A Portable, Encapsulated Microbial Whole-Cell Biosensing System for the Detection of Bioavailable Copper (II) in Soil.

A portable, field deployable whole-cell biosensor was developed that can withstand the complex matrices of soil and requires minimal to no sample preparation to monitor bioavailable concentrations of the essential micronutrient copper (II). Conventional measurement of micronutrients is often complex, laboratory-based, and not suitable for monitoring their bioavailable concentration. To address this need, we developed a fluorescence based microbial whole-cell biosensing (MWCB) system encoding for a Cu2+-responsive protein capable of generating a signal upon binding to Cu2+. The sensing-reporting protein was designed by performing circular permutation on the green fluorescent protein (GFP) followed by insertion of a Cu2+ binding motif into the structure of GFP. The design included insertion of several binding motifs and creating plasmids that encoded the corresponding sensing proteins. The signal generated by the sensing-reporting protein is directly proportional to the concentration of Cu2+ in the sample. Evaluation of the resulting biosensing systems carrying these plasmids was performed prior to selection of the optimal fluorescence emitting Cu2+-binding protein. The resulting optimized biosensing system was encapsulated in polyacrylate-alginate beads and embedded in soil for detection of the analyte. Once exposed to the soil, the beads were interrogated to measure the fluorescence signal emitted by the sensing-reporting protein using a portable imaging device. The biosensor was optimized for detection of Cu2+ in terms of selectivity, sensitivity, matrix effects, detection limits, and reproducibility in both liquid and soil matrices. The limit of detection (LoD) of the optimized encapsulated biosensor was calculated as 0.27 mg/L and 1.26 mg/kg of Cu2+ for Cu2+ in solution and soil, respectively. Validation of the portable imaging tools as a potential biosensing device in the field was performed.

Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida.

The inclusion of biosafety strategies into strain engineering pipelines is crucial for safe-by-design biobased processes. This in turn might enable a more rapid regulatory acceptance of bioengineered organisms in both industrial and environmental applications. For this reason, we equipped the industrially relevant microbial chassis Pseudomonas putida KT2440 with an effective biocontainment strategy based on a synthetic dependency on phosphite, which is generally not readily available in the environment. The produced PSAG-9 strain was first engineered to assimilate phosphite through the genome-integration of a phosphite dehydrogenase and a phosphite-specific transport complex. Subsequently, to deter the strain from growing on naturally assimilated phosphate, all native genes related to its transport were identified and deleted generating a strain unable to grow on media containing any phosphorous source other than phosphite. PSAG-9 exhibited fitness levels with phosphite similar to those of the wild type with phosphate, and low levels of escape frequency. Beyond biosafety, this strategy endowed P. putida with the capacity to be cultured under non-sterile conditions using phosphite as the sole phosphorous source with a reduced risk of contamination by other microbes, while displaying enhanced NADH regenerative capacity. These industrially beneficial features complement the metabolic advantages for which this species is known for, thereby strengthening it as a synthetic biology chassis with potential uses in industry, with suitability towards environmental release.

A survey of biosecurity and health management practices on Irish dairy farms engaged in contract-rearing.

A survey was conducted to investigate potential differences in biosecurity and health management practices on Irish dairy farms that sent their heifers for contract-rearing (source dairy farms, SDF; n = 62) and those rearing their own heifers (control farms, CF; n = 50). Participating farmers were surveyed by postal questionnaire between September and November 2018. The overall response rate was 93%. Results show that structurally, SDF were larger, less fragmented, and more specialized than CF. Outsourcing of labor-intensive activities to external contractors was more common among SDF than CF, exposing them to potentially increased biosecurity risks associated with animal movements, use of shared equipment, and increased frequency of farm visitors. The majority of SDF sent heifers to a single-origin rearing facility (70%), with heifers most commonly arriving at the rearing unit between 2 and 4 mo (53%) and returning to the dairy farm between 18 and 21 mo of age (56%). Despite the increased biosecurity risk associated with contract-rearing, implementation of disease prevention measures was not superior on SDF compared with CF. For both farm types, there was scope for improvement to visitor biosecurity protocols, quarantine procedures, colostrum feeding practices, and hygiene of calving areas. This research provides an overview of the demographics and farm management practices implemented by dairy farmers engaged in contract-rearing of replacement heifers, and will serve to inform farmers, veterinary advisors, and policy makers.

A pilot study of core body temperatures in healthcare workers wearing personal protective equipment in a high-level isolation unit.

Personal protective equipment used by healthcare workers to mitigate disease transmission risks while caring for patients with high-consequence infectious diseases can impair normal body cooling mechanisms and exacerbate physiological strain. Symptoms of heat strain (e.g., cognitive impairment, confusion, muscle cramping) are especially harmful in the high-risk environment of high-consequence infectious disease care. In this pilot study, the core body temperatures of healthcare workers were assessed using an ingestible, wireless-transmission thermometer while performing patient care tasks common to a high-level isolation unit setting in powered air purifying respirator (PAPR)-level. The objective was to determine the potential for occupational health hazard due to heat stress in an environmentally controlled unit. Maximum core temperatures of the six participants ranged from 37.4 °C (99.3 °F) to 39.9 °C (103.8°F) during the 4-hr shift; core temperatures of half (n = 3) of the participants exceeded 38.5 °C (101.3 °F), the upper core temperature limit. Future investigations are needed to identify other heat stress risks both in and outside of controlled units. The ongoing COVID-19 pandemic offers unique opportunities for field-based research on risks of heat stress related to personal protective equipment in healthcare workers that can lead to both short- and long-term innovations in this field.

Environmental Performance of Pseudomonas putida with a Uracylated Genome.

A variant of the soil bacterium Pseudomonas putida with a genome containing a ∼20 % replacement of the whole of thymine (T) by uracil (U) was made by deleting genes ung (uracil DNA glycosylase) and dut (deoxyuridine 5'-triphosphate nucleotide hydrolase). Proteomic comparisons revealed that, of 281 up-regulated and 96 down-regulated proteins in the Δung Δdut cells, as compared to the wild-type, many were involved in nucleotide metabolism. Unexpectedly, genome uracylation did not greatly change the gross environmental endurance profile of P. putida, increased spontaneous mutagenesis by only twofold and supported expression of heterologous proteins well. As U-enriched DNA is potentially degraded by the base excision repair of recipients encoding a uracil DNA glycosylase, we then tested the spread potential of genetic material originating in the Δung Δdut cells either within the same species or in a commonly used Escherichia coli strain. Transformation and conjugation experiments revealed that horizontal gene transfer of U-containing plasmids fared worse than those made of standard DNA by two orders of magnitude. Although this figure does not guarantee the certainty of containment, it suggests a general strategy for curbing the dispersal of recombinant genetic constructs.

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas.

Edible microalgae have potential as low-cost cell factories for the production and oral delivery of recombinant proteins such as vaccines, anti-bacterials and gut-active enzymes that are beneficial to farmed animals including livestock, poultry and fish. However, a major economic and technical problem associated with large-scale cultivation of microalgae, even in closed photobioreactors, is invasion by contaminating microorganisms. Avoiding this requires costly media sterilisation, aseptic techniques during set-up and implementation of 'crop-protection' strategies during cultivation. Here, we report a strain improvement approach in which the chloroplast of Chlamydomonas reinhardtii is engineered to allow oxidation of phosphite to its bio-available form: phosphate. We have designed a synthetic version of the bacterial gene (ptxD)-encoding phosphite oxidoreductase such that it is highly expressed in the chloroplast but has a Trp→Opal codon reassignment for bio-containment of the transgene. Under mixotrophic conditions, the growth rate of the engineered alga is unaffected when phosphate is replaced with phosphite in the medium. Furthermore, under non-sterile conditions, growth of contaminating microorganisms is severely impeded in phosphite medium. This, therefore, offers the possibility of producing algal biomass under non-sterile conditions. The ptxD gene can also serve as a dominant marker for genetic engineering of any C. reinhardtii strain, thereby avoiding the use of antibiotic resistance genes as markers and allowing the 'retro-fitting' of existing engineered strains. As a proof of concept, we demonstrate the application of our ptxD technology to a strain expressing a subunit vaccine targeting a major viral pathogen of farmed fish.

Feeding of adult Ornithodoros tartakovskyi ticks using a modified artificial membrane feeding system.

Development and maintenance of laboratory tick colonies provides reliable access to a variety of tick species at multiple life stages. Advances in techniques for the membrane feeding of ticks reduce the number of laboratory animals needed for colony maintenance. In the present study, modifications to the existing protocol for in vitro feeding of the argasid species Ornithodoros tartakovskyi were made. Adult O. tartakovskyi ticks of both sexes were allowed to feed to engorgement using a novel membrane feeding apparatus in a six-well plate format with well-inserts of laboratory-grade, wax sealing film. Of the 193 ticks placed on the membrane, 89% (n = 172) fed until engorgement and subsequently detached. The modified feeding method described will aid in future laboratory tick-based research because it allows for increased containment, ease of sorting, successful in vitro feeding, easy replacement of blood meals and a reduction in the total volume of blood meal required.

Guidance for building a dedicated health facility to contain the spread of the 2019 novel coronavirus outbreak.

Preparedness for the ongoing coronavirus disease 2019 (COVID-19) and its spread in India calls for setting up of adequately equipped and dedicated health facilities to manage sick patients while protecting healthcare workers and the environment. In the wake of other emerging dangerous pathogens in recent times, such as Ebola, Nipah and Zika, it is important that such facilities are kept ready during the inter-epidemic period for training of health professionals and for managing cases of multi-drug resistant and difficult-to-treat pathogens. While endemic potential of such critically ill patients is not yet known, the health system should have surge capacity for such critical care units and preferably each tertiary government hospital should have at least one such facility. This article describes elements of design of such unit (e.g., space, infection control, waste disposal, safety of healthcare workers, partners to be involved in design and plan) which can be adapted to the context of either a new construction or makeshift construction on top of an existing structure. In view of a potential epidemic of COVID-19, specific requirements to handle it are also given.

Effect of an Intervention Package and Teamwork Training to Prevent Healthcare Personnel Self-contamination During Personal Protective Equipment Doffing.

BACKGROUND: More than 28 000 people were infected with Ebola virus during the 2014-2015 West African outbreak, resulting in more than 11 000 deaths. Better methods are needed to reduce the risk of self-contamination while doffing personal protective equipment (PPE) to prevent pathogen transmission. METHODS: A set of interventions based on previously identified failure modes was designed to mitigate the risk of self- contamination during PPE doffing. These interventions were tested in a randomized controlled trial of 48 participants with no prior experience doffing enhanced PPE. Contamination was simulated using a fluorescent tracer slurry and fluorescent polystyrene latex spheres (PLSs). Self-contamination of scrubs and skin was measured using ultraviolet light visualization and swabbing followed by microscopy, respectively. Doffing sessions were videotaped and reviewed to score standardized teamwork behaviors. RESULTS: Participants in the intervention group contaminated significantly fewer body sites than those in the control group (median [interquartile range], 6 [3-8] vs 11 [6-13], P = .002). The median contamination score was lower for the intervention group than the control group when measured by ultraviolet light visualization (23.15 vs 64.45, P = .004) and PLS swabbing (72.4 vs 144.8, P = .001). The mean teamwork score was greater in the intervention group (42.2 vs 27.5, P < .001). CONCLUSIONS: An intervention package addressing the PPE doffing task, tools, environment, and teamwork skills significantly reduced the amount of self-contamination by study participants. These elements can be incorporated into PPE guidance and training to reduce the risk of pathogen transmission.

Design Strategies for Biocontainment Units to Reduce Risk During Doffing of High-level Personal Protective Equipment.

BACKGROUND: Few data exist to guide the physical design of biocontainment units, particularly the doffing area. This can impact the contamination risk of healthcare workers (HCWs) during doffing of personal protective equipment (PPE). METHODS: In phase I of our study, we analyzed simulations of a standard patient care task with 56 trained HCWs focusing on doffing of high-level PPE. In phase II, using a rapid cycle improvement approach, we tested different balance aids and redesigned doffing area layouts with 38 students. In phase III, we tested 1 redesigned layout with an additional 10 trained HCWs. We assessed the effectiveness of design changes on improving the HCW performance (measured by occurrence and number of risky behaviors) and reducing the physical and cognitive load by comparing the results from phase I and phase III. RESULTS: The physical load was highest when participants were removing their shoe covers without any balance aid; the use of a chair required the lowest physical effort, followed by horizontal and vertical grab bars. In the revised design (phase III), the overall performance of participants improved. There was a significant decrease in the number of HCW risky behaviors (P = .004); 5 risky behaviors were eliminated and 2 others increased. There was a significant decrease in physical load when removing disposable shoe covers (P = .04), and participants reported a similar workload in the redesigned doffing layout (P = .43). CONCLUSIONS: Through optimizing the design and layout of the doffing space, we reduced risky behaviors of HCWs during doffing of high-level PPE.

Biosafety and biosecurity requirements for Orientia spp. diagnosis and research: recommendations for risk-based biocontainment, work practices and the case for reclassification to risk group 2.

Scrub typhus is an important arthropod-borne disease causing significant acute febrile illness by infection with Orientia spp.Using a risk-based approach, this review examines current practice, the evidence base and regulatory requirements regarding matters of biosafety and biosecurity, and presents the case for reclassification from Risk Group 3 to Risk Group 2 along with recommendations for safe working practices of risk-based activities during the manipulation of Orientia spp. in the laboratory.We recommend to reclassify Orientia spp. to Risk Group 2 based on the classification for RG2 pathogens as being moderate individual risk, low community risk. We recommend that low risk activities, can be performed within a biological safety cabinet located in a Biosafety Level (BSL) 2 core laboratory using standard personal protective equipment. But when the risk assessment indicates, such as high concentration and volume, or aerosol generation, then a higher biocontainment level is warranted. For, the majority of animal activities involving Orientia spp., Animal BSL 2 (ABSL2) is recommended however where high risk activities are performed including necropsies, Animal BSL (ABSL3) is recommended.

Impact of air-handling system exhaust failure on dissemination pattern of simulant pathogen particles in a clinical biocontainment unit.

Biocontainment units (BCUs) are facilities used to care for patients with highly infectious diseases. However, there is limited guidance on BCU protocols and design. This study presents the first investigation of how HVAC (heating, ventilation, air-conditioning) operating conditions influence the dissemination of fluorescent tracer particles released in a BCU. Test conditions included normal HVAC operation and exhaust failure resulting in loss of negative pressure. A suspension of optical brightener powder and water was nebulized to produce fluorescent particles simulating droplet nuclei (0.5-5 µm). Airborne particle number concentrations were monitored by Instantaneous Biological Analyzers and Collectors (FLIR Systems). During normal HVAC operation, fluorescent tracer particles were contained in the isolation room (average concentration = 1 × 104 ± 3 × 103 /Lair ). Under exhaust failure, the automated HVAC system maximizes airflow into areas adjacent to isolation rooms to attempt to maintain negative pressure differential. However, 6% of the fluorescent particles were transported through cracks around doors/door handles out of the isolation room via airflow alone and not by movement of personnel or doors. Overall, this study provides a systematic method for evaluating capabilities to contain aerosolized particles during various HVAC scenarios. Recommendations are provided to improve situation-specific BCU safety.

Caring for patients with Ebola virus disease: Are U.S. biocontainment centers ready for the next outbreak?

The 2014 West African Ebola virus disease (EVD) outbreak is the largest and deadliest EVD epidemic to date, resulting in fivefold more cases than all other outbreaks combined. This outbreak was particularly devastating to healthcare workers in West Africa and resulted in several EVD patients being medically evacuated for treatment in the U.S. and Europe. Governmental agencies provide recommendations for triaging and testing patients with EVD, however best laboratory practices are still unknown and are very resource dependent.

U.S. High-Level Isolation Unit Clinical Laboratory Capabilities Update.

In late 2014, 56 hospitals in the United States were designated by state and federal public health authorities as specially designed high-level isolation units (HLIUs) equipped with advanced infrastructure, laboratory capabilities, and trained staff to care for patients with highly hazardous communicable diseases (HHCDs), such as Ebola virus disease. This survey describes the clinical laboratory support capabilities of U.S. HLIUs, including the specific test menus that HLIUs have identified to safely manage HHCD patients and the locations where such testing would be performed. In spring 2016, a survey was electronically distributed, as a fillable pdf file, to the 56 U.S. HLIUs. Site representatives completed the surveys, and data were coded and analyzed in an electronic spreadsheet, using descriptive statistics. Thirty-six HLIUs (64%) responded, and 33 completed the laboratory capabilities section. Thirty-one HLIUs (94%) had performed risk analyses for all laboratory procedures and equipment. Twenty-nine (88%) had decontamination procedures specified for all laboratory equipment used for patients with suspected or confirmed HHCDs. On-site laboratories in 27 HLIUs (81%) had the capacity to inventory and to securely store HHCD patient specimens. Ten HLIUs (31%) had at least one test they would conduct within the patient isolation room. The high-risk nature of HHCDs and the occupational exposures that may occur in clinical laboratories demand advanced preparation and risk assessment of work practices, laboratory equipment, and instrumentation by HLIU laboratories. Although risk analyses of clinical laboratory testing and equipment that HLIUs have conducted have likely focused on those for Ebola virus, HLIUs must be prepared to revise their current procedures for other HHCDs.

Building an Intelligent Hospital to Fight Contagion.

The idea of building hospitals to fight contagion was born with the lazarettos. At the time when the microorganisms were not yet known, the mechanisms of transmission of contagion were already well apprehended. Based on the same knowledge but thanks to new technologies, such hospitals have now been built downtown, next to the most highly performing technological plateau. Regrouping patient care, diagnostics, research, and development, the University Hospital Institute Méditerranée Infection building offers a wonderful tool to contain and understand contagion, in a well-designed setting, creating excellent working conditions that are attractive for interested scientists.

Synthetic biology and occupational risk.

Synthetic biology is an emerging interdisciplinary field of biotechnology that involves applying the principles of engineering and chemical design to biological systems. Biosafety professionals have done an excellent job in addressing research laboratory safety as synthetic biology and gene editing have emerged from the larger field of biotechnology. Despite these efforts, risks posed by synthetic biology are of increasing concern as research procedures scale up to industrial processes in the larger bioeconomy. A greater number and variety of workers will be exposed to commercial synthetic biology risks in the future, including risks to a variety of workers from the use of lentiviral vectors as gene transfer devices. There is a need to review and enhance current protection measures in the field of synthetic biology, whether in experimental laboratories where new advances are being researched, in health care settings where treatments using viral vectors as gene delivery systems are increasingly being used, or in the industrial bioeconomy. Enhanced worker protection measures should include increased injury and illness surveillance of the synthetic biology workforce; proactive risk assessment and management of synthetic biology products; research on the relative effectiveness of extrinsic and intrinsic biocontainment methods; specific safety guidance for synthetic biology industrial processes; determination of appropriate medical mitigation measures for lentiviral vector exposure incidents; and greater awareness and involvement in synthetic biology safety by the general occupational safety and health community as well as by government occupational safety and health research and regulatory agencies.

Containment Care Units for Managing Patients With Highly Hazardous Infectious Diseases: A Concept Whose Time Has Come.

The concept of containment care for patients with highly hazardous infectious diseases originated in conjunction with the development of sophisticated biosafety level 4 laboratories at the US Army Medical Research Institute of Infectious Diseases in the late 1960s. Over time, the original containment facility served as a model for the development of other facilities in the United States at government and academic centers. The Ebola outbreak of 2014-2015 brought the issue of containment care into the mainstream and led to the development of such capabilities at strategic points around the country. We describe the original concepts behind development of such facilities, how the concept and acceptance has evolved over time, and how the guidelines for managing patients infected with viral hemorrhagic fevers have evolved as new information has been learned about protecting medical care providers from highly hazardous infectious pathogens.

Synthetic biology: from mainstream to counterculture.

Existing at the interface of science and engineering, synthetic biology represents a new and emerging field of mainstream biology. However, there also exists a counterculture of Do-It-Yourself biologists, citizen scientists, who have made significant inroads, particularly in the design and development of new tools and techniques. Herein, I review the development and convergence of synthetic biology's mainstream and countercultures.

Successful delivery of RRT in Ebola virus disease.

AKI has been observed in cases of Ebola virus disease. We describe the protocol for the first known successful delivery of RRT with subsequent renal recovery in a patient with Ebola virus disease treated at Emory University Hospital, in Atlanta, Georgia. Providing RRT in Ebola virus disease is complex and requires meticulous attention to safety for the patient, healthcare workers, and the community. We specifically describe measures to decrease the risk of transmission of Ebola virus disease and report pilot data demonstrating no detectable Ebola virus genetic material in the spent RRT effluent waste. This article also proposes clinical practice guidelines for acute RRT in Ebola virus disease.

Biocontainment through reengineered genetic codes.

It's only unnatural: Reengineered genetic codes can achieve biocontainment by specifying different meanings for protein sequences in modified versus natural organisms; the synthetic bacterium discussed here is dependent on unnatural amino acids because its reengineered genetic code specifies unnatural amino acids in essential genes.