Qualitative detection of E. coli in distributed drinking water using real-time reverse transcription PCR targeting 16S rRNA: Validation and practical experiences.

Escherichia coli (E. coli) plays a central role as an indicator for fecal contamination to predict the possible presence of microbial pathogens in drinking water. Current detection methods for E. coli are based on time-consuming culture-based techniques. There is a strong need for methods to detect fecal contamination rapidly in distributed drinking water to prevent outbreaks of waterborne disease and support water utilities to efficiently manage their operations like actions to repair or maintain distribution pipes, to minimize impact on consumers. This study describes the validation and application of a qualitative real time reverse transcription PCR (RT-PCR) method targeting 16S ribosomal RNA (rRNA) for rapid detection of E. coli in distributed drinking water. The RT-PCR assay targets 16S rRNA, a highly abundant RNA in viable cells, enabling robust detection at the required sensitivity of 1 CFU/100 ml. The validation was performed by comparing the RT-PCR method with the culture-based chromogenic reference method (CCA) using the protocol and criteria described in ISO 16,140-2:2016. The validation demonstrated that this RT-PCR method can be used to specifically detect E. coli in a broad range of drinking water samples with at least the same limit of detection as the culture method (Relative Limit Of Detection = 0.75, range 0.43-1.43). The inclusivity study showed that the RT-PCR method was able to detect a broad range of E. coli strains derived from different sources and geographic areas, including pathogenic serotype O157 strains that are not detected with the culture method. The exclusivity study determined that other bacterial genera are not detected with this RT-PCR. However, Escherichia fergusonii was detected and, based on "in silico" analysis, it is expected that also E. albertii and E. marmotae and Shigella species will be detectable using this RT-PCR. An interlaboratory study confirmed that the RT-PCR and culture method have comparable sensitivities when tested by different participants at different laboratories. The application of RT-PCR to confirm the hygienic quality of distributed drinking water after actions to repair or maintain distribution pipes was compared with the culture method on 8076 routine samples, analyzed by the drinking water laboratories in the Netherlands. This comparison study showed a 96.4 % agreement between RT-PCR and culture. In 3.3 % of the samples E. coli was detected with RT-PCR and not with the culture method and in 0.1 % of the samples E. coli was only detected by culture confirming either a higher sensitivity for RT-PCR or the detection of RNA from uncultivable cells. Finally, the application of RT-PCR was highlighted during a contamination event in Belgium where we demonstrate the potency of RT-PCR as a tool to rapidly monitor the spread of microbial contamination and to monitor the effect of measures to remove the contamination This is the first fully validated rapid nucleic based method for detection of E. coli in distributed drinking water. These results demonstrate that this RT-PCR method can be used as a rapid alternative to the culture method to monitor E. coli in distributed drinking water. However, it should be emphasized that nucleic acid based detection methods rely on highly different detection principles (detection of captured nucleic acids present in a sample) than culture base methods (presence of cells cultivable on a selective medium) resulting in occasional different analysis results. Varying treatment and disinfection steps (UV, chlorine, monochloramine, Ozone) or environmental factors (decay) can influence the results and cause differences between RT-PCR and culture methods.

Cold spots in neonatal incubators are hot spots for microbial contamination.

Thermal stability is essential for the survival and well-being of preterm neonates. This is achieved in neonatal incubators by raising the ambient temperature and humidity to sufficiently high levels. However, potentially pathogenic microorganisms also can thrive in such warm and humid environments. We therefore investigated whether the level of microbial contamination (i.e., the bacterial load) inside neonatal incubators can be predicted on the basis of their average temperature and relative humidity settings, paying special attention to local temperature differences. Swab samples were taken from the warmest and coldest spots found within Caleo incubators, and these were plated to determine the number of microbial CFU per location. In incubators with high average temperature (≥ 34°C) and relative humidity (≥ 60%) values, the level of microbial contamination was significantly higher at cold spots than at hot spots. This relates to the fact that the local equilibrium relative humidity at cold spots is sufficiently high to sustain microbial growth. The abundance of staphylococci, which are the main causative agents of late-onset sepsis in preterm neonates, was found to be elevated significantly in cold areas. These findings can be used to improve basic incubator hygiene.

Biofouling control: the impact of biofilm dispersal and membrane flushing.

Pure culture studies have shown that biofilm dispersal can be triggered if the nutrient supply is discontinued by stopping the flow. Stimulating biofilm dispersal in this manner would provide a sustainable manner to control unwanted biofilm growth in industrial settings, for instance on synthetic membranes used to purify water. The response of multispecies biofilms to nutrient limitation has not been thoroughly studied. To assess biomass dispersal during nutrient limitation it is common practise to flush the biofilm after a stop-period. Hence, flow-stop-induced biomass removal could occur as a response to nutrient limitation followed by mechanical removal due to biofilm flushing (e.g. biofilm detachment). Here, we investigated the feasibility to reduce membrane biofouling by stopping the flow and flushing the membrane. Using a membrane fouling simulator, biomass removal from synthetic membranes after different stop-periods was determined, as well as biomass removal at different cross flow velocities. Biomass removal from membrane surfaces depended on the nutrient limiting period and on the flow velocity during the biofilm flush. When flushed at a low flow velocity (0.1 m.s-1), the duration of the stop-period had a large effect on the biomass removal rate, but when the flow velocity was increased to 0.2 m.s-1, the length of the stop period became less considerable. The flow velocity during membrane flushing has an effect on the bacterial community that colonized the membranes afterwards. Repetition of the stop-period and biofilm flushing after three repetitive biofouling cycles led to a stable bacterial community. The increase in bacterial community stability coincided with a decrease in cleaning effectivity to restore membrane performance. This shows that membrane cleaning comes at the costs of a more stable bacterial community that is increasingly difficult to remove.

A designer FG-Nup that reconstitutes the selective transport barrier of the nuclear pore complex.

Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure selective binding of the artificial FG-Nup brushes to the transport receptor Kap95 over cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create selective NPCs.

Biodiversity and ecology of microorganisms in high pressure membrane filtration systems.

High-pressure membrane filtration (reverse osmosis and nanofiltration) is used to purify different water sources, including wastewater, surface water, groundwater and seawater. A major concern in membrane filtration is the accumulation and growth of micro-organisms and their secreted polymeric substances, leading to reduced membrane performance and membrane biofouling. The fundamental understanding of membrane biofouling is limited despite years of research, as the means of microbial interactions and response to the conditions on the membrane surface are complicated. Here, we discuss studies that investigated the microbial diversity of fouled high-pressure membranes. High-throughput amplicon sequencing of the 16S rRNA gene have shown that Burkholderiales, Pseudomonadales, Rhizobiales, Sphingomonadales and Xanthomonadales frequently obtain a high relative abundance on fouled membranes. The bacterial communities present in the diverse feed water types and in pre-treatment compartments are different from the communities on the membrane, because high-pressure membrane filtration provides a selective environment for certain bacterial groups. The biofilms that form within the pre-treatment compartments do not commonly serve as an inoculum for the subsequent high-pressure membranes. Besides bacteria also fungi are detected in the water treatment compartments. In contrast to bacteria, the fungal community does not change much throughout membrane cleaning. The stable fungal diversity indicates that they are more significant in membrane biofouling than previously thought. By reviewing the biodiversity and ecology of microbes in the whole high pressure membrane filtration water chain, we have been able to identify potentials to improve biofouling control. These include modulation of hydrodynamic conditions, nutrient limitation and the combination of cleaning agents to target the entire membrane microbiome.

A questionnaire to assess rehabilitation patients' experiences with motivational interviewing consultation in the context of physical activity stimulation.

Purpose: The current article provides a description of the construction process of a short questionnaire that measures patients' experiences with a motivational interviewing consultation by a (sports) counselor in rehabilitation. Subsequently, results from confirmatory factor analyses are presented to investigate a first perspective on factorial construct validity of the questionnaire.Material and methods: Based on motivational interviewing literature, an initial item pool was created. All items were critically reviewed, resulting in the new "Evaluation of Motivational Interviewing Consultation on Active Lifestyle and Sports" questionnaire. The final items were determined by a confirmatory factor analysis based on 890 completed questionnaires.Results: The initial 26-item questionnaire consisted of four inter-correlated subscales. Factor analyses underpinned the proposed factors: acceptance, evocation, partnership, and non-adherent. After removing six items, an alternative model remained and showed an acceptable model fit. The internal consistency of the subscales derived varied from 0.70 to 0.90.Conclusion: The shortened questionnaire provides a feasible and easy to administer tool and may provide a cost saving method of assessing motivational interviewing fidelity from a patient's perspective in disability and rehabilitation.Implications for rehabilitationMany persons with a physical disability do not obtain the recommended amount of physical activity in order to maintain health.Stimulation of a physically active lifestyle through motivational interviewing is promising.Measuring motivational interviewing treatment fidelity is time consuming and often from a counselor's perspective.We developed a short questionnaire facilitating the assessment of treatment fidelity at the side of the client.Our questionnaire provides a feasible and easy to administer tool for assessing MI fidelity in daily rehabilitation practice.

Isolation and characterization of Sphingomonadaceae from fouled membranes.

Membrane filtration systems are widely applied for the production of clean drinking water. However, the accumulation of particles on synthetic membranes leads to fouling. Biological fouling (i.e., biofouling) of reverse osmosis and nanofiltration membranes is difficult to control by existing cleaning procedures. Improved strategies are therefore needed. The bacterial diversity on fouled membranes has been studied, especially to identify bacteria with specialized functions and to develop targeted approaches against these microbes. Previous studies have shown that Sphingomonadaceae are initial membrane colonizers that remain dominant while the biofilm develops. Here, we characterized 21 Sphingomonadaceae isolates, obtained from six different fouled membranes, to determine which physiological traits could contribute to colonization of membrane surfaces. Their growth conditions ranged from temperatures between 8 and 42 oC, salinity between 0.0 and 5.0% w/v NaCl, pH from 4 and 10, and all isolates were able to metabolize a wide range of substrates. The results presented here show that Sphingomonadaceae membrane isolates share many features that are uncommon for other members of the Sphingomonadaceae family: all membrane isolates are motile and their tolerance for different temperatures, salt concentrations, and pH is high. Although relative abundance is an indicator of fitness for a whole group, for the Sphingomonadaceae it does not reveal the specific physiological traits that are required for membrane colonization. This study, therefore, adds to more fundamental insights in membrane biofouling.

Self-Regenerating Soft Biophotovoltaic Devices.

This paper describes the fabrication of soft, stretchable biophotovoltaic devices that generate photocurrent from photosystem I (PSI) complexes that are self-assembled onto Au electrodes with a preferred orientation. Charge is collected by the direct injection of electrons into the Au electrode and the transport of holes through a redox couple to liquid eutectic gallium-indium (EGaIn) electrodes that are confined to microfluidic pseudochannels by arrays of posts. The pseudochannels are defined in a single fabrication step that leverages the non-Newtonian rheology of EGaIn. This strategy is extended to the fabrication of reticulated electrodes that are inherently stretchable. A simple shadow evaporation technique is used to increase the surface area of the Au electrodes by a factor of approximately 106 compared to planar electrodes. The power conversion efficiency of the biophotovoltaic devices decreases over time, presumably as the PSI complexes denature and/or detach from the Au electrodes. However, by circulating a solution of active PSI complexes the devices self-regenerate by mass action/self-assembly. These devices leverage simple fabrication techniques to produce complex function and prove that photovoltaic devices comprising PSI can retain the ability to regenerate, one of the most important functions of photosynthetic organisms.

The impact of post-resuscitation feedback for paramedics on the quality of cardiopulmonary resuscitation.

PURPOSE: The Guidelines place emphasis on high-quality cardiopulmonary resuscitation (CPR). This study aims to measure the impact of post-resuscitation feedback on the quality of CPR as performed by ambulance personnel. MATERIALS AND METHODS: Two ambulances are dispatched for suspected cardiac arrest. The crew (driver and paramedic) of the first arriving ambulance is responsible for the quality of CPR. The crew of the second ambulance establishes an intravenous access and supports the first crew. All resuscitation attempts led by the ambulance crew of the study region were reviewed by two research paramedics and structured feedback was given based on defibrillator recording with impedance signal. A 12-months period before introduction of post-resuscitation feedback was compared with a 19-months period after introduction of feedback, excluding a six months run-in interval. Quality parameters were chest compression fraction (CCF), chest compression rate, longest peri-shock pause and longest non-shock pause. RESULTS: In the pre-feedback period 55 cases were analyzed and 69 cases in the feedback period. Median CCF improved significantly in the feedback period (79% vs 86%, p<0.001). The mean chest compression rate was within the recommended range of 100-120/min in 87% of the cases in the pre-feedback period and in 90% of the cases in the feedback period (p=0.65). The duration of longest non-shock pause decreased significantly (40s vs 19s, p<0.001), the duration of the longest peri-shock pause did not change significantly (16s vs 13s, p=0.27). CONCLUSION: Post-resuscitation feedback improves the quality of resuscitation, significantly increasing CCF and decreasing the duration of longest non-shock pauses.

Draft Genome Sequence of Sphingomonas sp. Strain Sph1(2015), Isolated from a Fouled Membrane Filter Used To Produce Drinking Water.

We report here the high-quality draft genome sequence of Sphingomonas sp. strain Sph1(2015), isolated from a fouled reverse osmosis membrane used for the production of high-quality drinking water. The draft sequence provides insights into the modus operandi of this strain to form biofilms on membrane surfaces. This knowledge offers tools to develop novel antifouling strategies.

One-day low-intensity combined arm-leg (Cruiser) ergometer exercise intervention: cardiorespiratory strain and gross mechanical efficiency in one-legged and two-legged exercise.

This study aims to research whether there is a difference in cardiorespiratory variables and gross mechanical efficiency (GE) in healthy individuals during low-intensity one-legged and two-legged exercise on the combined arm-leg (Cruiser) ergometer and whether motor learning occurs. The outcome of this study will support the use of the Cruiser ergometer in future as a testing and training instrument in the rehabilitation of patients with a lower limb amputation. Twenty-eight healthy men participated in this randomized-controlled trial. One group (n=14) used one leg and both arms during the exercise and the other group (n=14) used both legs and both arms. All participants performed a 1-day low-intensity exercise protocol. This included a standardized pretest and post-test of three bouts of 4 min exercise at 40 W and an exercise intervention of seven bouts of 2×4 min exercise at 40 W. The one-legged and two-legged group differed significantly in the heart rate and GE between the pretest and post-test. At the post-test, the one-legged group showed motor learning. GE improved significantly in both groups over the duration of the three exercise bouts of the pretest, but it did not improve during the post-test. There are differences in cardiorespiratory variables and GE between one-legged and two-legged exercise on the Cruiser ergometer. When using this ergometer in the rehabilitation of patients with a lower limb amputation, it is important to consider these differences and the occurrence of motor learning.

The LifeLines Cohort Study: Prevalence and treatment of cardiovascular disease and risk factors.

BACKGROUND: The LifeLines Cohort Study is a large three-generation prospective study and Biobank. Recruitment and data collection started in 2006 and follow-up is planned for 30years. The central aim of LifeLines is to understand healthy ageing in the 21st century. Here, the study design, methods, baseline and major cardiovascular phenotypes of the LifeLines Cohort Study are presented. METHODS AND RESULTS: Baseline cardiovascular phenotypes were defined in 9700 juvenile (8-18years) and 152,180 adult (≥18years) participants. Cardiovascular disease (CVD) was defined using ICD-10 criteria. At least one cardiovascular risk factor was present in 73% of the adult participants. The prevalence, adjusted for the Dutch population, was determined for risk factors (hypertension (33%), hypercholesterolemia (19%), diabetes (4%), overweight (56%), and current smoking (19%)) and CVD (myocardial infarction (1.8%), heart failure (1.0%), and atrial fibrillation (1.3%)). Overall CVD prevalence increased with age from 9% in participants<65years to 28% in participants≥65years. Of the participants with hypertension, hypercholesterolemia and diabetes, respectively 75%, 96% and 41% did not receive preventive pharmacotherapy. CONCLUSIONS: The contemporary LifeLines Cohort Study provides researchers with unique and novel opportunities to study environmental, phenotypic, and genetic risk factors for CVD and is expected to improve our knowledge on healthy ageing. In this contemporary Western cohort we identified a remarkable high percentage of untreated CVD risk factors suggesting that not all opportunities to reduce the CVD burden are utilised.