Active efflux systems in the solvent-tolerant bacterium Pseudomonas putida S12

The aim of the research presented in this thesis was to study the molecular mechanisms of organic solvent tolerance in Pseudomonas putida S12. This bacterium is capable of growth at saturated solvent concentrations, which are lethal to normal bacteria. Organic solvent-tolerant bacteria have potential advantages in either the remediation of highly polluted waste streams or biocatalytic applications for the production of specialty chemicals. The use of these bacteria in biocatalysis would allow the introduction of an organic phase to dissolve water-insoluble substrates or to remove toxic products. As a first step in the identification of genes involved in solvent tolerance, toluene-sensitive transposon mutants of P. putida S12 were generated. As described in Chapter 3, we were able to isolate the genes involved in toluene efflux using the toluene-sensitive strain P. putida JK1. The deduced amino acid sequences encoded by the srpABC genes isolated were highly homologous to proteins involved in proton-dependent efflux. Transfer of the genes for the toluene efflux pump to a normally toluene-sensitive P. putida strain resulted in the acquisition of toluene tolerance. From these results we conclude that organic solvent efflux is the key factor in solvent tolerance. In Chapter 4 it was found that the induction of the membrane associated efflux system SrpABC of P. putida S12 is inducible. Using a reporter vector, containing the srp promoter, it was determined that aromatic and aliphatic solvents and alcohols were capable of inducing the transcription the srpABC genes. However, antibiotics, heavy metals and general stress conditions (pH, temperature, NaCl, and organic acids) did not induce srp transcription. From the results presented in Chapter 4 we conclude that SrpABC-mediated efflux of organic solvents is solely induced by solvent stress. The high levels of antibiotic resistance of P. putida S12 and the relationship between solvent tolerance and antibiotic resistance triggered us to study multidrug resistance in this strain. In analogy to the results presented in Chapter 3 the first step in the identification of genes involved in multidrug resistance was to generate transposon mutants of P. putida S12. In Chapter 5 we describe the isolation the arp genes involved in chloramphenicol efflux, using the isolated chloramphenicol-sensitive P. putida strains CM1 and CM2. Moreover, the ArpABC efflux system was involved in the resistance towards tetracycline, carbenicillin, streptomycin, erythromycin, and novobiocin. Surprisingly, the deduced amino acid sequences encoded by the isolated arpABC genes were highly homologous to proteins involved in proton-dependent efflux of organic solvents. By constructing an arp-srp double mutant it was concluded that arpABC was not involved in efflux of organic solvents. In Chapter 6 octanol-sensitive mutants of P. putida S12 were isolated, which were interrupted in genes for the flagella biosynthetic pathway. These mutants were nonmotile and the formation of the flagellum was totally impaired. The expression of the SrpABC efflux pump in the nonmotile mutants was decreased, possibly due to general regulatory mechanisms. Several genes involved in multidrug resistance and solvent tolerance in P. putida S12 have been isolated and characterized. It would now be interesting to investigate the complex regulation of these systems and to identify new genes using the mutants described in this thesis

Isolation and characterization of Alicycliphilus denitrificans strain BC, which grows on benzene with chlorate as the electron acceptor

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 microm wide and 1 to 2 microm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplie

Smart Emission - Building a Spatial Data Infrastructure for an Environmental Citizen Sensor Network

Item does not contain fulltextSmart Emission is a citizen sensor network using low-cost sensors that enables citizens to gather data about environmental quality, like air quality, noise load, vibrations, light intensities and heat stress. This paper introduces the design and development of the data infrastructure for the Smart Emission initiative and discusses challenges for the future. The Spatial Data Infrastructure (SDI) is open and accessible on the Internet using open geospatial standards and (Web-) client applications. Smart Emission as a citizen sensor network offers several possibilities for heterogonous applications, from health determination to spatial planning purposes, environmental monitoring for sustainable traffic management, climate adaptation in cities and city planning.Geospatial Sensor Webs Conference 2016 (GSW 2016), 29 augustus 201

Identification and molecular characterization of an efflux system involved in Pseudomonas putida 12 multidrug resistance

The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in P. putida S12 but is solely involved in multidrug resistance

The trajectory of caregiver burden and risk factors in dementia progression:A systematic review

Background: Caring for patients with dementia at home is often a long-term process, in which the independence of the patient declines, and more responsibility and supervision time is required from the informal caregiver. Objective: In order to minimize and reduce caregiver burden, it is important to explore its trajectory and the accompanying risk factors as dementia progresses; the objective of this systematic review. Methods: PRISMA guidelines were followed in this systematic review. Three databases, PubMed, PsycINFO, and EMbase, were systematically searched in November 2019 using specific keywords. Results: 1,506 hits emerged during the systematic search but only eleven articles actually met the inclusion criteria for this review. The trajectory of caregiver burden is highly variable and depends on multiple factors. Important risk factors included: patients’ behavioral and neuropsychiatric symptoms, and their decline in functioning in (I)ADL; the caregiver’s age, gender, and physical and mental health; and, within the dyads (patient/caregiver), cohabitation and kinship. Conclusion: There is no one-size-fits-all for predicting how caregiver burden will change over time, but specific factors (like being a spouse and increased behavioral impairment and decline in functional status in the patient) may heighten the risk. Other factors, not yet comprehensively included in the published studies, might also prove to be important risk factors. Future research in the field of reducing caregiver burden is recommended to integrate the patient, caregiver, and context characteristics in the trajectory of caregiver burden, and to assess more clearly the phase of the dementia progression and use of external resources

Outcome and risk factors for recurrence of early onset fracture-related infections treated with debridement, antibiotics and implant retention:Results of a large retrospective multicentre cohort study

Introduction: Early Fracture-Related Infections (FRIs) are a common entity in hospitals treating trauma patients and are often treated with a Debridement, Antibiotics and Implant Retention (DAIR) procedure. Aims of this study were to 1) evaluate the recurrence rate after DAIR procedures for early onset FRI, 2) establish the number of surgical procedures to gain control of the initial infection and 3) identify independent predictors for recurrence in this cohort. Methods: A retrospective multicentre cohort study was conducted in two level 1 trauma centres. Consecutive patients who underwent a DAIR procedure between January 1st 2015 and July 1st 2020 for confirmed FRI with an onset of <6 weeks after the latest osseous operation were included. Recorded data included patient demographics, treatment characteristics and follow-up. Univariate and multivariate logistic regression analyses were performed to assess predictors for recurrent FRI. Results: A total of 141 patients with early FRI were included in this study with a median age of 54.0 years (interquartile range (IQR) 34.5–64.0). The recurrence rate of FRI was 13% (n = 19) at one year follow-up and 18% (n = 25) at 23.1 months (IQR 15.3–36.4) follow-up. Infection control was achieved in 94% (n = 127/135) of cases. In total, 73 patients (52%) underwent at least two surgical procedures to treat the ongoing initial episode of FRI, of whom 54 patients (74%) required two to three procedures and 17 patients (23%) four to five procedures. Predictors for recurrent FRI were use of an intramedullary nail during index operation (odds ratio (OR) 4.0 (95% confidence interval (CI) 1.1–13.8)), need for additional surgical procedures to treat ongoing infection during the treatment period following the first presentation of early FRI (OR 1.9 (95% CI 1.1–3.5)) and a decreased Injury Severity Score (ISS) (inverted OR 1.1 (95% CI 1.0–1.1)). Conclusion: The recurrence rate after treatment of early onset FRI in patients treated with a DAIR procedure was 18% at 23.1 months follow-up. At least two surgical procedures to gain control of the initial infection were needed in 52% of patients. Independent predictors for recurrent FRI were the use of an intramedullary nail during index operation, need for additional surgical procedures and a decreased ISS

Subtalar versus triple arthrodesis after intra-articular calcaneal fractures

Depending upon initial treatment, between 2 and 30% of patients with a displaced intra-articular calcaneal fracture require a secondary arthrodesis. The aim of this study was to investigate the effect of subtalar versus triple arthrodesis on functional outcome. A total of 33 patients with 37 secondary arthrodeses (17 subtalar and 20 triple) with a median follow-up of 116 months were asked to complete questionnaires regarding disease-specific functional outcome (Maryland Foot Score, MFS), quality of life (SF-36) and overall satisfaction with the treatment (Visual Analogue Scale, VAS). Patient groups were comparable considering median age at fracture, initial treatment (conservative or operative), time to arthrodesis, median follow-up, and post-arthrodesis radiographic angles. The MFS score was similar after subtalar versus triple arthrodesis (59 vs. 56 points; P = 0.79). No statistically significant difference was found for the SF-36 (84 vs. 83 points; P = 0.67) and the VAS (5 vs. 6; P = 0.21). Smoking was statistically significantly associated with a non-union (χ2 = 6.60, P = 0.017). The current study suggests that there is no significant difference in functional outcome between an in situ subtalar or triple arthrodesis as a salvage technique for symptomatic arthrosis after an intra-articular calcaneal fracture. Smoking is a risk factor for non-union

Outcome after resuscitation beyond 30 minutes in drowned children with cardiac arrest and hypothermia:Dutch nationwide retrospective cohort study

OBJECTIVES To evaluate the outcome of drowned children with cardiac arrest and hypothermia, and to determine distinct criteria for termination of cardiopulmonary resuscitation in drowned children with hypothermia and absence of spontaneous circulation. DESIGN Nationwide retrospective cohort study. SETTING Emergency departments and paediatric intensive care units of the eight university medical centres in the Netherlands. PARTICIPANTS Children aged up to 16 with cardiac arrest and hypothermia after drowning, who presented at emergency departments and/or were admitted to intensive care. MAIN OUTCOME MEASURE Survival and neurological outcome one year after the drowning incident. Poor outcome was defined as death or survival in a vegetative state or with severe neurological disability (paediatric cerebral performance category (PCPC) >= 4). RESULTS From 1993 to 2012, 160 children presented with cardiac arrest and hypothermia after drowning. In 98 (61%) of these children resuscitation was performed for more than 30 minutes (98/160, median duration 60 minutes), of whom 87 (89%) died (95% confidence interval 83% to 95%; 87/98). Eleven of the 98 children survived (11%, 5% to 17%), but all had a PCPC score >= 4. In the 62 (39%) children who did not require prolonged resuscitation, 17 (27%, 16% to 38%) survived with a PCPC score CONCLUSIONS Drowned children in whom return of spontaneous circulation is not achieved within 30 minutes of advanced life support have an extremely poor outcome. Good neurological outcome is more likely when spontaneous circulation returns within 30 minutes of advanced life support, especially when the drowning incident occurs in winter. These findings question the therapeutic value of resuscitation beyond 30 minutes in drowned children with cardiac arrest and hypothermia

Non-conservative Evolution of Cataclysmic Variables

We suggest a new mechanism to account for the loss of angular momentum in binaries with non-conservative mass exchange. It is shown that in some cases the loss of matter can result in increase of the orbital angular momentum of a binary. If included into consideration in evolutionary calculations, this mechanism appreciably extends the range of mass ratios of components for which mass exchange in binaries is stable. It becomes possible to explain the existence of some observed cataclysmic binaries with high donor/accretor mass ratio, which was prohibited in conservative evolution models.Comment: LaTeX, 32 pages, to be published in Astron. Z