on the revised and final version of the algorithm. CONCLUSIONS: We present a validated algorithm for intavenous fluid management in pediatric trauma patients that focuses on the de-escalation of fluids. Focusing on this time point of fluid therapy will help minimize iatrogenic complications of crystalloid fluids within this patient population.

Derivation, Validation, and Clinical Relevance of a Pediatric Sepsis Phenotype With Persistent Hypoxemia, Encephalopathy, and Shock.

OBJECTIVES: Untangling the heterogeneity of sepsis in children and identifying clinically relevant phenotypes could lead to the development of targeted therapies. Our aim was to analyze the organ dysfunction trajectories of children with sepsis-associated multiple organ dysfunction syndrome (MODS) to identify reproducible and clinically relevant sepsis phenotypes and determine if they are associated with heterogeneity of treatment effect (HTE) to common therapies. DESIGN: Multicenter observational cohort study. SETTING: Thirteen PICUs in the United States. PATIENTS: Patients admitted with suspected infections to the PICU between 2012 and 2018. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We used subgraph-augmented nonnegative matrix factorization to identify candidate trajectory-based phenotypes based on the type, severity, and progression of organ dysfunction in the first 72 hours. We analyzed the candidate phenotypes to determine reproducibility as well as prognostic, therapeutic, and biological relevance. Overall, 38,732 children had suspected infection, of which 15,246 (39.4%) had sepsis-associated MODS with an in-hospital mortality of 10.1%. We identified an organ dysfunction trajectory-based phenotype (which we termed persistent hypoxemia, encephalopathy, and shock) that was highly reproducible, had features of systemic inflammation and coagulopathy, and was independently associated with higher mortality. In a propensity score-matched analysis, patients with persistent hypoxemia, encephalopathy, and shock phenotype appeared to have HTE and benefit from adjuvant therapy with hydrocortisone and albumin. When compared with other high-risk clinical syndromes, the persistent hypoxemia, encephalopathy, and shock phenotype only overlapped with 50%-60% of patients with septic shock, moderate-to-severe pediatric acute respiratory distress syndrome, or those in the top tier of organ dysfunction burden, suggesting that it represents a nonsynonymous clinical phenotype of sepsis-associated MODS. CONCLUSIONS: We derived and validated the persistent hypoxemia, encephalopathy, and shock phenotype, which is highly reproducible, clinically relevant, and associated with HTE to common adjuvant therapies in children with sepsis.

Early post-transplant recurrence of ANCA vasculitis while on belatacept maintenance immunosuppression.

Post-transplant recurrence of ANCA-associated vasculitis (AAV) is infrequent, with recurrence within weeks of transplantation being even rarer. We describe an unusual case of AAV recurrence within 2 weeks post-transplant. Our patient received a deceased donor kidney transplant (KDPI 60%) after 6 years on hemodialysis for end-stage renal disease from AAV. She was induced with thymoglobulin and steroids, and maintained on belatacept, mycophenolate and prednisone. Time-zero biopsy showed acute tubular injury. Due to persistent delayed graft function by post-operative day 14, she underwent repeat biopsy, which showed focal segmental necrotizing and crescentic glomerulonephritis, with positive MPO, PR3 and negative anti-glomerular basement membrane antibodies. As her findings were in keeping with recurrent AAV, she underwent induction with rituximab, prednisone and intravenous immunoglobulin, with repeat rituximab 14 days later because of increasing B-lymphocyte counts. Belatacept was replaced with tacrolimus due to concerns with autoimmunity. Fortunately, renal function began to recover 4 days after treatment. In addition to highlighting potential immunologic mechanisms in AAV and the use of rituximab in post-transplant recurrence, our case suggests that for systemic autoimmune disease, patients maintained on belatacept must be monitored closely for recurrence, particularly in the setting of delayed graft function.

Derivation and Validation of Vasoactive Inotrope Score Trajectory Groups in Critically Ill Children With Shock.

OBJECTIVES: To determine whether there are clinically relevant and reproducible Vasoactive Inotrope Score (VIS) trajectories in children with shock during the acute phase of critical illness. DESIGN: Retrospective, observational cohort study. SETTING: Two tertiary, academic PICUs. PATIENTS: Children (< 18 yr old) who required vasoactive infusions within 24 hours of admission to the PICU. Those admitted post cardiac surgery were excluded. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: An hourly VIS was calculated for the first 72 hours after initiation of vasoactives. Group-based trajectory modeling (GBTM) was applied to a derivation set (75% of encounters) and compared with the trajectories in a validation set (25% of encounters) using the same variables. The primary outcome was in-hospital mortality, and the secondary outcome was multiple organ dysfunction syndrome (MODS) on day 7. A total of 1,828 patients met inclusion criteria, and 309 (16.9%) died. GBTM identified four subgroups that were reproducible in the validation set: "Mild, fast resolving shock" ( n = 853 [47%]; mortality 9%), "Moderate, slow resolving shock" ( n = 422 [23%]; mortality 15%), "Moderate, prolonged shock" ( n = 312 [17%]; mortality 21%), and "Severe, prolonged shock" ( n = 241 [13%]; mortality 40%). There was a significant difference in mortality, MODS on day 7, and suspected infection ( p < 0.001) across groups. The "Mild, fast resolving shock" and "Severe, prolonged shock" groups were identifiable within the first 24 hours. The "Moderate, slow resolving" and "Moderate, prolonged shock" groups were indistinguishable in the first 24 hours after initiation of vasoactives but differed in in-hospital mortality and MODS on day 7. Hydrocortisone administration was independently associated with poor outcomes in the "Mild, fast resolving shock" group. CONCLUSIONS: We uncovered four distinct and reproducible VIS trajectory groups that were associated with different risk factors, response to therapy, and outcomes in children with shock. Characterizing VIS trajectory groups in the acute phase of critical illness may enable better prognostication and more targeted management.

Single molecule tracking of bacterial cell surface cytochromes reveals dynamics that impact long-distance electron transport.

Using a series of multiheme cytochromes, the metal-reducing bacterium Shewanella oneidensis MR-1 can perform extracellular electron transfer (EET) to respire redox-active surfaces, including minerals and electrodes outside the cell. While the role of multiheme cytochromes in transporting electrons across the cell wall is well established, these cytochromes were also recently found to facilitate long-distance (micrometer-scale) redox conduction along outer membranes and across multiple cells bridging electrodes. Recent studies proposed that long-distance conduction arises from the interplay of electron hopping and cytochrome diffusion, which allows collisions and electron exchange between cytochromes along membranes. However, the diffusive dynamics of the multiheme cytochromes have never been observed or quantified in vivo, making it difficult to assess their hypothesized contribution to the collision-exchange mechanism. Here, we use quantum dot labeling, total internal reflection fluorescence microscopy, and single-particle tracking to quantify the lateral diffusive dynamics of the outer membrane-associated decaheme cytochromes MtrC and OmcA, two key components of EET in S. oneidensis. We observe confined diffusion behavior for both quantum dot-labeled MtrC and OmcA along cell surfaces (diffusion coefficients DMtrC = 0.0192 ± 0.0018 µm2/s, DOmcA = 0.0125 ± 0.0024 µm2/s) and the membrane extensions thought to function as bacterial nanowires. We find that these dynamics can trace a path for electron transport via overlap of cytochrome trajectories, consistent with the long-distance conduction mechanism. The measured dynamics inform kinetic Monte Carlo simulations that combine direct electron hopping and redox molecule diffusion, revealing significant electron transport rates along cells and membrane nanowires.

Excessive Oxygen Supplementation in the First Day of Mechanical Ventilation Is Associated With Multiple Organ Dysfunction and Death in Critically Ill Children.

OBJECTIVES: To determine if greater cumulative exposure to oxygen despite adequate oxygenation over the first 24 hours of mechanical ventilation is associated with multiple organ dysfunction syndrome at 7 days and inhospital mortality in critically ill children. DESIGN: Retrospective, observational cohort study. SETTING: Two urban, academic PICUs. PATIENTS: Patients less than 18 years old who required mechanical ventilation within 3 days of admission between 2010 and 2018 (Lurie Children's Hospital) or 2010 and 2016 (Comer Children's Hospital). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: There were 5,406 mechanically ventilated patients, of which 960 (17.8%) had multiple organ dysfunction syndrome on day 7 of admission and 319 died (5.9%) during their hospitalization. Cumulative exposure to greater amounts of supplemental oxygen, while peripheral oxygen saturation was 95% or more during the first 24 hours of mechanical ventilation was independently associated with an increased risk of both multiple organ dysfunction syndrome on day 7 and inhospital mortality after adjusting for confounders. Patients in the highest quartile of cumulative oxygen exposure had an increased odds of multiple organ dysfunction syndrome on day 7 (adjusted odds ratio, 3.9; 95% CI, 2.7-5.9) and inhospital mortality (adjusted odds ratio, 1.7; 95% CI, 1.1-2.9), when compared with those in the lowest quartile of cumulative oxygen exposure after adjusting for age, presence of multiple organ dysfunction syndrome on day 1 of mechanical ventilation, immunocompromised state, and study site. CONCLUSIONS: Greater cumulative exposure to excess supplemental oxygen in the first 24 hours of mechanical ventilation is independently associated with an increased risk of multiple organ dysfunction syndrome on day 7 of admission and inhospital mortality in critically ill children.

Hypernatremia subgroups among hospitalized patients by machine learning consensus clustering with different patient survival.

BACKGROUND: The objective of this study was to characterize hypernatremia patients at hospital admission into clusters using an unsupervised machine learning approach and to evaluate the mortality risk among these distinct clusters. METHODS: We performed consensus cluster analysis based on demographic information, principal diagnoses, comorbidities, and laboratory data among 922 hospitalized adult patients with admission serum sodium of > 145 mEq/L. We calculated the standardized difference of each variable to identify each cluster's key features. We assessed the association of each hypernatremia cluster with hospital and 1-year mortality. RESULTS: There were three distinct clusters of patients with hypernatremia on admission: 318 (34%) patients in cluster 1, 339 (37%) patients in cluster 2, and 265 (29%) patients in cluster 3. Cluster 1 consisted of more critically ill patients with more severe hypernatremia and hypokalemic hyperchloremic metabolic acidosis. Cluster 2 consisted of older patients with more comorbidity burden, body mass index, and metabolic alkalosis. Cluster 3 consisted of younger patients with less comorbidity burden, higher baseline eGFR, hemoglobin, and serum albumin. Compared to cluster 3, odds ratios for hospital mortality were 15.74 (95% CI 3.75-66.18) for cluster 1, and 6.51 (95% CI 1.48-28.59) for cluster 2, whereas hazard ratios for 1-year mortality were 6.25 (95% CI 3.69-11.46) for cluster 1 and 4.66 (95% CI 2.73-8.59) for cluster 2. CONCLUSION: Our cluster analysis identified three clinically distinct phenotypes with differing mortality risk in patients hospitalized with hypernatremia.

Non-resuscitation fluid in excess of hydration requirements is associated with higher mortality in critically ill children.

BACKGROUND: Large volumes of non-resuscitation fluids are often administered to critically ill children. We hypothesize that excess maintenance fluid is a significant contributor to non-resuscitation fluid and that non-resuscitation fluid administered beyond hydration requirements is associated with worse clinical outcomes in critically ill children. METHODS: We evaluated all patients admitted to two large urban pediatric intensive care units (PICU) between January 2010-August 2016 and January 2010-August 2018, respectively, who survived and remained in the hospital for at least 3 days following PICU admission. The primary outcome was in-hospital mortality. Association of excess fluid with outcomes was adjusted for confounders (age, Pediatric Risk of Mortality III score, study site, day 3 acute kidney injury, PICU era, resuscitation volume, and volume output) using multivariable regression. RESULTS: We evaluated 14,483 patients; 52% received non-resuscitation fluid in excess of hydration requirements. Non-resuscitation fluid in excess of hydration requirements was associated with higher in-hospital mortality after adjustment for confounders (adjusted odds ratio 1.01 per 10 mL/kg in excess fluid, 95% confidence interval: 1.002-1.02). CONCLUSIONS: Non-resuscitation fluid in excess of hydration requirements is associated with increased mortality in critically ill children. Excess maintenance fluid is a modifiable contributor to this fluid volume. Strategies to reduce excess maintenance fluids warrant further study. IMPACT: Critically ill children frequently receive non-resuscitation fluid in excess of their estimated hydration requirements. Non-resuscitation fluid volume in excess of estimated hydration requirements is associated with higher morbidity and mortality in critically ill children. Critically ill children receive a large volume burden from maintenance fluid. Maintenance fluid represents a modifiable contributor of non-resuscitation fluid in excess of hydration requirements. Strategies focused on limitation of maintenance fluid warrant further study.

Fulminant myocarditis and atrial fibrillation in child with acute COVID-19.

COVID-19 has manifested with ventricular dysfunction and cardiac arrhythmias, most commonly atrial fibrillation (AFib), in adults. However, very few pediatric patients with acute COVID-19 have had cardiac involvement. AFib, an exceedingly rare arrhythmia in otherwise healthy children, has not been reported in children with COVID-19. We report a 15 year-old girl with acute COVID-19, fulminant myocarditis and AFib.

A bacterial membrane sculpting protein with BAR domain-like activity.

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.

Hyperferritinemia: A Diagnostic Marker for Disseminated Neonatal Herpes Simplex Virus Infection?

Although the prognosis of neonatal herpes simplex virus (HSV) infection has improved with intravenous acyclovir, the morbidity and mortality of disseminated disease remains high. Transaminitis and thrombocytopenia have been reported to be sensitive markers of neonatal HSV disease; however, early diagnosis remains a challenge due to a lack of specific clinical and laboratory indicators for this disease process. Ferritin, an acute phase reactant known for its use in diagnosing hemophagocytic lymphohistiocytosis, has recently been reported as extremely elevated in neonates with disseminated HSV due to its high inflammatory nature. We report three cases of neonates at a single institution with hyperferritinemia in the setting of disseminated HSV. Based on this case series, we discuss whether ferritin can be used as an early diagnostic marker in the setting of suspected neonatal HSV disease. [Pediatric Annals. 2021;50(6):e264-e267.].

Pediatric Intensive Care Unit Resident Educational Curriculum.

Introduction: Consistent medical knowledge acquisition while caring for the critically ill can be challenging for learners and educators in the pediatric intensive care unit (PICU), a unit often distinguished by fluctuating acuity and severity. We implemented a standardized didactic curriculum for PICU residents to facilitate their acquisition and retention of knowledge in core PICU topics. Methods: We developed a comprehensive standardized curriculum for PGY 2-PGY 4 PICU pediatric and internal medicine-pediatric residents. Thirteen core topics were administered as 30-minute didactic sessions during the rotation, using either PowerPoint slides or a dry-erase board. Residents were tested to assess knowledge acquisition and retention. Results: Seventy-eight residents participated, 86% of whom completed posttests. Seventeen percent completed follow-up tests. Of the learners who participated, 60 (77%) completed pretests and posttests, indicating their confidence level each time. The pretest mean was 55% (SD = 14.4%), and the posttest mean was 64% (SD = 15.6%). This 9% increase was statistically significant (p = .001; CI, 3.9% to 14.8%). The follow-up test at 3 months, completed by 15% of this subgroup, demonstrated a mean score of 62% (SD = 14.5%). When matched with posttest scores (mean score of 64%, SD = 13.3%), there was no significant difference (p = .7398; CI, -11.7% to 16.2%), suggesting retention of previously acquired knowledge. Discussion: Our standardized didactic curriculum effectively facilitated the acquisition and retention of the medical knowledge of core PICU topics among PICU residents, in addition to their usual experiential learning.

Online Learning and Residents' Acquisition of Mechanical Ventilation Knowledge: Sequencing Matters.

OBJECTIVE: Rapid advancements in medicine and changing standards in medical education require new, efficient educational strategies. We investigated whether an online intervention could increase residents' knowledge and improve knowledge retention in mechanical ventilation when compared with a clinical rotation and whether the timing of intervention had an impact on overall knowledge gains. DESIGN: A prospective, interventional crossover study conducted from October 2015 to December 2017. SETTING: Multicenter study conducted in 33 PICUs across eight countries. SUBJECTS: Pediatric categorical residents rotating through the PICU for the first time. We allocated 483 residents into two arms based on rotation date to use an online intervention either before or after the clinical rotation. INTERVENTIONS: Residents completed an online virtual mechanical ventilation simulator either before or after a 1-month clinical rotation with a 2-month period between interventions. MEASUREMENTS AND MAIN RESULTS: Performance on case-based, multiple-choice question tests before and after each intervention was used to quantify knowledge gains and knowledge retention. Initial knowledge gains in residents who completed the online intervention (average knowledge gain, 6.9%; SD, 18.2) were noninferior compared with those who completed 1 month of a clinical rotation (average knowledge gain, 6.1%; SD, 18.9; difference, 0.8%; 95% CI, -5.05 to 6.47; p = 0.81). Knowledge retention was greater following completion of the online intervention when compared with the clinical rotation when controlling for time (difference, 7.6%; 95% CI, 0.7-14.5; p = 0.03). When the online intervention was sequenced before (average knowledge gain, 14.6%; SD, 15.4) rather than after (average knowledge gain, 7.0%; SD, 19.1) the clinical rotation, residents had superior overall knowledge acquisition (difference, 7.6%; 95% CI, 2.01-12.97;p = 0.008). CONCLUSIONS: Incorporating an interactive online educational intervention prior to a clinical rotation may offer a strategy to prime learners for the upcoming rotation, augmenting clinical learning in graduate medical education.

Nature's conductors: what can microbial multi-heme cytochromes teach us about electron transport and biological energy conversion?

Microorganisms can acquire energy from the environment by extending their electron transport chains to external solid electron donors or acceptors. This process, known as extracellular electron transfer (EET), is now being heavily pursued for wiring microbes to electrodes in bioelectrochemical renewable energy technologies. Recent studies highlight the crucial role of multi-heme cytochromes in facilitating biotic-abiotic EET both for cellular electron export and uptake. Here we explore progress in understanding the range and function of these biological electron conduits in the context of fuel-to-electricity and electricity-to-bioproduct conversion. We also highlight emerging topics, including the role of multi-heme cytochromes in inter-species electron transfer and in inspiring the design and synthesis of a new generation of protein-based bioelectronic components.

Quorum Sensing Signal Synthesis May Represent a Selective Advantage Independent of Its Role in Regulation of Bioluminescence in Vibrio fischeri.

The evolution of biological signalling systems and apparently altruistic or cooperative traits in diverse organisms has required selection against the subversive tendencies of self-interested biological entities. The bacterial signalling and response system known as quorum sensing or Acylated Homoserine Lactone (AHL) mediated gene expression is thought to have evolved through kin selection. In this in vitro study on the model quorum sensing bioluminescent marine symbiont Vibrio fischeri, competition and long-term sub culturing experiments suggest that selection for AHL synthesis (encoded by the AHL synthase gene luxI) is independent of the quorum sensing regulated phenotype (bioluminescence encoded by luxCDABE). Whilst results support the hypothesis that signal response (AHL binding and transcriptional activation encoded by the luxR gene) is maintained through indirect fitness benefits (kin selection), signal synthesis is maintained in the V. fischeri genome over evolutionary time through direct fitness benefits at the individual level from an unknown function.

The presence and role of bacterial quorum sensing in activated sludge.

Activated sludge used for wastewater treatment globally is composed of a high-density microbial community of great biotechnological significance. In this study the presence and purpose of quorum sensing via N-acylated-l-homoserine lactones (AHLs) in activated sludge was explored. The presence of N-heptanoyl-l-homoserine lactone in organic extracts of sludge was demonstrated along with activation of a LuxR-based AHL monitor strain deployed in sludge, indicating AHL-mediated gene expression is active in sludge flocculates but not in the bulk aqueous phase. Bacterial isolates from activated sludge were screened for AHL production and expression of phenotypes commonly but not exclusively regulated by AHL-mediated gene transcription. N-acylated-l-homoserine lactone and exoenzyme production were frequently observed among the isolates. N-acylated-l-homoserine lactone addition to sludge upregulated chitinase activity and an AHL- and chitinase-producing isolate closely related to Aeromonas hydrophila was shown to respond to AHL addition with upregulation of chitinase activity. N-acylated-l-homoserine lactones produced by this strain were identified and genes ahyI/R and chiA, encoding AHL production and response and chitinase activity respectively, were sequenced. These experiments provide insight into the relationship between AHL-mediated gene expression and exoenzyme activity in activated sludge and may ultimately create opportunities to improve sludge performance.