Glycerol Metabolism Contributes to Competition by Oral Streptococci through Production of Hydrogen Peroxide.

As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. Here we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II ( manL ), glycerol metabolism ( glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H 2 O 2 ), transcription, and competition with Streptococcus mutans . Biochemical assays identified the glp pathway as a novel source of H 2 O 2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased expression of spxB and a second, H 2 O 2 -non-producing glycerol metabolic pathway ( dha ), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate for growth, benefit from catabolism of glycerol through production of both ATP and H 2 O 2 . Importance: Glycerol is an abundant carbohydrate found in oral cavity, both due to biological activities of humans and microbes, and as a common ingredient of foods and health care products. However, very little is understood regarding the metabolism of glycerol by some of the most abundant oral bacteria, commensal streptococci. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. Here we show that Streptococcus sanguinis , an oral commensal associated with dental health, can degrade glycerol for persistence and competition through two independent pathways, one of which generates hydrogen peroxide at levels capable of inhibiting a dental pathobiont, Streptococcus mutans . Preliminary studies suggest that several other commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are being actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis which warrants further exploration.

Glucose Phosphotransferase System Modulates Pyruvate Metabolism, Bacterial Fitness, and Microbial Ecology in Oral Streptococci.

Spontaneous mutants with defects in the primary glucose phosphotransferase permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. These observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of acetate, formate, pyruvate, and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide metabolism, and arginine deiminase and pathways for metabolism of acetoin, ethanolamine, ascorbate, and formate, along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm cocultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis cocultures increased the proportions of UA159 in the biofilms. Reducing the buffer capacity or increasing the concentration of glucose benefited UA159 when cocultured with SK36, but not with SK36/manL, likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that phosphotransferase system (PTS)-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. IMPORTANCE Dental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia and improved persistence under acidic conditions. A metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-Phosphotransferase System Show Enhanced Post-Exponential-Phase Fitness.

Genetic truncations in a gene encoding a putative glucose-phosphotransferase system (PTS) protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. To understand the emergence of these mutants, we engineered deletion mutants of manL and showed that the ManL-deficient strain had improved bacterial viability in the stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small but significant percentage (∼10%) of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. To our surprise, significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e., excess carbohydrates and low pH, are those associated with caries development, we propose that glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2. IMPORTANCE A health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of the central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

Simulation-based mastery learning for endoscopy using the endoscopy training system: a strategy to improve endoscopic skills and prepare for the fundamentals of endoscopic surgery (FES) manual skills exam.

INTRODUCTION: The fundamentals of endoscopic surgery (FES) program has considerable validity evidence for its use in measuring the knowledge, skills, and abilities required for competency in endoscopy. Beginning in 2018, the American Board of Surgery will require all candidates to have taken and passed the written and performance exams in the FES program. Recent work has shown that the current ACGME/ABS required case volume may not be enough to ensure trainees pass the FES skills exam. The aim of this study was to investigate the feasibility of a simulation-based mastery-learning curriculum delivered on a novel physical simulation platform to prepare trainees to pass the FES manual skills exam. METHODS: The newly developed endoscopy training system (ETS) was used as the training platform. Seventeen PGY 1 (10) and PGY 2 (7) general surgery residents completed a pre-training assessment consisting of all 5 FES tasks on the GI Mentor II. Subjects then trained to previously determined expert performance benchmarks on each of 5 ETS tasks. Once training benchmarks were reached for all tasks, a post-training assessment was performed with all 5 FES tasks. RESULTS: Two subjects were lost to follow-up and never returned for training or post-training assessment. One additional subject failed to complete any portion of the curriculum, but did return for post-training assessment. The group had minimal endoscopy experience (median 0, range 0-67) and minimal prior simulation experience. Three trainees (17.6%) achieved a passing score on the pre-training FES assessment. Training consisted of an average of 48 ± 26 repetitions on the ETS platform distributed over 5.1 ± 2 training sessions. Seventy-one percent achieved proficiency on all 5 ETS tasks. There was dramatic improvement demonstrated on the mean post-training FES assessment when compared to pre-training (74.0 ± 8 vs. 50.4 ± 16, p < 0.0001, effect size = 2.4). The number of ETS tasks trained to proficiency correlated moderately with the score on the post-training assessment (r = 0.57, p = 0.028). Fourteen (100%) subjects who trained to proficiency on at least one ETS task passed the post-training FES manual skills exam. CONCLUSIONS: This simulation-based mastery learning curriculum using the ETS is feasible for training novices and allows for the acquisition of the technical skills required to pass the FES manual skills exam. This curriculum should be strongly considered by programs wishing to ensure that trainees are prepared for the FES exam.

Do Resident Case Logs Meet ACGME Requirements? A Comparison Between Acute Care and Elective Cases.

PURPOSE: Operative experience is at the core of general surgery residency, and recently operative volume requirements for graduating residents were increased. The ACGME has outlined 4 areas of required resident participation and documentation in order for a surgical case to be logged: determination or confirmation of the diagnosis, provision of preoperative care, selection and accomplishment of the operative procedure, and direction of the postoperative care. The purpose of this study was to examine whether general surgery residents are currently meeting the required care participation documentation standard and to examine the effect of acute care vs. elective cases on documentation. METHODS: The operative case logs of 7 PGY-3 and 7 PGY-5 general surgery residents from March 2016 were retrospectively reviewed and compared to the electronic medical record (EMR) to verify documentation of resident participation in each of the 4 required areas. Chart review was also utilized to classify cases as either acute care or elective. RESULTS: A total of 339 cases were reviewed (159 PGY-3 and 180 PGY-5). Of these, 251 cases were classified as elective and 88 were classified as acute care. Overall, documentation of comprehensive care (participation in all four required areas) was found for 44% of cases, with residents reporting participation in a higher percentage of comprehensive care (all 4 domains completed) than was actually documented in the EMR (71.9% vs. 44.4%, t[13] = 2.57, p = 0.023, d = 1.13). Comprehensive care was documented more frequently in elective cases than acute care cases (49.7% vs. 38.3%), and there was less discrepancy between perceived and documented comprehensive care within elective cases (67% vs. 49.7%, t[13] = 1.17, p = 0.27) than acute care cases (80.9% vs. 38.3%, t[13] = 4.40, p = 0.001). CONCLUSIONS: Despite ACGME requirements, the majority of cases logged by general surgery residents do not have documentation by the operating resident in the EMR verifying provision of comprehensive care. Elective cases were more likely to meet documentation requirements than acute care cases, and we purport that this is possibly secondary to restricted work hours. We expect that other programs would find similar compliance in the documentation of comprehensive care. These results question whether the requirement for documenting comprehensive care to log a surgical case is practical in surgical residency training, particularly with an increasing demand for operative volume in the setting of limited work hours.

Low Grade Lymphoma Mimicking Metastatic Urothelial Carcinoma: When Do We Need Further Histologic Staging?

Introduction. Patients with urothelial carcinoma of the bladder often present with metastases to regional lymph nodes, with lymphadenopathy on physical examination or radiographic imaging. Case Presentation. We present the case of a 73-year-old Caucasian man with presumed metastatic urothelial carcinoma of the bladder to regional pelvic and retroperitoneal lymph nodes. He underwent systemic chemotherapy for treatment of urothelial carcinoma and was discovered on restaging to have findings suggestive of disease progression but ultimately was found to have a concurrent secondary malignancy. Conclusion. Our case suggests that in patients with urothelial carcinoma, the concurrent presentation of regional lymphadenopathy may not be metastatic urothelial carcinoma and may warrant further investigation.

A Novel Case of Penile Gangrene in a Patient Treated with Ibrutinib for Chronic Lymphocytic Leukemia.

Introduction. Ibrutinib is commonly used for the treatment of patients with CLL in either first-line or relapsed/refractory settings. Case Presentation. We present the case of a 74-year-old Caucasian man with CLL who presented with penile gangrene upon initiation of ibrutinib treatment. Our case is the first showing the complication of penile gangrene associated with ibrutinib use. The gangrene was self-limited upon discontinuing ibrutinib. Conclusion. Our finding describes a very rare yet important adverse event associated with ibrutinib use.

Rare cause of acute hepatitis: a common energy drink.

A previously healthy man aged 50 years presented with malaise, anorexia, abdominal pain, nausea, vomiting, generalised jaundice, scleral icterus and dark urine. He was not on any prescription or over-the-counter medications, but reported drinking 4-5 energy drinks daily for 3 weeks prior to presentation. Physical examination revealed jaundice and right upper quadrant abdominal tenderness. Laboratory studies were remarkable for transaminitis and evidence of chronic hepatitis C infection. Ultrasound scan demonstrated an echogenic liver and diffuse gallbladder wall thickening. Liver biopsy showed severe acute hepatitis with bridging necrosis and marked cholestasis. The patient was treated supportively with complete resolution of his symptoms and marked improvement in his laboratory abnormalities. The development of acute hepatitis in this patient was likely secondary to excessive energy drink consumption. Energy drinks as well as other herbal/over-the-counter supplements should be considered by clinicians in the workup of patients with acute hepatitis, particularly once other aetiologies have been excluded.