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Bioregenerative food systems that routinely produce fresh, safe-to-eat crops onboard spacecraft can supplement the nutrition and variety of shelf-stable spaceflight food systems for use during future exploration missions (i.e., low earth orbit, Mars transit, lunar, and Martian habitats). However, current space crop production systems are not yet sustainable because they primarily utilize consumable granular media and, to date, operate like single crop cycle, space biology experiments where root modules are sanitized prior to launch and discarded after each grow-out. Moreover, real-time detection of the cleanliness of crops produced in spacecraft is not possible. A significant paradigm shift is needed in the design of future space crop production systems, as they transition from operating as single grow-out space biology experiments to becoming sustainable over multiple cropping cycles. Soilless nutrient delivery systems have been used to demonstrate post-harvest sanitization and inflight microbial monitoring technologies to enable sequential cropping cycles in spacecraft. Post-harvest cleaning and sanitization prevent the buildup of biofilms and ensure a favorable environment for seedling establishment of the next crop. Inflight microbial monitoring of food and watering systems ensures food safety in spaceflight food systems. A sanitization protocol, heat sterilization at 60°C for 1 h, and soaking for 12 h in 1% hydrogen peroxide, developed in this study, was compared against a standard hydroponic sanitization protocol during five consecutive crop cycles. Each cropping cycle included protocols for the cultivation of a crop to maturity, followed by post-harvest cleaning and inflight microbial monitoring. Microbial sampling of nutrient solution reservoirs, root modules, and plants demonstrated that the sanitization protocol could be used to grow safe-to-eat produce during multiple crop cycles. The cleanliness of the reservoir and root module surfaces measured with aerobic plate counts was verified in near real time using a qPCR-based inflight microbial monitoring protocol. Post-harvest sanitization and inflight microbial monitoring are expected to significantly transform the design of sustainable bioregenerative food and life support systems for future exploration missions beyond low earth orbit (LEO). [ABSTRACT FROM AUTHOR]

Bioregenerative food systems that routinely produce fresh, safe-to-eat crops onboard spacecraft can supplement the nutrition and variety of shelf-stable spaceflight food systems for use during future exploration missions (i.e., low earth orbit, Mars transit, lunar, and Martian habitats). However, current space crop production systems are not yet sustainable because they primarily utilize consumable granular media and, to date, operate like single crop cycle, space biology experiments where root modules are sanitized prior to launch and discarded after each grow-out. Moreover, real-time detection of the cleanliness of crops produced in spacecraft is not possible. A significant paradigm shift is needed in the design of future space crop production systems, as they transition from operating as single grow-out space biology experiments to becoming sustainable over multiple cropping cycles. Soilless nutrient delivery systems have been used to demonstrate post-harvest sanitization and inflight microbial monitoring technologies to enable sequential cropping cycles in spacecraft. Post-harvest cleaning and sanitization prevent the buildup of biofilms and ensure a favorable environment for seedling establishment of the next crop. Inflight microbial monitoring of food and watering systems ensures food safety in spaceflight food systems. A sanitization protocol, heat sterilization at 60°C for 1 h, and soaking for 12 h in 1% hydrogen peroxide, developed in this study, was compared against a standard hydroponic sanitization protocol during five consecutive crop cycles. Each cropping cycle included protocols for the cultivation of a crop to maturity, followed by post-harvest cleaning and inflight microbial monitoring. Microbial sampling of nutrient solution reservoirs, root modules, and plants demonstrated that the sanitization protocol could be used to grow safe-to-eat produce during multiple crop cycles. The cleanliness of the reservoir and root module surfaces measured with aerobic plate counts was verified in near real time using a qPCR-based inflight microbial monitoring protocol. Post-harvest sanitization and inflight microbial monitoring are expected to significantly transform the design of sustainable bioregenerative food and life support systems for future exploration missions beyond low earth orbit (LEO).

A protocol for responding to Legionella pneumophila detections in distribution system samples is presented and justified. The protocol was developed using existing protocols for building water systems, vetted in workshops, and provided to utilities participating in a research project. The protocol provides action levels and actions that utilities can take when L. pneumophila is detected in distribution system samples. Action levels are based on the best available science and expert judgment and are similar to those in protocols for assessing building water system monitoring results. Action levels should be reassessed as additional data and knowledge on the occurrence and growth potential of L. pneumophila in distribution systems become available. All positive detections trigger assessments that focus first on conditions local to the sample location and then extend further into the distribution system. Higher concentration or more frequent detections initiate more assertive responses and communication than lower concentration and frequency detections. The protocol provides a starting point for the development of protocols for purposes such as L. pneumophila operational surveillance or within an outbreak investigation. [ABSTRACT FROM AUTHOR]

Landfill sites are subjected to long-term risks of accidental spill of leachate through the soil and consequential contamination of the groundwater. Wide areas surrounding the landfill can seriously be threatened with possible consequences to human health and the environment. Given the potential impact of different coexisting anthropic pollution sources (i.e., agriculture and cattle farming) on the same site, the perturbation of the groundwater quality may be due to multiple factors. Therefore, it is a challenging issue to correctly establish the pollution source of an aquifer where the landfill is not isolated from other anthropic land uses, especially in the case of a karstic coastal aquifer. The present study is aimed at setting in place an integrated environmental monitoring system that included microbiological, chemical, and isotope methods to evaluate potential groundwater pollution in a landfill district in the south of Italy located in Murgia karstic aquifer. Conventional (microbial plate count and physical–chemical analyses) and advanced methods (PCR-ARISA, isotope analysis of δ18O, δ2H, 3H, δ 13C, δ 15N-NO3−, and δ 18O-NO3−) were included in the study. Through data integration, it was possible to reconstruct a scenario in which agriculture and other human activities along with seawater intrusion in the karst aquifer were the main drivers of groundwater pollution at the monitored site. The microbiological, chemical, and isotope results confirmed the absence of leachate effects on groundwater quality, showing the decisive role of fertilizers as potential nitrate sources. The next goal will be to extend long-term integrated monitoring to other landfill districts, with different geological and hydrogeological characteristics and including different sources of pollution, to support the ecological restoration of landfills. [ABSTRACT FROM AUTHOR]

Abstract Marine bacterioplankton underpin the health and function of coral reefs and respond in a rapid and sensitive manner to environmental changes that affect reef ecosystem stability. Numerous meta-omics surveys over recent years have documented persistent associations of opportunistic seawater microbial taxa, and their associated functions, with metrics of environmental stress and poor reef health (e.g. elevated temperature, nutrient loads and macroalgae cover). Through positive feedback mechanisms, disturbance-triggered heterotrophic activity of seawater microbes is hypothesised to drive keystone benthic organisms towards the limit of their resilience and translate into shifts in biogeochemical cycles which influence marine food webs, ultimately affecting entire reef ecosystems. However, despite nearly two decades of work in this space, a major limitation to using seawater microbes in reef monitoring is a lack of a unified and focused approach that would move beyond the indicator discovery phase and towards the development of rapid microbial indicator assays for (near) real-time reef management and decision-making. By reviewing the current state of knowledge, we provide a comprehensive framework (defined as five phases of research and innovation) to catalyse a shift from fundamental to applied research, allowing us to move from descriptive to predictive reef monitoring, and from reactive to proactive reef management.

Marine bacterioplankton underpin the health and function of coral reefs and respond in a rapid and sensitive manner to environmental changes that affect reef ecosystem stability. Numerous meta-omics surveys over recent years have documented persistent associations of opportunistic seawater microbial taxa, and their associated functions, with metrics of environmental stress and poor reef health (e.g. elevated temperature, nutrient loads and macroalgae cover). Through positive feedback mechanisms, disturbance-triggered heterotrophic activity of seawater microbes is hypothesised to drive keystone benthic organisms towards the limit of their resilience and translate into shifts in biogeochemical cycles which influence marine food webs, ultimately affecting entire reef ecosystems. However, despite nearly two decades of work in this space, a major limitation to using seawater microbes in reef monitoring is a lack of a unified and focused approach that would move beyond the indicator discovery phase and towards the development of rapid microbial indicator assays for (near) real-time reef management and decision-making. By reviewing the current state of knowledge, we provide a comprehensive framework (defined as five phases of research and innovation) to catalyse a shift from fundamental to applied research, allowing us to move from descriptive to predictive reef monitoring, and from reactive to proactive reef management. [ABSTRACT FROM AUTHOR]

The pressing need to safeguard the health of astronauts aboard the International Space Station (ISS) necessitates constant and rigorous microbial monitoring. Recognizing the shortcomings of traditional culture-based methods, NASA is deliberating the incorporation of molecular-based techniques. The challenge, however, lies in developing and validating effective methods for concentrating samples to facilitate this transition. This study is dedicated to investigating the potential of an ISS Smart Sample Concentrator (iSSC) as an innovative concentration method. First, the iSSC system and its components were tested and optimized for microgravity, including various testing environments: a drop tower, parabolic flight, and the ISS itself. Upon confirming the system's compatibility with microgravity, we further evaluated its proficiency and reliability in concentrating large volumes (i.e., 1 L) of water samples inoculated with different microbes. The samples carried 102 to 105 colony-forming units (CFUs) of Sphingomonas paucimobilis, Ralstonia pickettii, or Cupriavidus basilensis per liter, aligning with NASA's acceptable limit of 5 × 104 CFU/L. The average retrieved volume post-concentration was ≈450 µL, yielding samples that were ≈2200 times more concentrated for subsequent quantitative PCR (qPCR) and CFU analysis. The average microbial percent recovery, as assessed with CFU counts, demonstrated consistency for C. basilensis and R. pickettii at around 50% and 45%, respectively. For S. paucimobilis, the efficiency oscillated between 40% and 80%. Interestingly, when we examined microbial recovery using qPCR, the results showed more variability across all tested species. The significance of these findings lies not merely in the successful validation of the iSSC but also in the system's proven consistency, as evidenced by its alignment with previous validation-phase results. In conclusion, conducted research underscored the potential of the iSSC in monitoring microbial contamination in potable water aboard the ISS, heralding a paradigm shift from culture-based to molecular-based monitoring methods. [ABSTRACT FROM AUTHOR]

Abstract Background The International Space Station (ISS) is a unique and complex built environment with the ISS surface microbiome originating from crew and cargo or from life support recirculation in an almost entirely closed system. The Microbial Tracking 1 (MT-1) project was the first ISS environmental surface study to report on the metagenome profiles without using whole-genome amplification. The study surveyed the microbial communities from eight surfaces over a 14-month period. The Microbial Tracking 2 (MT-2) project aimed to continue the work of MT-1, sampling an additional four flights from the same locations, over another 14 months. Methods Eight surfaces across the ISS were sampled with sterile wipes and processed upon return to Earth. DNA extracted from the processed samples (and controls) were treated with propidium monoazide (PMA) to detect intact/viable cells or left untreated and to detect the total DNA population (free DNA/compromised cells/intact cells/viable cells). DNA extracted from PMA-treated and untreated samples were analyzed using shotgun metagenomics. Samples were cultured for bacteria and fungi to supplement the above results. Results Staphylococcus sp. and Malassezia sp. were the most represented bacterial and fungal species, respectively, on the ISS. Overall, the ISS surface microbiome was dominated by organisms associated with the human skin. Multi-dimensional scaling and differential abundance analysis showed significant temporal changes in the microbial population but no spatial differences. The ISS antimicrobial resistance gene profiles were however more stable over time, with no differences over the 5-year span of the MT-1 and MT-2 studies. Twenty-nine antimicrobial resistance genes were detected across all samples, with macrolide/lincosamide/streptogramin resistance being the most widespread. Metagenomic assembled genomes were reconstructed from the dataset, resulting in 82 MAGs. Functional assessment of the collective MAGs showed a propensity for amino acid utilization over carbohydrate metabolism. Co-occurrence analyses showed strong associations between bacterial and fungal genera. Culture analysis showed the microbial load to be on average 3.0 × 105 cfu/m2 Conclusions Utilizing various metagenomics analyses and culture methods, we provided a comprehensive analysis of the ISS surface microbiome, showing microbial burden, bacterial and fungal species prevalence, changes in the microbiome, and resistome over time and space, as well as the functional capabilities and microbial interactions of this unique built microbiome. Data from this study may help to inform policies for future space missions to ensure an ISS surface microbiome that promotes astronaut health and spacecraft integrity. Video Abstract

In order to intensify and guarantee the agricultural productivity and thereby to be able to feed the world's rapidly growing population, irrigation has become very important. In parallel, the limited water resources lead to an increase in usage of poorly characterized sources of water, which is directly linked to a higher prevalence of foodborne diseases. Therefore, analyzing the microorganisms or even the complete microbiome of irrigation water used for food production can prevent the growing numbers of such cases. In this study, we compared the efficacy of MALDI-TOF Mass spectrometry (MALDI TOF MS) identification to 16S rRNA gene Sanger sequencing of waterborne microorganisms. Furthermore, we analyzed the whole microbial community of irrigation water using high-throughput 16S rRNA gene amplicon sequencing. The identification results of MALDI-TOF MS and 16S rRNA gene Sanger sequencing were almost identical at species level (66.7%; 64.3%). Based on the applied cultivation techniques, Acinetobacter spp., Enterobacter spp., Pseudomonas spp., and Brevundimonas spp. were the most abundant cultivable genera. In addition, the uncultivable part of the microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidota, Patescibacteria, and Verrucomicrobiota. Our findings indicate that MALDI-TOF MS offers a fast, reliable identification method and can act as an alternative to 16S rRNA gene Sanger sequencing of isolates. Moreover, the results suggest that MALDI-TOF MS paired with 16S rRNA gene amplicon sequencing have the potential to support the routine monitoring of the microbiological quality of irrigation water. [ABSTRACT FROM AUTHOR]

The pressing need to safeguard the health of astronauts aboard the International Space Station (ISS) necessitates constant and rigorous microbial monitoring. Recognizing the shortcomings of traditional culture-based methods, NASA is deliberating the incorporation of molecular-based techniques. The challenge, however, lies in developing and validating effective methods for concentrating samples to facilitate this transition. This study is dedicated to investigating the potential of an ISS Smart Sample Concentrator (iSSC) as an innovative concentration method. First, the iSSC system and its components were tested and optimized for microgravity, including various testing environments: a drop tower, parabolic flight, and the ISS itself. Upon confirming the system’s compatibility with microgravity, we further evaluated its proficiency and reliability in concentrating large volumes (i.e., 1 L) of water samples inoculated with different microbes. The samples carried 102 to 105 colony-forming units (CFUs) of Sphingomonas paucimobilis, Ralstonia pickettii, or Cupriavidus basilensis per liter, aligning with NASA’s acceptable limit of 5 × 104 CFU/L. The average retrieved volume post-concentration was ≈450 µL, yielding samples that were ≈2200 times more concentrated for subsequent quantitative PCR (qPCR) and CFU analysis. The average microbial percent recovery, as assessed with CFU counts, demonstrated consistency for C. basilensis and R. pickettii at around 50% and 45%, respectively. For S. paucimobilis, the efficiency oscillated between 40% and 80%. Interestingly, when we examined microbial recovery using qPCR, the results showed more variability across all tested species. The significance of these findings lies not merely in the successful validation of the iSSC but also in the system’s proven consistency, as evidenced by its alignment with previous validation-phase results. In conclusion, conducted research underscored the potential of the iSSC in monitoring microbial contamination in potable water aboard the ISS, heralding a paradigm shift from culture-based to molecular-based monitoring methods.

Background: The International Space Station (ISS) is a unique and complex built environment with the ISS surface microbiome originating from crew and cargo or from life support recirculation in an almost entirely closed system. The Microbial Tracking 1 (MT-1) project was the first ISS environmental surface study to report on the metagenome profiles without using whole-genome amplification. The study surveyed the microbial communities from eight surfaces over a 14-month period. The Microbial Tracking 2 (MT-2) project aimed to continue the work of MT-1, sampling an additional four flights from the same locations, over another 14 months. Methods: Eight surfaces across the ISS were sampled with sterile wipes and processed upon return to Earth. DNA extracted from the processed samples (and controls) were treated with propidium monoazide (PMA) to detect intact/viable cells or left untreated and to detect the total DNA population (free DNA/compromised cells/intact cells/viable cells). DNA extracted from PMA-treated and untreated samples were analyzed using shotgun metagenomics. Samples were cultured for bacteria and fungi to supplement the above results. Results: Staphylococcus sp. and Malassezia sp. were the most represented bacterial and fungal species, respectively, on the ISS. Overall, the ISS surface microbiome was dominated by organisms associated with the human skin. Multi-dimensional scaling and differential abundance analysis showed significant temporal changes in the microbial population but no spatial differences. The ISS antimicrobial resistance gene profiles were however more stable over time, with no differences over the 5-year span of the MT-1 and MT-2 studies. Twenty-nine antimicrobial resistance genes were detected across all samples, with macrolide/lincosamide/streptogramin resistance being the most widespread. Metagenomic assembled genomes were reconstructed from the dataset, resulting in 82 MAGs. Functional assessment of the collective MAGs showed a propensity for amino acid utilization over carbohydrate metabolism. Co-occurrence analyses showed strong associations between bacterial and fungal genera. Culture analysis showed the microbial load to be on average 3.0 × 105 cfu/m2 Conclusions: Utilizing various metagenomics analyses and culture methods, we provided a comprehensive analysis of the ISS surface microbiome, showing microbial burden, bacterial and fungal species prevalence, changes in the microbiome, and resistome over time and space, as well as the functional capabilities and microbial interactions of this unique built microbiome. Data from this study may help to inform policies for future space missions to ensure an ISS surface microbiome that promotes astronaut health and spacecraft integrity. 4WcFw6bAGHFuUU3jmVVd3X Video Abstract [ABSTRACT FROM AUTHOR]

In order to intensify and guarantee the agricultural productivity and thereby to be able to feed the world’s rapidly growing population, irrigation has become very important. In parallel, the limited water resources lead to an increase in usage of poorly characterized sources of water, which is directly linked to a higher prevalence of foodborne diseases. Therefore, analyzing the microorganisms or even the complete microbiome of irrigation water used for food production can prevent the growing numbers of such cases. In this study, we compared the efficacy of MALDI-TOF Mass spectrometry (MALDI TOF MS) identification to 16S rRNA gene Sanger sequencing of waterborne microorganisms. Furthermore, we analyzed the whole microbial community of irrigation water using high-throughput 16S rRNA gene amplicon sequencing. The identification results of MALDI-TOF MS and 16S rRNA gene Sanger sequencing were almost identical at species level (66.7%; 64.3%). Based on the applied cultivation techniques, Acinetobacter spp., Enterobacter spp., Pseudomonas spp., and Brevundimonas spp. were the most abundant cultivable genera. In addition, the uncultivable part of the microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidota, Patescibacteria, and Verrucomicrobiota. Our findings indicate that MALDI-TOF MS offers a fast, reliable identification method and can act as an alternative to 16S rRNA gene Sanger sequencing of isolates. Moreover, the results suggest that MALDI-TOF MS paired with 16S rRNA gene amplicon sequencing have the potential to support the routine monitoring of the microbiological quality of irrigation water.

Background and objectives: The transmission of infection from the equipment and the environment to patients is widely carried out through contact. The aim of this study was to determine the health status of medical equipment and pseudo-homogeneous environmental levels in hospitals in Ardebil, in observational and microbiological hospitals in year 1396. Methods: in this descriptive cross-sectional study, 17 levels of hospitalization in Imam Khomeini Hospital (RA) were evaluated in a two month observational stage (by completing the checklist) and microbial. Data analysis was done by using SPSS software and by comparing it with British standards. Results: This study showed that the rate of contamination in different parts of the hospital was microbial 75.75% and 29.15%, respectively. According to statistical analyzes in the microbial method, the most infected ventilators were among the most polluted medical equipment and among the peripheral levels of the most infectious patient ward. At the observation station, among the medical equipment levels, the cliché was the most contaminated case, and among the pseudo-environmental environmental levels, the most infected fridge was determined. The most contaminated part in terms of cardiothoracic microbial monitoring was determined in terms of male in-patient observation. Conclusion: Therefore, it is suggested that by systematically planning and applying appropriate and preventive methods, implementing disinfection programs, developing guidelines for cleaning and disinfection, training and continuous monitoring of the standard methods of contamination will be minimized.

Abstract Background Coral reefs are facing unprecedented pressure on local and global scales. Sensitive and rapid markers for ecosystem stress are urgently needed to underpin effective management and restoration strategies. Although the fundamental contribution of microbes to the stability and functioning of coral reefs is widely recognised, it remains unclear how different reef microbiomes respond to environmental perturbations and whether microbiomes are sensitive enough to predict environmental anomalies that can lead to ecosystem stress. However, the lack of coral reef microbial baselines hinders our ability to study the link between shifts in microbiomes and ecosystem stress. In this study, we established a comprehensive microbial reference database for selected Great Barrier Reef sites to assess the diagnostic value of multiple free-living and host-associated reef microbiomes to infer the environmental state of coral reef ecosystems. Results A comprehensive microbial reference database, originating from multiple coral reef microbiomes (i.e. seawater, sediment, corals, sponges and macroalgae), was generated by 16S rRNA gene sequencing for 381 samples collected over the course of 16 months. By coupling this database to environmental parameters, we showed that the seawater microbiome has the greatest diagnostic value to infer shifts in the surrounding reef environment. In fact, 56% of the observed compositional variation in the microbiome was explained by environmental parameters, and temporal successions in the seawater microbiome were characterised by uniform community assembly patterns. Host-associated microbiomes, in contrast, were five-times less responsive to the environment and their community assembly patterns were generally less uniform. By applying a suite of indicator value and machine learning approaches, we further showed that seawater microbial community data provide an accurate prediction of temperature and eutrophication state (i.e. chlorophyll concentration and turbidity). Conclusion Our results reveal that free-living microbial communities have a high potential to infer environmental parameters due to their environmental sensitivity and predictability. This highlights the diagnostic value of microorganisms and illustrates how long-term coral reef monitoring initiatives could be enhanced by incorporating assessments of microbial communities in seawater. We therefore recommend timely integration of microbial sampling into current coral reef monitoring initiatives.

As the use of hydrogen gas (H 2) as a renewable energy carrier experiences a major boost, one of the key challenges for a constant supply is safe and cost-efficient storage of surplus H 2 to bridge periods with low energy demand. For this purpose, underground gas storage (UGS) in salt caverns, deep aquifers and oil-/gas reservoirs has been proposed, which are environments with potentially high microbial abundance and activity. Subsurface microorganisms can use H 2 in their metabolism and thus may lead to a variety of undesired side effects such as H 2 loss, H 2 S formation, methane formation, acid formation, clogging and corrosion. In this review, we summarize the current knowledge and methodologies on microbial related risks related to H 2 UGS, including basic research on subsurface microbiology, results from field trials, and experience from other industries including oil & gas. For safe underground storage of H 2 gas we recommend to analyze the relevant microbiological and geochemical characteristics of each site to be able to predict the most suitable storage strategy and establish a good monitoring and mitigation approach to follow and counter potential microbial side effects. • Microorganisms can be present in all locations intended for H 2 underground storage. • Microbial-associated risks include H 2 loss, souring, corrosion and clogging. • Microbial activity is field specific and should be assessed prior to implementation. • More experience from field-specific tests is needed to predict microbial influence. [ABSTRACT FROM AUTHOR]

NASA has made great strides in the past five years to develop a suite of instruments for the International Space Station in order to perform molecular biology in space. However, a key piece of equipment that has been lacking is an instrument that can extract nucleic acids from an array of complex human and environmental samples. The Omics in Space team has developed the μTitan (simulated micro(μ) gravity tested instrument for automated nucleic acid) system capable of automated, streamlined, nucleic acid extraction that is adapted for use under microgravity. The μTitan system was validated using a whole cell microbial reference (WCMR) standard comprised of a suspension of nine bacterial strains, titrated to concentrations that would challenge the performance of the instrument, as well as to determine the detection limits for isolating DNA. Quantitative assessment of system performance was measured by comparing instrument input challenge dose vs recovery by Qubit spectrofluorometry, qPCR, Bioanalyzer, and Next Generation Sequencing. Overall, results indicate that the μTitan system performs equal to or greater than a similar commercially available, earth-based, automated nucleic acid extraction device. The μTitan system was also tested in Yellowstone National Park (YNP) with the WCMR, to mimic a remote setting, with limited resources. The performance of the device at YNP was comparable to that in a laboratory setting. Such a portable, field-deployable, nucleic extraction system will be valuable for environmental microbiology, as well as in health care diagnostics.

Exposure to microbial contamination through drinking water is a major global health concern. Effective management of microbial drinking water quality requires rapid detection equipment. Currently, microbial quality is monitored using time‐consuming laboratory methods, which delay any response. This study demonstrates the development of an automated and high‐throughput method for the measurement of viable biomass in water through the quantification of cellular adenosine triphosphate (ATP). The developed method was able to efficiently and accurately quantify cellular ATP in multiple water samples simultaneously. In addition, it proved to be 5× faster and as accurate as the Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water (ASTM D4012). The developed method has the potential to represent a significant advancement for microbial monitoring and could benefit utilities interested in measuring viable biomass in water to monitor the health of biofilters and the effectiveness of disinfection strategies. [ABSTRACT FROM AUTHOR]

NASA has made great strides in the past five years to develop a suite of instruments for the International Space Station in order to perform molecular biology in space. However, a key piece of equipment that has been lacking is an instrument that can extract nucleic acids from an array of complex human and environmental samples. The Omics in Space team has developed the μTitan (simulated micro(μ) gravity t ested i ns t rument for a utomated n ucleic acid) system capable of automated, streamlined, nucleic acid extraction that is adapted for use under microgravity. The μTitan system was validated using a whole cell microbial reference (WCMR) standard comprised of a suspension of nine bacterial strains, titrated to concentrations that would challenge the performance of the instrument, as well as to determine the detection limits for isolating DNA. Quantitative assessment of system performance was measured by comparing instrument input challenge dose vs recovery by Qubit spectrofluorometry, qPCR, Bioanalyzer, and Next Generation Sequencing. Overall, results indicate that the μTitan system performs equal to or greater than a similar commercially available, earth-based, automated nucleic acid extraction device. The μTitan system was also tested in Yellowstone National Park (YNP) with the WCMR, to mimic a remote setting, with limited resources. The performance of the device at YNP was comparable to that in a laboratory setting. Such a portable, field-deployable, nucleic extraction system will be valuable for environmental microbiology, as well as in health care diagnostics. [ABSTRACT FROM AUTHOR]

Closed environments such as the International Space Station (ISS) and spacecraft for other planned interplanetary destinations require sustainable environmental control systems for manned spaceflight and habitation. These systems require monitoring for microbial contaminants and potential pathogens that could foul equipment or affect the health of the crew. Technological advances may help to facilitate this environmental monitoring, but many of the current advances do not function as expected in reduced gravity conditions. The microbial monitoring system (RAZOR® EX) is a compact, semi-quantitative rugged PCR instrument that was successfully tested on the ISS using station potable water. After a series of technical demonstrations between ISS and ground laboratories, it was determined that the instruments functioned comparably and provided a sample to answer flow in approximately 1 hour without enrichment or sample manipulation. Post-flight, additional advancements were accomplished at Kennedy Space Center, Merritt Island, FL, USA, to expand the instrument’s detections of targeted microorganisms of concern such as water, food-borne, and surface microbes including Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Escherichia coli, and Aeromonas hydrophilia. Early detection of contaminants and bio-fouling microbes will increase crew safety and the ability to make appropriate operational decisions to minimize exposure to these contaminants.

The Black Sea is acknowledged to be a unique aquatic ecosystem, characterized by complex aquaculture - environment interactions, whereby a tailored approach of this field is absolutely necessary. Black Sea aquaculture, although not as developed as in the Mediterranean, is exposed to the spread of aquatic diseases and pathologies that can cause heavy losses to farmers, as well as generate public health problems. The 10th session of the Scientific Advisory Committee on Aquaculture (CAQ) of the General Fisheries Commission for the Mediterranean (GFCM) (Turkey, March 2017) recognized that environmental protection is a major concern and stressed the importance of tackling all environmental aspects related to aquaculture at sea, including the use of risk analysis and data recording to assess aquaculture - environment interactions, as well as the vulnerability of ecosystems and the ecological services they provide. Furthermore, the GFCM adopted, at its 41st Session (Montenegro, October 2017), a strategy for the sustainable development of Mediterranean and Black Sea aquaculture (Resolution GFCM/41/2017/1). Target 2 of this strategy aims in particular at enhancing interactions between aquaculture and the environment, while ensuring animal health and welfare in order to minimize potential negative externalities, ensure adequate and responsible aquaculture management in the region and guarantee food safety and food quality. In this context, in the frame of NIMRD's Shellfish Aquaculture Demonstrative Center (S-ADC), a dedicated training course was organized during 6-10 May 2019, focusing on preventing and controlling mussel pathologies by acquiring knowledge of bacterial contamination, as well as early diagnosis. The course included both theoretical and practical modules (sampling, laboratory analyses, field visits). Also, complex issues were addressed, such as legislative and institutional gaps in the classification of mollusks from the veterinary point of view for consumption and Hazard Analysis and Critical Control Points (HACCP). The trainees represented institutions from Bulgaria, Georgia, Turkey, Russia, Ukraine and Romania, and the lecturers were both NIMRD and foreign experts in shellfish pathology. [ABSTRACT FROM AUTHOR]

Aims: We constantly interact with our surrounding microbiome, including the micro‐organisms present in highly populated public places. However, data on everyday exposure to background levels of micro‐organisms are limited. To address this, bacteria and fungi were collected and enumerated in settled dust from railway stations. Methods and Results: Samples were collected weekly for 52 weeks, from up to three pre‐determined surfaces in each of 17 railway stations in England and Scotland. Trained staff at each station took surface wipes, sending them to the laboratory for culture‐based analysis for total bacteria and fungi. Maximum yields of bacteria at the stations were 107–108 colony forming units (CFU) per cm2, and 104–105CFU per cm2 for fungi. Conclusions: There was evidence of seasonal trends, with bacterial numbers rising from spring through to winter, while fungal numbers peaked in autumn. Microbial numbers were similar in samples taken at the same time at a given station. Influences on contamination levels were likely to be a combination of passenger numbers and station layout, with dust generated from construction work also contributing. Significance and Impact of the Study: A baseline of typical human exposure to micro‐organisms in public transport hubs was established through the generation of a comprehensive database. [ABSTRACT FROM AUTHOR]

Short-term fluctuations in bacterial concentrations in drinking water systems, occurring on time scales of hours-to-weeks, are essentially unexplored due to a lack of microbial monitoring tools that allow high frequency measurements. Here, we applied fully automated online flow cytometry to measure the total cell concentrations (TCC) in both raw water (karstic groundwater) and treated water (flocculation – ultrafiltration (UF) – ozonation – granular active carbon (GAC) filtration) during a period of 70 days at high temporal resolution (n > 4000 for both water types). We detected and characterized in considerable detail aperiodic fluctuations in the raw water following regional precipitation, with TCC increasing up to 50-fold from a dry weather baseline of approximately 120 cells μl −1 to an event peak of > 5000 cells μl −1 . Moreover, we observed the buffering of the treatment plant against these fluctuations, but in addition we recorded a completely unexpected periodic fluctuation of TCC in the treated water after GAC filtration. We concluded that the latter was the result of fluctuating water abstraction from the treatment plant reservoir by two connected water utilities, which resulted in variations in water throughput in the plant. This in turn influenced bacterial detachment and dilution in the GAC filter. This study provides strong evidence of multiple different microbial dynamics occurring in a drinking water treatment system. Given numerous possible sources of natural and operational fluctuations in raw water and drinking water treatment plants, such microbial fluctuations should be expected in many systems. The high-frequency monitoring approach presented herein can improve the understanding and eventual mitigation of such fluctuations. [ABSTRACT FROM AUTHOR]

Loop-mediated isothermal amplification (LAMP) is rapidly being established as the preferred method for field-based, rapid and cost-effective detection of DNA sequences, but a deficiency of current methods for diagnostic purposes is the prevalence of non-specific amplification. We have previously demonstrated the applicability of the LAMP method for pollen from a variety of plant species with a resolution down to a single grain, based on the application of an extraction-free methodology. Here, we show how this method can be applied to detect pollen with sequence-specific accuracy and without the danger of false positives. We establish the lower limits of detection and efficiency of this approach, and in addition, we show that this method does not lose robustness for pollen material stored for at least 8 years. [ABSTRACT FROM AUTHOR]

Accounts of drinking water-borne disease outbreaks have always captured the interest of the public, elected and health officials, and the media. During the twentieth century, the drinking water community and public health organizations have endeavored to craft regulations and guidelines on treatment and management practices that reduce risks from drinking water, specifically human pathogens. During this period there also evolved misunderstandings as to potential health risk associated with microorganisms that may be present in drinking waters. These misunderstanding or 'myths' have led to confusion among the many stakeholders. The purpose of this article is to provide a scientific- and clinically-based discussion of these 'myths' and recommendations for better ensuring the microbial safety of drinking water and valid public health decisions. [ABSTRACT FROM AUTHOR]

The Magoura Cave contains an impressive display of prehistoric art. The different ecological niches in cave caverns and galleries may be exploited by a large variety of microorganisms and their growth can lead to both aesthetic and structural damages of the paintings mentioned. For this reason the microbial diversity in the cave and the role of microbial communities in rock paintings destruction is of great interest. The main objective of this research is connected with investigation of the microbial diversity of the rock paintings in the cave. The investigations are focused on characterization of the microbial communities and determination of the dominant microbial groups. Eleven different physiological groups of microorganisms have been tested by using of the elective nutrient media and specific conditions of cultivation. The quantitative analysis of the each group was done by the classical methods. The microbial studies reveals that on the rock paintings present continuously stable microbial population with stable qualitative and quantitative composition. In some samples are found high levels of sulphate-reducing bacteria, denitrifying bacteria, ammonifying bacteria and silicate bacteria. These taxonomic groups are involve in the biodeterioration processes. These results must be taken in mind during the conservation activities connected with paintings in the cave. [ABSTRACT FROM AUTHOR]

The microbial diversity in caves and the role of microbial communities in rock paintings destruction is a topic of present interest. The aim of the recent study is focused on the characterization of the microbial communities inhabiting the Magura Cave in order to develop a strategy for conservation of the unique paintings in the cave. Aimed at estimating the dynamics of microbial populations inhabiting the Magura Cave, four subsequent samplings were done in the course of twelve months. The comparative analysis of the microbial communities clearly demonstrates that each sample, respectively location, possess a unique microbial population structure and specific ratios between the different target groups, slightly changed during the monitoring investigation. The analysis revealed the presence of all tested physiological groups with the predominance of psychrophiles and oligiocarbophiles. Slightly variations in the number of actinomyces and fungi were observed among the different locations. [ABSTRACT FROM AUTHOR]

The collection of microbiological samples represents an important aspect of care both for doctors as well as nurses. It is important to recognise and identify some key points, to avoid performing 'unnecessary' or 'incorrect' sampling, which may give useless or even misleading results, these are: the moment at which the sample is collected, the collection method and timing (if indicated). The comparison between the various nursing members of the Italian National bone marrow transplant group (GITMO), showed diversity of practice across all fields. A formal survey was therefore conducted within GITMO centres looking at the methods of microbiological sample collection. These results were compared with the literature, and in addition to the lack of homogeneity of practice within the centres, a lack of compliance with the recommendations was also observed. To evaluate the effectiveness of this survey in highlighting awareness of this issue and the presence of relevant guidelines, the questionnaire was repeated (with the same centres responding), which demonstrated no major changes in care practices. Conclusion: The survey has allowed us to highlight many critical issues regarding common procedures which are not commonly discussed. Considerable differences were noted between different transplant centres, which may be attributable to the lack of Italian guidelines that can be used as a starting point for clinical practice. The plenary discussion allowed for an exchange of findings with the medical staff, who are usually responsible for requesting microbiological samples. The ideal solution would be a unique field-based training programme, associated with the dissemination of a common procedural document for ensuring evidence-based practice. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) has been identified as one of the major foodborne pathogenic bacteria. The development of rapid detection methods for S. aureus is needed for assuring food safety. In this study, quantum dots were used as fluorescent labels in an immunoassay for quantitative detection of S. aureus. Firstly, biotin-labeled anti-S. aureus antibody was conjugated with streptavidin-coated magnetic nanobeads (180 nm diameter) and used to separate S. aureus cells. Then streptavidin coated quantum dots (QDs) were conjugated with biotin-labeled anti-S. aureus antibody and used as the fluorescence labels to mix with the separated S. aureus. Finally the fluorescence intensity of the bead-cell-QD complexes was measured at a wavelength of 620 nm. A linear relationship between S. aureus cell number (X) and fluorescence intensity (Y) was found for cell numbers ranging from 10 ³ to 106 CFU (Colony Forming Unit)/mL, and the detection limit was 10³ CFU/mL. The regression model can be expressed as Y = 7.68X + 35.06 with R² =0.94. The detection of S. aureus in food sample was explored initially. The fluorescence intensity of food sample was close to the background, so it was not satisfied. Further study will focus on the application of the method for detection of S. aureus in food sample. [ABSTRACT FROM AUTHOR]

Microorganism contamination is a permanent concern in a wide range of fields, including the water-treatment, food and pharmaceutical industries, in which fast detection is critical to prevent microbial outbreaks. In water monitoring, current procedures for water-quality analysis are based on periodic sampling and detection by culture methods, which are slow, requiring 24–48h for completion, so that, when first results reach the decision-takers and trigger an alarm, significant time has already passed and the population may have been exposed to a health hazard. There is a need for rapid, reliable detection of contaminants in a broad spectrum of water-management situations. For real-time detection, on-line monitoring seems to be the ideal approach, but the need to adjust the available techniques to autonomous operation and the optimization of response time are substantial challenges. This review presents the findings of an identification study about the state-of-the-art of technologies and commercial devices for on-line biomonitoring of water quality, specifically for the detection of fecal contamination. We also include studies dealing with verification or use of these devices. [Copyright &y& Elsevier]

Summary: Background: The purpose of microbial monitoring of the inanimate environment surrounding a patient can be two-fold; to monitor hygiene standards and also to examine for the presence of specific nosocomial pathogens which may be the source of an outbreak. While both purposes involve routine culture of microorganisms, the methods used for each can differ in order to provide optimal results. The main difference between both purposes is the need for enumeration, site specificity for an aerobic colony count (ACC) for hygiene assessments, and the need to simply detect the presence or absence of multi-resistant nosocomial pathogens for infection control surveillance. Aim: To access current methods used in research studies and during outbreak investigations to detect nosocomial pathogens in the inanimate environment in the clinical setting. Methods: A Pubmed search of published literature was performed. Findings: Microbial monitoring of the environment can involve the use of swabs, sponges, contact plates and dip slides coupled with a variety of enrichment broths and selective media. The use of molecular methods such as polymerase chain reaction (PCR) can potentially provide a faster turnaround time, resulting in the quicker implementation of infection prevention and control cleaning and disinfection regimens. However, the optimal methods for performing a microbial hygiene evaluation or detecting specific bacterial pathogens are not generally agreed. Conclusion: There is a need for agreed standards on the optimal methods, frequency of environmental sampling and acceptable levels of surface contamination within the healthcare system. [ABSTRACT FROM AUTHOR]

Summary: Background: Microbial air monitoring in operating theatres has been a subject of interest and debate. No generally accepted sampling methods and threshold values are available. Aim: To assess microbial air contamination in empty and working conventionally ventilated operating theatres over a three-year period at the University Hospital of Parma, Italy. Methods: Air sampling was performed in 29 operating theatres. Both active and passive sampling methods were used to assess bacterial and fungal contamination. Findings: In empty theatres, median bacterial values of 12 colony-forming units (cfu)/m3 [interquartile range (IQR) 4–32] and 1 index of microbial air contamination (IMA) (IQR 0–3) were recorded. In working theatres, these values increased significantly (P < 0.001) to 80 cfu/m3 (IQR 42–176) and 7 IMA (IQR 4–13). Maximum recorded values were 166 cfu/m3 and 8 IMA for empty theatres, and 798 cfu/m3 and 42 IMA for working theatres. Combining active and passive samplings, fungi were isolated in 39.13% of samples collected in empty theatres and 56.95% of samples collected in working theatres. Over the three-year study period, bacterial contamination decreased in both empty and working theatres, and the percentage of samples devoid of fungi increased. In working theatres, a significant correlation was found between the bacterial contamination values assessed using passive and active sampling methods (P < 0.001). Conclusion: Microbiological monitoring is a useful tool for assessment of the contamination of operating theatres in order to improve air quality. [Copyright &y& Elsevier]

Abstract: The 14th-century fresco Stories of the Holy Fathers (size 6.10 × 15.65 m), at Camposanto Monumentale in Pisa, Italy, was painted by Buonamico Buffalmacco. The building housing it was damaged by a bomb during World War II and the fresco was quickly removed from the original walls under extremely dangerous conditions. It was detached using the “tear-off” technique with gauze and a layer of warm animal glue, and it was then stored by rolling it up, without adding any rigid support. In a 1960s restoration, an asbestos-cement support (eternit) was applied to the back and the gauze and thick layer of animal glue were removed from the front of the fresco. Early alteration phenomena such as swelling and detachment of the paint layer were noticed. In 2008, the fresco was again detached from the Camposanto wall for further restoration, which would include removal of residual traces of casein and animal glue from previous restorations and the reattachment of the paintings to a more suitable support. On this fresco, the combined removal in one step of casein and animal glue was based on the fast application of whole, viable bacterial cells of Pseudomonas stutzeri, A29 strain, to the fresco surface for a period of 2 h. An assessment of the effectiveness of the biological cleaning test was carried out using analytical pyrolysis. The results confirmed the success of this advanced biological approach for recovering valuable frescoes, and gave insight into selecting the optimum conditions for fast-treatment efficiency. Data on short- and medium-term microbial monitoring confirm both that viable cells are not present in the fresco after biotreatment and the absence of any potentially negative effects that could have been caused by metabolism. The conclusion was that the procedure was safe, non-invasive, and risk-free. The relatively low-cost of this biological cleaning process means that this biotechnological application represents a highly competitive, cost-effective solution. [Copyright &y& Elsevier]

Abstract: This study characterized the composition and activity of the autochthonous microbial community in formation fluids of a saline CO2 storage aquifer during CO2 injection and during an N2 lift. The clean-up of the wells prior CO2 injection by N2 lift decreased the total microbial cell numbers, and the number of sulphate reducing bacteria (SRB) was reduced by at least two orders of magnitude. Fluorescence in situ Hybridisation (FISH) and molecular fingerprinting demonstrated that the microbial community was strongly influenced by the CO2 injection. Before CO2 arrival, up to 106 cells ml-1 were detected by DAPI-staining at a depth of 647 m below the surface. The microbial community was dominated by fermentative halophilic bacteria and sulphate reducing bacteria. Both the FISH and fingerprinting analyses revealed quantitative and qualitative changes after CO2 arrival. An enhanced activity and quantity of the microbial population after five months of CO2 storage indicated that the community was able to adapt to the extreme conditions of the deep biosphere and to the extreme changes of these anthropogenically modified conditions. [Copyright &y& Elsevier]

Abstract: In this study the first results of the microbial monitoring within the framework of the CLEAN project are described. The microbial community of a 3.5 km deep depleted gas reservoir in Altmark, Germany was analyzed by molecular genetic techniques. Sequence analyses indicated the presence of microorganisms similar to previously identified microbes from saline, thermophilic and anaerobic environments in the deep hypersaline and hot reservoir environments. The results of the phylogenetic analyses revealed the presence of the different H2-oxidising bacteria (Hydrogenophaga sp., Acidovorax sp., Ralstonia sp., Pseudomonas sp.), thiosulfate-oxidising bacteria (Diaphorobacter sp.) and biocorrosive thermophilic microorganisms. [Copyright &y& Elsevier]

Abstract: A company, which produces non-carbonated water in 18.9l bottles, sold to supermarkets through out Crete, Greece, was evaluated. Sampling was carried out in the bottling plant, and at supermarkets where bottled water was stored for 1, 2, and 4 months. In total, 300 samples from final product, 240 samples from raw or filtered water, and 60 samples of empty bottles were analyzed. The results of these analyses indicated the need to improve Hazard Analysis Critical Control Points (HACCP) systems, in order to continuously monitor the water supply source in bottling plants, and to fully implement the correct storage conditions, hygiene procedures, and customer training at supermarkets. [Copyright &y& Elsevier]

Abstract: Sourdough technology is widely used; it is employed in bread making and for the production of cakes. Sourdough is characterized by a complex microbial ecosystem, mainly represented by lactic acid bacteria and yeasts, whose fermentation confers to the resulting bread its characteristic features such as palatability and high sensory quality. Investigation of the microbial composition of sourdough is relevant in order to determine the potential activities of sourdough microorganisms. This review focuses on the role of the most important group of sourdough fermenting bacteria that consists of lactobacilli; species that belong to the Lactobacillus genus are the main responsible of flavor development, improvement of nutritional quality as well as stability over consecutive refreshments of sourdough. Lactobacilli also establish some durable microbial associations. An overview of the tools for monitoring predominant species is also reported. [Copyright &y& Elsevier]

Aims: Flow cytometry offers rapid and reliable analyses of bacteria in milk. However, a flow cytometer is relatively expensive and operation is rather complicated for an unskilled operator. We applied flow cytometry using a microfluidic device (on-chip flow cytometry) in detection of small amounts of milk-spoiling bacteria. Methods and Results: Pseudomonas cells in milk were in situ hybridized with Cy5-labelled probe specific for Pseudomonas spp. under optimized condition. Numbers of Pseudomonas cells in the stationary phase and in the starved state determined by on-chip flow cytometry were compared with those determined by conventional plate counting, and on-chip flow cytometry detected targeted cells in milk that were undetectable as colony forming units(CFU) on Standards Methods Agar. Conclusions: The contamination in milk with fewer than 10 CFU ml−1 of targeted cells in starved state was detectable with simple procedure (0·5 h milk-clearing, 1 h fixation, 2 h hybridization and 0·5 h on-chip flow cytometry following 12 h enrichment of cells). Significance and Impact of the Study: On-chip flow cytometry following fluorescence in situ hybridization could be applicable to simple detection of milk-spoiling bacteria. [ABSTRACT FROM AUTHOR]

Microbial consortia in a two-phase, anaerobic bioreactor using a mixture of fruit and vegetable wastes were established. Bacterial and archaeal communities obtained by a culture-independent approach based on single strand conformation polymorphism analysis of total 16S rDNA showed the adaptation of the microflora to the process parameters. Throughout the 90 d of the study, the species composition of the bacterial community changed significantly. Bacterial 16S rDNA showed at least 7 different major species with a very prominent one corresponding to a Megasphaera elsdenii whereas bacterial 16S rDNA of a methanization bioreactor showed 10 different major species. After two weeks, Prevotella ruminicola became major and its dominance increased continuously until day 50. After an acid shock at pH 5, the 16S rDNA archaeal patterns in the acidogenic reactor showed two major prominent species corresponding to Methanosphaera stadtmanii and Methanobrevibacter wolinii, a hydrogenotrophic bacterium. [ABSTRACT FROM AUTHOR]