Dispersal of bacteria and stimulation of permafrost decomposition by Collembola

Contrary to most soils, permafrost soils have the atypical feature of being almost entirely deprived of soil fauna. Abiotic constraints on the fate of permafrost carbon after thawing are increasingly understood, but biotic constraints remain scarcely investigated. Incubation studies, essential to estimate effects of permafrost thaw on carbon cycling, typically measure the consequences of permafrost thaw in isolation from the topsoil and thus do not account for the effects of altered biotic interactions because of e.g. colonization by soil fauna. Microarthropods facilitate the dispersal of microorganisms in soil, both on their cuticle (ectozoochory) and through their digestive tract (endozoochory), which may be particularly important in permafrost soils, considering that microbial community composition can strongly constrain permafrost biogeochemical processes.Here we tested how a model species of microarthropod (the CollembolaFolsomia candida) affected aerobic CO2 production of permafrost soil over a 25 d incubation. By using Collembola stock cultures grown on permafrost soil or on an arctic topsoil, we aimed to assess the potential for endo- and ectozoochory of soil bacteria, while cultures grown on gypsum and sprayed with soil suspensions would allow the observation of only ectozoochory.The presence of Collembola introduced bacterial amplicon sequence variants (ASVs) absent in the no-Collembola control, regardless of their microbiome manipulation, when considering presence–absence metrics (unweighted UniFrac metrics), which resulted in increased species richness. However, these introduced ASVs did not induce changes in bacterial community composition as a whole (accounting for relative abundances, weighted UniFrac), which might only become detectable in the longer term.CO2 production was increased by 25.85 % in the presence of Collembola, about half of which could be attributed to Collembola respiration based on respiration rates measured in the absence of soil. We argue that the rest of the CO2 being respired can be considered a priming effect of the presence of Collembola, i.e. a stimulation of permafrost CO2 production in the presence of active microarthropod decomposers. Overall, our findings underline the importance of biotic interactions in permafrost biogeochemical processes and the need to explore the additive or interactive effects of other soil food web groups of which permafrost soils are deprived.

Cell and Organism Technologies for Assessment of the SARS-CoV-2 Infectivity in Fluid Environment.

Under conditions of COVID-19 pandemic, considerable amounts of SARS-CoV-2 contained in household, municipal, and medical wastewaters inevitably reach natural water bodies. Possible preservation of virus infectivity in liquid environment is of a paramount epidemiological importance. Experiments demonstrated that SARS-CoV-2 is resistant to multiple freezing/thawing cycles and retains its infectivity in tap and river water for up to 2 days at 20°C and 7 days at 4°C. In natural milk, its viability is preserved in a refrigerator for 6 days. The exposure of aquarium fish to the virus-containing water fails to cause any infection.

Preparation and effect evaluation of positive quality control reference for nucleic acid detection of 2019 Novel Corona Virus

Objective To prepare the indoor positive quality control reference for nucleic acid detection of 2019 Novel Corona Virus (2019-nCoV), investigate the feasibility of self-made quality control reference and evaluate the effect. Methods The positive control samples in the test kit from a third party were collected, diluted doubly with the virus preservation solution in which healthy human nasopharyngeal swabs were preserved, test samples to make a dilution, and determined for the Ct value of ORFlab gene by real-time fluorescent quantitative PCR. The dilutions with Ct values of about 31 and 34 were served as high-(nCov-H) and low-value quality control references (nCov-L) respectively and prepared in large quantities, then filled into small aliquots and stored at-80 °C to evaluate the uniformity and stability. Results The self-made indoor positive quality control reference for nucleic acid detection of 2019-nCoV showed good uniformity and good stability at various temperatures for storage, which could be kept stably at-80 °C for at least 6 months and could be controlled after repeated freezing and thawing for 3 times. Conclusion The self-made indoor positive quality control reference for nucleic acid detection of 2019-nCoV is simple to prepare, which has good uniformity and stability, and may be used as a substitute for commercial quality control for clinical test.

PC-578-02 JUNCTIONAL TACHYCARDIA

Background: Junctional ectopic tachycardia (JET) is a rare tachyarrhythmia in adults. The precise site of origin within the AV junction is unknown. Objective: N/A Methods: N/A Results: A 71-year-old male presented with dyspnea on exertion and recently diagnosed tachycardia in March 2021. He had a history of diabetes mellitus, obesity, hypertension, obstructive sleep apnea, and COVID-19 in 2020. A 14-day monitor demonstrated 43% supraventricular ectopy SVE burden and short runs of SVT. He presented for an electrophysiology (EP) study. He presented to the EP lab in sinus rhythm with frequent SVE. Multipolar catheters were placed in the His bundle region, right atrium, coronary sinus, and right ventricle. The SVE beats had the same QRS morphology, and an identical HV interval and His-right bundle activation sequence as in sinus rhythm and no retrograde conduction, consistent with premature junctional complexes (PJCs). Occasional short bursts of junctional tachycardia were noted. Isoproterenol was titrated to a maximum dose of 8 mcg/min. No other SVT was inducible with atrial overdrive pacing or programmed stimulation or with isoproterenol infusion. A 6 mm tip cryoablation catheter was advanced to the right atrium to the anatomical location of the slow pathway in the inferior triangle of Koch using an electroanatomic mapping system (EnSite NavX). Signals immediately prior to ablation (Figure 1) were notable for a pre-potential 26 ms prior to the His with PJCs. Cryoablation was performed at this site (Figure 2) with resolution of the PJCs at the onset of the freeze. After thawing, a second freeze was administered. No further PJCs were noted at baseline or with isoproterenol infusion. Conclusion: JET could originate from anywhere within the AV node or proximal His bundle. The application of cryoablation at a typical AV nodal slow pathway location with a preceding pre- potential and immediate obliteration of PJCs suggests that the origin in this case was from this region rather than a true His bundle extrasystole. Identification of pre-potentials to the junctional ectopy can guide safe ablation of this dysrhythmia. [Formula presented] [Formula presented]

Evaluating the Use of Alternative Normalization Approaches on SARS-CoV-2 Concentrations in Wastewater: Experiences from Two Catchments in Northern Sweden

The detection of SARS-CoV-2 RNA fragments in feces has paved the way for wastewater-based epidemiology to contribute to COVID-19 mitigation measures, with its use in a public health context still under development. As a way to facilitate data comparison, this paper explores the impact of using alternative normalization approaches (wastewater treatment plant (WWTP) flow, population size estimates (derived using total nitrogen (TN), total phosphorus (TP) and census data) and pepper mild mottle virus (PMMoV)) on the relationship between viral wastewater data and clinical case numbers. Influent wastewater samples were collected at two WWTPs in Luleå, northern Sweden, between January and March 2021. TN and TP were determined upon sample collection, with RNA analysis undertaken on samples after one freeze–thaw cycle. The strength of the correlation between normalization approaches and clinical cases differed between WWTPs (r ≤ 0.73 or r ≥ 0.78 at the larger WWTP and r ≤ 0.23 or r ≥ 0.43 at the smaller WWTP), indicating that the use of wastewater as an epidemiological tool is context-dependent. Depending on the normalization approach utilized, time-shifted analyses imply that wastewater data on SARS-CoV-2 RNA pre-dated a rise in clinical cases by 0–2 and 5–8 days, for the lager and smaller WWTPs, respectively. SARS-CoV-2 viral loads normalized to the population or PMMoV better reflect the number of clinical cases when comparing wastewater data between sewer catchments.

Evaluation of detection efficiency by SARS-CoV-2 Assay performed on the VitaPCRTM Instrument in samples collected in UTM

Background The diagnosis of SARS-CoV-2 infection is essential for the control of the epidemic, the establishment of protecting measures and the therapeutic management of patients. Among diagnostic tests utilizing a real-time reverse transcription polymerase chain reaction (RTPCR) amplification technology, VitaPCRTM SARS-CoV-2 Assay was used in our Laboratory for the rapid qualitative detection and discrimination of SARS-CoV-2 viral RNAs in direct nasopharyngeal (NP) or oropharyngeal (OP) swabs. Aim of the study To test SARS-CoV-2 Assay on NP or OP swabs collected in UTM instead of directly melted in the provided buffer of the kit in order to assess the detection efficiency of the system in relation to the potential interference of UTM in the PCR reaction and to viral RNA concentration. Methods A pool of ten SARS-CoV-2 positive samples collected in UTM, previously tested by quantitative real-time RT-PCR, was assayed on the VitaPCRTM Instrument at several dilutions (from 1:10 to 1:100000) in the provided buffer of the kit, immediately and after a cycle of freezing and thawing at -20°C. Results VitaPCRTM Instrument was able to detect SARSCoV- 2 viral RNA at four out of five tested dilutions, with a good efficiency especially at 1:10 and 1:100 dilutions, while at 1:1000000 the result was doubtful. After freezing and thawing the detection efficiency sensibly decreased and the Instrument was able to detect the viral RNA only at 1:10, 1:100 and 1:1000 dilutions, while at 1:10000 and 1:1000000 the result was doubtful. Conclusions These preliminary results are promising suggesting the potential use of SARS-CoV-2 Assay on the VitaPCRTM Instrument even with swabs collected in UTM, increasing the versatility of this system. Moreover, the reduced performance of VitaPCRTM Instrument after freezing and thawing the sample suggests a consistent degradation of viral RNA, advising against the use of this type of frozen samples with this assay. Further investigations are recommended.

Stability of Viral RNA in an Environmental Sampling Kit and its Detection with Molecular Biology Techniques

Introduction: The detection of SARS-CoV-2 on inanimate surfaces lead to an increasing demand for environmental sampling solutions to detect microbiological contaminations. Monitoring of surface contamination was used to monitor potential implication for viral transmission and possible mitigation strategies using effective cleaning and disinfection procedures and precaution. Objectives: The purpose of this study was to evaluate viral RNA sample stability using Copan SRK®, an environmental sampling kit consisting of a pre-moistened swab for sample collection and a reagent able to neutralize antimicrobial agents, for the detection of RNA Influenza A virus with molecular biology techniques. Methods: For the study, 100 μL of Influenza A virus (H3N2) (ATCC® VR-1679™) viral suspension, chosed as representative of a ssRNA virus, were inoculated in 1 mL SRK tubes. To simulate product performances upon shipment, some SRK tubes were previously stressed performing a thermal shock. Additional 100 μL were inoculated in Copan UTM as reference. Viral nucleic acids were extracted with the ThermoFisher PrepSEQ™ Residual DNA kit and the Applied Biosystem Automate express at different timepoints from inoculation. Samples were extracted at T0, after 2 and 7 days of storage at 2-8 °C and room temperature. Nucleic acids were stored at.20 °C until test (all samples were subjected to a freeze/thaw cycle before test). Detection of viral RNA was performed with the RIDA® GENE real time PCR Flu/RSV. Additionally, to assess long term preservation, samples were extracted after 28 days at both 2-8 °C and RT starting from Copan SRK tubes. Results: Data obtained (Table 1) showed that Copan SRK is suitable to preserve inoculated samples and viral RNA from Influenza A virus can be detected up to 7 days from collection when stored at 2-8 °C and RT and up to 28 days at 2-8 °C, with a difference in Ct from T0 lower than 1 Ct (Fig. 1). As reference, samples stored at RT up to 7 days in UTM showed the same variation in Ct values from T0. No significant differences were observed when variation in Ct values is compared between SRK with and without the stress procedure. Conclusion: Copan SRK is compatible with the detection of Influenza A viral RNA with molecular biology techniques and samples are stable up to 7 days of storage at 2-8 °C and RT and up to 28 days at 2-8 °C.

"On-Chip"-Cryopreservation For Cell-Based Biosensors and Lab-On-A-Chip Systems

There is an increased number of publications on biosensors addressing substances such as pollutants, toxins, or viruses, like SARS-CoV-2. Cell-based biosensors, which use cells as biorecognition elements (bioreceptor), offer outstanding advantages regarding a fast and sensitive detection of these substances. However, there are only a few cell-based biosensors available on the market because such sensor chip contains a ‘living’ component (e.g., adherent mammalian cells), which is very vulnerable and demanding for certain vital parameters (e.g., oxygen, nutrients, pH) as well as temperature during storage and transportation to the end-user. This challenge often hinders the commercialization and practical applicability of cell-based biosensors. There is still an urgent need for preservation tools and methods to enable “off-the-shelf” format as well as ready-to-use on-site systems. Herein, we studied a strategy named “on-sensor cryopreservation (OSC)”, a method for preserving this living component by freezing it (-80 °C) directly on the biosensor surface. However, it is found that cryo-injury on cells occurs over a freeze-thaw cycle, which is most likely due to a mismatch in the coefficient of linear thermal expansion between the frozen cell membrane and the typically rigid sensor surface in contact. Thus, to protect the cells from cryo-injury by absorbing this mismatch energy, the rigid sensor surface was modified with elastic electrospun fibers composed of a polymer (polyethylene vinyl acetate), which has a low glass-transition temperature and a high thermal expansion coefficient. The modified sensor chip is then integrated into a microfluidic system to obtain a so-called cryo-chip. The presented cryo-chip is found to be effective for keeping cells viable during cryopreservation as well as for post-thaw monitoring of extracellular acidification, exemplarily demonstrated for CHO-K1 cells. Cryopreservation of the chips containing cells at the manufacturing stage and transporting them through cold-chain transport can pave the way for “all-in-one” format and “off-the-shelf” applicability. Funding: DÖ would like to acknowledge the Ph.D. research scholarship grant from the Scientific and Technological Research Council of Turkey (TÜBITAK). The authors gratefully thank the Federal Ministry of Education and Research of Germany (Opto-Switch FKZ: 13N12585). Conflict of Interest: None to disclose

Microbiological analysis of frozen profiteroles and mini chocolate eclairs implicated in a national salmonellosis outbreak.

Between November 2018 and May 2019, Canada experienced a nationwide salmonellosis outbreak linked to the presence of Salmonella enterica ser. Enteritidis in frozen profiteroles. Analysis of the implicated food products revealed low levels of Salmonella ranging from 0.2 to 0.7 MPN/100g. Water activity and pH of the food samples ranged from 0.9479 to 0.9867 and 4.6-6.8 respectively indicating conditions conducive to bacterial growth. Higher levels of the hygiene indicators Enterobacteriaceae and coliforms were associated with Salmonella positive samples compared to Salmonella negative samples. Investigation of the relationship between storage conditions, temperature, and pathogen levels during thawing revealed that the profiteroles reached temperatures permissive to pathogen growth (≥5 °C) much sooner than pathogen growth was observed and that the composition of the food matrix can influence bacterial levels upon thawing. Collectively these data can be used to inform guidance to minimize the risk of infection from the consumption of contaminated cream-filled frozen desserts.