Norwich COVID-19 testing initiative pilot: evaluating the feasibility of asymptomatic testing on a university campus.

BACKGROUND: There is a high prevalence of COVID-19 in university-age students, who are returning to campuses. There is little evidence regarding the feasibility of universal, asymptomatic testing to help control outbreaks in this population. This study aimed to pilot mass COVID-19 testing on a university research park, to assess the feasibility and acceptability of scaling up testing to all staff and students. METHODS: This was a cross-sectional feasibility study on a university research park in the East of England. All staff and students (5625) were eligible to participate. All participants were offered four PCR swabs, which they self-administered over two weeks. Outcome measures included uptake, drop-out rate, positivity rates, participant acceptability measures, laboratory processing measures, data collection and management measures. RESULTS: 798 (76%) of 1053 who registered provided at least one swab; 687 (86%) provided all four; 792 (99%) of 798 who submitted at least one swab had all negative results and 6 participants had one inconclusive result. There were no positive results. 458 (57%) of 798 participants responded to a post-testing survey, demonstrating a mean acceptability score of 4.51/5, with five being the most positive. CONCLUSIONS: Repeated self-testing for COVID-19 using PCR is feasible and acceptable to a university population.

Microarray-based detection of Salmonella enterica serovar Enteritidis genes involved in chicken reproductive tract colonization.

Salmonella enterica serovar Enteritidis has developed the potential to contaminate table eggs internally, by colonization of the chicken reproductive tract and internalization in the forming egg. The serotype Enteritidis has developed mechanisms to colonize the chicken oviduct more successfully than other serotypes. Until now, the strategies exploited by Salmonella Enteritidis to do so have remained largely unknown. For that reason, a microarray-based transposon library screen was used to identify genes that are essential for the persistence of Salmonella Enteritidis inside primary chicken oviduct gland cells in vitro and inside the reproductive tract in vivo. A total of 81 genes with a potential role in persistence in both the oviduct cells and the oviduct tissue were identified. Major groups of importance include the Salmonella pathogenicity islands 1 and 2, genes involved in stress responses, cell wall, and lipopolysaccharide structure, and the region-of-difference genomic islands 9, 21, and 40.

A genome-wide screen identifies Salmonella Enteritidis lipopolysaccharide biosynthesis and the HtrA heat shock protein as crucial factors involved in egg white persistence at chicken body temperature.

Eggs contaminated with Salmonella Enteritidis are an important source of human foodborne Salmonella infections. Salmonella Enteritidis is able to contaminate egg white during formation of the egg within the chicken oviduct, and it has developed strategies to withstand the antimicrobial properties of egg white to survive in this hostile environment. The mechanisms involved in the persistence of Salmonella Enteritidis in egg white are likely to be complex. To address this issue, a microarray-based transposon library screen was performed to identify genes necessary for survival of Salmonella Enteritidis in egg white at chicken body temperature. The majority of identified genes belonged to the lipopolysaccharide biosynthesis pathway. Additionally, we provide evidence that the serine protease/heat shock protein (HtrA) appears essential for the survival of Salmonella Enteritidis in egg white at chicken body temperature.

Effect of microbial loading on the efficiency of cold atmospheric gas plasma inactivation of Salmonella enterica serovar Typhimurium.

In recent years the application of cold atmospheric gas plasma (CAP) aimed at the removal of microbial contamination from fresh and minimally processed food has received increased attention. For CAP to be successfully adopted by the food production industry, factors which affect its potential for microbial inactivation must be evaluated. In this study, we examined the effects of initial microbial concentration, present on filter discs, on the inactivation of Salmonella enterica serovar Typhimurium (S. Typhimurium) with nitrogen CAP. It was found that the rate of inactivation of S. Typhimurium is inversely proportional to initial bacterial concentration, with the D-value observed at the highest cell concentration assayed (10(8) CFU/filter) being 14 fold higher than seen at the lowest starting concentration (10(5) CFU/filter). Addition of increasing concentrations of Pseudomonas fluorescens cells to a Salmonella population of 10(5) CFU/filter resulted in an exponential decrease in the rate of killing of the Salmonella cells. However, whilst the addition of heat-killed S. Typhimurium cells to 10(5) CFU/filter live S. Typhimurium cells resulted in a significant decrease in the killing rate, this effect was dose independent. This suggests that although biomass plays a role in the protection against CAP inactivation seen at high cell densities, dead cells and their components released during the heating period are not as effective as viable cells. Fluorescence microscopy showed that, unlike the single dispersed cells observed at low cell densities, at higher cell densities bacteria were present in a multilayered structure. This phenomenon could explain the reduced inactivation by the plasma, since the top layer may present a physical barrier that protects underlying cells. In conclusion, this work clearly shows a link between bacterial cell density and the efficacy of CAP inactivation, making an important contribution to the understanding of this alternative food processing technology, which should be taken into account in both further studies and in the practical application of this technique to the food industry.

Genomics of thermophilic campylobacter species.

The thermophilic Campylobacter species C. jejuni and C. coli are important human pathogens, which are major causes of bacterial gastroenteritis. The recent progress in genomics techniques has allowed for a rapid increase in our knowledge of the molecular biology of Campylobacter species, but needs to be matched by concurrent increases in our understanding of the unique biology of these organisms. Campylobacter species display significant levels of genomic variation via natural transformation, phase variation, plasmid transfer and infection with bacteriophages, and this poses a continuous challenge for studies on pathogenesis, physiology, epidemiology and evolution of Campylobacter. In this chapter we will review the current state of the art of the genomics of thermophilic Campylobacter species, and opportunities where genomics can further contribute to our understanding of the biology of these successful human pathogens.

Nitrogen fixation: key genetic regulatory mechanisms.

The necessity to respond to the level of fixed nitrogen and external oxygen concentrations and to provide sufficient energy for nitrogen fixation imposes common regulatory principles amongst diazotrophs. The NifL-NifA system in Azotobacter vinelandii integrates the signals of redox, fixed-nitrogen and carbon status to regulate nif transcription. Multidomain signalling interactions between NifL and NifA are modulated by redox changes, ligand binding and interaction with the signal-transduction protein GlnK. Under adverse redox conditions (excess oxygen) or when fixed nitrogen is in excess, NifL forms a complex with NifA in which transcriptional activation is prevented. Oxidized NifL forms a binary complex with NifA to inhibit NifA activity. When fixed nitrogen is in excess, the non-covalently modified form of GlnK interacts with NifL to promote the formation of a GlnK-NifL-NifA ternary complex. When the cell re-encounters favourable conditions for nitrogen fixation, it is necessary to deactivate the signals to ensure that the NifL-NifA complex is dissociated so that NifA is free to activate transcription. This is achieved through interactions with 2-oxoglutarate, a key metabolic signal of the carbon status, which binds to the N-terminal GAF (cGMP-specific and stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domain of NifA.

Heterologous NNR-mediated nitric oxide signaling in Escherichia coli.

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO(+) donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.

Anaerobic growth of Paracoccus denitrificans requires cobalamin: characterization of cobK and cobJ genes.

A pleiotropic mutant of Paracoccus denitrificans, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated. This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B(12)) or cobinamide to the media or by growth on rich media. Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to cobK of Pseudomonas denitrificans, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway. Convergently transcribed with cobK is a gene homologous to cobJ of Pseudomonas denitrificans, which encodes precorrin-3b methyltransferase. The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type P. denitrificans (which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions. Comparison of the cobK and cobJ genes with their orthologues suggests that P. denitrificans uses the aerobic pathway for cobalamin synthesis. It is paradoxical that under anaerobic growth conditions, P. denitrificans appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis. Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media. These observations provide evidence that P. denitrificans expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions.