Comparison of Surface Persistence of SARS-CoV-2 Alpha and Delta Variants on Stainless Steel at 4°C and 24°C.

Most studies on surface persistence of SARS-CoV-2 have been conducted at temperatures between 20°C and 30°C. There is limited data on the survival of SARS-CoV-2 at low temperatures. In this study, the stability of SARS-CoV-2 Alpha and Delta variants on stainless steel was investigated at two temperatures (4°C and 24°C). The results show that both variants decayed more rapidly at 24°C compared with 4°C. At 24°C, Alpha and Delta variants showed reductions of 0.33 log10 and 1.02 log10, respectively, within the first 2.5 h. However, at 4°C, Alpha variant showed a reduction of 0.16 log10 within the first 2.5 h while no reduction was observed with Delta variant. After remaining in situ for 24 h at 24°C, log10 reductions of 2.66 (Alpha) and 3.11 (Delta) were observed. No viable Alpha and Delta variant was recovered after 48 h and 72 h, respectively. After 24 h in a refrigerated environment (4°C) log10 reductions of 1.16 (Alpha) and 0.95 (Delta) were observed. Under these experimental conditions, both viruses survived on stainless steel for at least 1 week. No viable Alpha and Delta variant was recovered after 10 days. These findings support the potential for increased fomite transmission of SARS-CoV-2 during winter months in colder regions worldwide and in some industrial sectors. IMPORTANCE Human transmission is believed to occur primarily through direct transfer of infectious droplets or aerosols. However, fomite transmission through contact with contaminated surfaces may also play an important role. This study provides novel evidence comparing the stability of Alpha and Delta variants on stainless steel surfaces at 4°C and 24°C. At 4°C both variants were found to be still detectable for up to 7 days. At 24°C Delta variant could be recovered over 2 days compared with Alpha variant which could not be recovered after 2 days. This has implications for fomite transmission interventions for people living and working in cold environments.

Characterisation of Particle Size and Viability of SARS-CoV-2 Aerosols from a Range of Nebuliser Types Using a Novel Sampling Technique.

Little is understood about the impact of nebulisation on the viability of SARS-CoV-2. In this study, a range of nebulisers with differing methods of aerosol generation were evaluated to determine SARS-CoV-2 viability following aerosolization. The aerosol particle size distribution was assessed using an aerosol particle sizer (APS) and SARS-CoV-2 viability was determined after collection into liquid media using All-Glass Impingers (AGI). Viable particles of SARS-CoV-2 were further characterised using the Collison 6-jet nebuliser in conjunction with novel sample techniques in an Andersen size-fractioning sampler to predict lung deposition profiles. Results demonstrate that all the tested nebulisers can generate stable, polydisperse aerosols (Geometric standard deviation (GSD) circa 1.8) in the respirable range (1.2 to 2.2 µm). Viable fractions (VF, units PFU/particle, the virus viability as a function of total particles produced) were circa 5 × 10-3. VF and spray factors were not significantly affected by relative humidity, within this system where aerosols were in the spray tube an extremely short time. The novel Andersen sample collection methods successfully captured viable virus particles across all sizes; with most particle sizes below 3.3 µm. Particle sizes, in MMAD (Mass Median Aerodynamic Diameters), were calculated from linear regression of log10-log10 transformed cumulative PFU data, and calculated MMADs accorded well with APS measurements and did not differ across collection method types. These data will be vital in informing animal aerosol challenge models, and infection prevention and control policies.

Persistence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Virus and Viral RNA in Relation to Surface Type and Contamination Concentration.

The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used reverse transcription-PCR (RT-PCR) analysis to detect SARS-CoV-2 RNA on surfaces, but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered and whether virus viability is concentration dependent. Viable virus persisted for the longest time on surgical mask material and stainless steel, with a 99.9% reduction in viability by 122 and 114 h, respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 h. Viability on the bank note was reduced second fastest, with 99.9% reduction in 75 h. RNA on all surfaces exhibited a 1-log reduction in genome copy number recovery over 21 days. The findings show that SARS-CoV-2 is most stable on nonporous hydrophobic surfaces. RNA is highly stable when dried on surfaces, with only 1-log reduction in recovery over 3 weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within 2 days. Therefore, when RNA is detected on surfaces, it does not directly indicate the presence of viable virus, even at low cycle threshold values. IMPORTANCE This study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface, where RT-PCR is not an appropriate technique to determine viable virus. Unless sampled immediately after contamination, it is difficult to align RNA copy numbers to quantity of viable virus on a surface.

Long-Term Exposure to Octenidine in a Simulated Sink Trap Environment Results in Selection of Pseudomonas aeruginosa, Citrobacter, and Enterobacter Isolates with Mutations in Efflux Pump Regulators.

Octenidine-based disinfection products are becoming increasingly popular for infection control of multidrug-resistant (MDR) Gram-negative isolates. When a waste trap was removed from a hospital and allowed to acclimatize in a standard tap rig in our laboratory, it was shown that Klebsiella pneumoniae, Pseudomonas aeruginosa, and Citrobacter and Enterobacter spp. were readily isolated. This study aimed to understand the potential impact of prolonged exposure to low doses of a commercial product containing octenidine on these bacteria. Phenotypic and genotypic analyses showed that P. aeruginosa strains had increased tolerance to octenidine, which was characterized by mutations in the Tet repressor SmvR. Enterobacter species demonstrated increased tolerance to many other cationic biocides, although not octenidine, as well as the antibiotics ciprofloxacin, chloramphenicol, and ceftazidime, through mutations in another Tet repressor, RamR. Citrobacter species with mutations in RamR and MarR were identified following octenidine exposure, and this is linked to development of resistance to ampicillin, piperacillin, and chloramphenicol, as well as an increased MIC for ciprofloxacin. Isolates were able to retain fitness, as characterized by growth, biofilm formation, and virulence in Galleria mellonella, after prolonged contact with octenidine, although there were strain-to-strain differences. These results demonstrate that continued low-level octenidine exposure in a simulated sink trap environment selects for mutations that affect smvR It may also promote microbial adaptation to other cationic biocides and cross-resistance to antibiotics, while not incurring a fitness cost. This suggests that hospital sink traps may act as a reservoir for more biocide-tolerant organisms.IMPORTANCE Multidrug-resistant (MDR) strains of bacteria are a major clinical problem, and several reports have linked outbreaks of MDR bacteria with bacterial populations in hospital sinks. Biocides such as octenidine are used clinically in body washes and other products, such as wound dressings for infection control. Therefore, increased tolerance to these biocides would be detrimental to infection control processes. Here, we exposed bacterial populations originally from hospital sink traps to repeated dosing with an octenidine-containing product over several weeks and observed how particular species adapted. We found mutations in genes related to biocide and antibiotic susceptibility, which resulted in increased tolerance, although this was species dependent. Bacteria that became more tolerant to octenidine also showed no loss of fitness. This shows that prolonged octenidine exposure has the potential to promote microbial adaptation in the environment and that hospital sink traps may act as a reservoir for increased biocide- and antibiotic-tolerant organisms.