Transposon Mutagenesis Identification of Polymicrobial Interaction Mechanisms Between Prokaryotic and Eukaryotic Microorganisms

Antibiotic resistance occurs when bacteria change in response to selective pressures induced by antibiotics, which has become a major concern worldwide and one of the biggest threats to global health. Antibiotic resistance can occur naturally, but the misuse and overuse of antibiotics is accelerating the process. One way to combat this process is to understand the different relationships between microbes, also known as polymicrobial interactions. Bacteria can interact with one another synergistically or antagonistically and understanding the mechanisms behind these interactions can lead to the discovery of new therapeutics or targets to fight and kill pathogenic microbes. The rarely pathogenic Gram-negative bacterium, Alcaligenes faecalis, has previously been shown in our lab as playing an important role in potentially fighting antibiotic and antifungal resistance due to its competitiveness during polymicrobial interaction. Our research has found that A. faecalis kills Bacillus cereus, Staphylococcus aureus, and Candida albicans. This is a unique characteristic as these targets encompass both prokaryotic (bacteria) and eukaryotic (fungi) microbes. These three species are known to cause numerous infections in humans and have increased cases of antibiotic and antifungal resistance. In the present study, we investigated the genetic elements A. faecalis utilizes to inhibit growth when interacting with B. cereus, S. aureus, and C. albicans. Transposon mutagenesis was performed to create a genetic library of A. faecalis loss-of-function mutants. These strains were then screened against all three microorganisms to determine which mutants no longer inhibited growth. The mutants that lacked zones-of-inhibition were sequenced to determine the gene that had been interrupted. BLAST analysis of these sequences identified a MFS transporter, a 2FE-2S iron sulfur binding protein, a mechanosensitive ion channel, and a glucose-6-phosphate isomerase as instrumental in this inhibitory mechanism. Results from this research study can be used to further study polymicrobial interactions and potentially discover new therapeutics to combat antimicrobial resistance

"Digging deeper, reaching further: libraries empowering users to mine the HathiTrust Digital Library resources" curriculum

Modern academic librarianship demands reconfigured skillsets and expertise to meet the rapidly evolving, data-driven needs of today’s students, faculty, and researchers as they pursue digital scholarship and produce immense amounts of research data. These rapid developments in user needs and research services speak to an urgent need to provide training for academic librarians to support digital scholarship. We seek to address this need in “Digging Deeper, Reaching Further: Libraries Empowering Users to Mine the HathiTrust Digital Library Resources,” a three-year project funded by a Laura Bush 21st Century Librarian grant award from the Institute of Museum and Library Services. The curriculum developed by the DDRF project leverages the tools and data from HathiTrust Research Center to provide a “train the trainer” curriculum for library and information professionals on text mining and digital scholarship methods.IMLS #RE-00-15-0112-15Ope

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Population‐based cohort study of outcomes following cholecystectomy for benign gallbladder diseases

Background The aim was to describe the management of benign gallbladder disease and identify characteristics associated with all‐cause 30‐day readmissions and complications in a prospective population‐based cohort. Methods Data were collected on consecutive patients undergoing cholecystectomy in acute UK and Irish hospitals between 1 March and 1 May 2014. Potential explanatory variables influencing all‐cause 30‐day readmissions and complications were analysed by means of multilevel, multivariable logistic regression modelling using a two‐level hierarchical structure with patients (level 1) nested within hospitals (level 2). Results Data were collected on 8909 patients undergoing cholecystectomy from 167 hospitals. Some 1451 cholecystectomies (16·3 per cent) were performed as an emergency, 4165 (46·8 per cent) as elective operations, and 3293 patients (37·0 per cent) had had at least one previous emergency admission, but had surgery on a delayed basis. The readmission and complication rates at 30 days were 7·1 per cent (633 of 8909) and 10·8 per cent (962 of 8909) respectively. Both readmissions and complications were independently associated with increasing ASA fitness grade, duration of surgery, and increasing numbers of emergency admissions with gallbladder disease before cholecystectomy. No identifiable hospital characteristics were linked to readmissions and complications. Conclusion Readmissions and complications following cholecystectomy are common and associated with patient and disease characteristics

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

The Cholecystectomy As A Day Case (CAAD) Score: A Validated Score of Preoperative Predictors of Successful Day-Case Cholecystectomy Using the CholeS Data Set

Background Day-case surgery is associated with significant patient and cost benefits. However, only 43% of cholecystectomy patients are discharged home the same day. One hypothesis is day-case cholecystectomy rates, defined as patients discharged the same day as their operation, may be improved by better assessment of patients using standard preoperative variables. Methods Data were extracted from a prospectively collected data set of cholecystectomy patients from 166 UK and Irish hospitals (CholeS). Cholecystectomies performed as elective procedures were divided into main (75%) and validation (25%) data sets. Preoperative predictors were identified, and a risk score of failed day case was devised using multivariate logistic regression. Receiver operating curve analysis was used to validate the score in the validation data set. Results Of the 7426 elective cholecystectomies performed, 49% of these were discharged home the same day. Same-day discharge following cholecystectomy was less likely with older patients (OR 0.18, 95% CI 0.15–0.23), higher ASA scores (OR 0.19, 95% CI 0.15–0.23), complicated cholelithiasis (OR 0.38, 95% CI 0.31 to 0.48), male gender (OR 0.66, 95% CI 0.58–0.74), previous acute gallstone-related admissions (OR 0.54, 95% CI 0.48–0.60) and preoperative endoscopic intervention (OR 0.40, 95% CI 0.34–0.47). The CAAD score was developed using these variables. When applied to the validation subgroup, a CAAD score of ≤5 was associated with 80.8% successful day-case cholecystectomy compared with 19.2% associated with a CAAD score >5 (p < 0.001). Conclusions The CAAD score which utilises data readily available from clinic letters and electronic sources can predict same-day discharges following cholecystectomy

Utilizing Transposon Mutagenesis to Identify Genetic Elements Important for Polymicrobial Interactions

Microorganisms interact in symbiotic and antagonistic relationships. These unique interactions can be used to identify novel targets and understand microbial diseases. A genetic library of A. faecalis strains was constructed by transposon mutagenesis and screened to determine the loss-of-function of A. faecalis’ inhibition of Bacillus, Candida, and Staphylococcus. These results are important in determining the genetic mechanism in which A. faecalis inhibits growth and how this information can be used to thwart common human pathogens at a clinical level

From cultivation to cancer: Formation of N-nitrosamines and other carcinogens in smokeless tobacco and their mutagenic implications

Tobacco use is a major cause of preventable morbidity and mortality globally. Tobacco products, including smokeless tobacco (ST), generally contain tobacco-specific N-nitrosamines (TSNAs), such as N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK), which are potent carcinogens that cause mutations in critical genes in human DNA. This review covers the series of biochemical and chemical transformations, related to TSNAs, leading from tobacco cultivation to cancer initiation. A key aim of this review is to provide a greater understanding of TSNAs: their precursors, the microbial and chemical mechanisms that contribute to their formation in ST, their mutagenicity leading to cancer due to ST use, and potential means of lowering TSNA levels in tobacco products. TSNAs are not present in harvested tobacco but can form due to nitrosating agents reacting with tobacco alkaloids present in tobacco during certain types of curing. TSNAs can also form during or following ST production when certain microorganisms perform nitrate metabolism, with dissimilatory nitrate reductases converting nitrate to nitrite that is then released into tobacco and reacts chemically with tobacco alkaloids. When ST usage occurs, TSNAs are absorbed and metabolized to reactive compounds that form DNA adducts leading to mutations in critical target genes, including the RAS oncogenes and the p53 tumor suppressor gene. DNA repair mechanisms remove most adducts induced by carcinogens, thus preventing many but not all mutations. Lastly, because TSNAs and other agents cause cancer, previously documented strategies for lowering their levels in ST products are discussed, including using tobacco with lower nornicotine levels, pasteurization and other means of eliminating microorganisms, omitting fermentation and fire-curing, refrigerating ST products, and including nitrite scavenging chemicals as ST ingredients.Fil: Stanfill, Stephen B.. National Center For Environmental Health; Estados UnidosFil: Hecht, Stephen S.. University of Minnesota; Estados UnidosFil: Joerger, Andreas C.. Goethe Universitat Frankfurt; AlemaniaFil: González, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Maia, Luisa B.. Universidade Nova de Lisboa; PortugalFil: Rivas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Moura, José J. G.. Universidade Nova de Lisboa; PortugalFil: Gupta, Alpana K.. Independent Research Consultant; IndiaFil: Le Brun, Nick E.. University of East Anglia; Reino UnidoFil: Crack, Jason C.. University of East Anglia; Reino UnidoFil: Hainaut, Pierre. Universite Grenoble Alpes; FranciaFil: Sparacino Watkins, Courtney. Univeristy of Pittsburgh. School of Medicine; Estados Unidos. Vascular Medicine Institute; Estados UnidosFil: Tyx, Robert E.. National Center For Environmental Health; Estados UnidosFil: Pillai, Suresh D.. Texas A&M University; Estados UnidosFil: Zaatari, Ghazi S.. American University Of Beirut; LíbanoFil: Henley, S. Jane. Centers for Disease Control and Prevention; Estados UnidosFil: Blount, Benjamin C.. National Center For Environmental Health; Estados UnidosFil: Watson, Clifford H.. National Center For Environmental Health; Estados UnidosFil: Kaina, Bernd. University Medical Center; AlemaniaFil: Mehrotra, Ravi. Innovation And Policy Foundation; Indi