Inactivation of Group I and Group II Clostridium botulinum spores by ultraviolet irradiation in water.

Spores of Clostridium botulinum are widely distributed in the environment, including in foods. Prevention of foodborne botulism relies on the inhibition of spore germination and subsequent growth and toxin production, or the destruction of viable spores in food and beverages. This study examined the lethality of 254 nm UV radiation (UV-C) to spores of Group I and Group II C. botulinum. Spores of C. botulinum were inactivated by UV-C, with doses required for incremental log reduction (D10) values calculated using linear regression ranging from 2.87 to 3.70 mJ/cm2 for Group I strains and 4.46 to 6.15 mJ/cm2 for Group II strains. The measured D10 value for spores of C. sporogenes ATCC 19404 was 8.27 mJ/cm2 indicating it was more resistant than the strains of C. botulinum used in this study. Calculation of dose per log using a Weibull model resulted in higher D10 values of 6.67 to 8.81 mJ/cm2 for Group I strains and 9.24 to 10.7 mJ/cm2 for Group II strains. Spores of C. sporogenes possessed a D10 value of 14.4 mJ/cm2. The higher values for the Weibull model indicate the Weibull model to be more conservative as a result as it factors in the lag prior to inactivation and the tailing observed with very low numbers of survivors. Spores of both Group I and Group II C. botulinum strains tended to form large aggregates, visible with phase contrast microscopy, that resulted in severe tailing. Disruption of aggregates by ultrasonication was necessary to obtain linear destruction curves extending beyond 5 log reduction. All strains from Group I and Group II required <55 mJ/cm2 to achieve 5 log inactivation. The strain of C. sporogenes used in this work can therefore be a conservative non-pathogenic surrogate, having higher UV-C resistance than the C. botulinum strains used in this study. Overall, this study is the first detailed study to demonstrate UV-C as an effective treatment method to inactivate C. botulinum spores in a suspending medium. In addition, the study paves the way for further studies towards the applications of this technology to inactivate C. botulinum spores in beverages or other liquids.

SARS-CoV-2 Ultraviolet Radiation Dose-Response Behavior.

Ultraviolet (UV) radiation in the wavelength range 200 nm ≤ λ ≤ 320 nm, which includes both the UV-C and UV-B portions of the spectrum, is known to be effective for inactivation of a wide range of microbial pathogens, including viruses. Previous research has indicated UV-C radiation to be effective for inactivation of severe acute respiratory syndrome coronavirus (SARS-CoV), the virus that caused an outbreak of SARS in 2003. Given the structural similarities of SARS-CoV and SARS-CoV-2, the cause of coronavirus disease 2019 (COVID-19), it is anticipated that UV radiation should be effective for inactivation of SARS-CoV-2 too. Recently published data support this assertion, but only for a narrow set of exposure and matrix conditions. Models based on genomic and other characteristics of viruses have been developed to provide predictions of viral inactivation responses to UV exposure at λ = 254 nm. The predictions of these models are consistent with reported measurements of viral inactivation, including for SARS-CoV-2. As such, current information indicates that UV-C irradiation should be effective for control of SARS-CoV-2, as well as for control of other coronaviruses; however, additional research is needed to quantify the effects of several important process variables, including the wavelength of radiation, the effects of relative humidity on airborne and surface-associated viruses, and the effects of the medium of exposure.

Phytoplankton growth characterization in short term MPN culture assays using 18S metabarcoding and qRT-PCR.

The most probable number dilution-culture assay (MPN) is used to enumerate viable phytoplankton in regulatory tests of ballast water treatment systems. However the United States Coast Guard has not yet accepted MPN, in part due to concerns of biased results due to cells being viable but not growing. MPN does not assess the fate of every cell, and thus the bias can only be evaluated by a companion method that assesses the ability of the various taxa to grow. This growth ability ("growability") is the complement of the bias, and has been evaluated by microscopic taxonomy of before-culture and after-culture samples. However, microscopic taxonomy is extremely laborious and few data have been produced for phytoplankton growability in MPN assays. To address the need for more and more reliable growability data, a method was developed using next-generation sequencing (NGS) and quantitative real time PCR (qRT-PCR) techniques that target the V9 region of the 18S rRNA gene for the taxonomic identification and growth assessment of eukaryotic phytoplankton, respectively. This growability method was applied to MPN samples from a ballast water management system test that were incubated with two different enrichment media at two different temperatures. DNA was extracted from filters of before-culture and after-culture samples, and assessed for taxonomy by NGS and for PCR template DNA concentration by qRT-PCR. Growth ratios based on changes in 18S template concentration over the incubation period were calculated for each taxon, and dead-cell DNA persistence through a 14 day incubation was verified to be <1% and did not influence the growth calculations. In total, 95 of 97 eukaryotic phytoplankton in the before-culture sample demonstrated growth, with definitive growth ratios ranging from 4.0 × 101-2.6 × 105. An additional 13 taxa demonstrated growth from non-detect in before-culture samples. Taxa-based growability values were 87-88% in individual incubation conditions with no statistical differences among conditions, and 98% for all conditions combined. When growability was weighted by the before-culture abundance of each taxa, relevant to regulations based on all organisms regardless of taxa, community-based growability was >99% in each condition and in all conditions combined because the most abundant taxa all exhibited growth. This study verifies that conventional phytoplankton MPN assays produce accurate results with low bias from undetected viable cells, regardless of enrichments and incubation temperatures. This work can provide regulatory confidence for broader acceptance of MPN assays without limitations.

The Biological Basis for Ballast Water Performance Standards: "Viable/Non-Viable" or "Live/Dead"?

The shipping industry is critical to international commerce; however, contemporary shipping practices involve uptake and discharge of ballast water, which introduces the potential for transfer of nonindigenous, invasive species among geographically distinct habitats. To counteract this hazard, regulations for ballast water management have been implemented by the International Maritime Organization (IMO) and by regulatory agencies such as the United States Coast Guard (USCG). IMO and USCG discharge standards are numerically identical, but involve different definitions of treatment end points, which are based on fundamentally different biological assays for quantification of ballast water treatment effectiveness. Available assays for quantification of the responses of organisms in the 10-50 µm size range include vital stains based on fluorescein diacetate (FDA), sometimes used in combination with 5-chloromethylfluorescein diacetate (CMFDA), observations of motility, and the most probable number dilution culture method (MPN). The mechanisms and implications of these assays are discussed relative to the Type Approval process, which quantitatively evaluates compliance with ballast water discharge standards (BWDSs) under controlled shipboard and land-based tests. For antimicrobial processes that accomplish treatment by preventing subsequent replication of the target species, the FDA/CMFDA and MPN methods can yield dramatically different results. An important example of a treatment process that is affected by the choice of assay is ultraviolet (UV) irradiation. Results of laboratory and field experiments have demonstrated UV-based technologies to be effective for accomplishing the objectives of ballast water treatment (inactivation of cellular reproduction), when the MPN assay is used as the basis for evaluation. The FDA, CMFDA, motility, and MPN methods are subject to well recognized sources of error; however, the MPN method is based on a response that is consistent with the objectives of ballast water management as well as the mechanism of action of UV-based inactivation. Complementary assays are available for use in compliance testing; however, the development of relevant indicative tests remains as a research priority. Historical lessons learned from applications of vital stains (and other indirect methods) for quantification of microbial responses to UV irradiation in other settings also support the use of assays that provide a direct measure of growth and reproduction, such as MPN. Collectively, these observations point to the use of MPN assays as the standard for type testing, especially when UV-based treatment is employed.

Enumerating viable phytoplankton using a culture-based Most Probable Number assay following ultraviolet-C treatment.

Ballast water management systems (BWMS) must be tested to assess their compliance with standards for the discharge of organisms, for example in the ≥ 10- and < 50-µm size category, which is dominated by phytoplankton. Assessment of BWMS performance with the vital stains fluorescein diacetate + 5-chlorofluorescein diacetate, required by regulations in the USA, is problematic in the case of ultraviolet-C (UVC) radiation. This is because UVC targets nucleotides-and thus reproduction, hence viability-rather than membrane integrity, which is assayed by the stains. The Serial Dilution Culture-Most Probable Number (SDC-MPN) method, long used to enumerate fragile phytoplankton from natural communities, is appropriate for counting viable phytoplankton. We developed QA/QC "best practice" criteria for its application as a robust and repeatable assay of viable cells in cultures of phytoplankton before and after experimental treatment, then constructed dose-response curves for UVC-induced loss of viable cells in 12 species of phytoplankton from seven divisions. Sensitivity to UVC, expressed as the dose required to reduce viability by 99%-the criterion for type approval of treatment systems-varied more than 10-fold and was not correlated with cell size. The form of the dose-response curves varied between taxa, with most having a threshold dose below which there was no reduction in viability. Analysis of the patterns of growth indicates that if recovery from treatment occurred, it was complete in 1 or 2 days in > 80% of cases, long before the assays were terminated. We conclude that the SDC-MPN assay as described is robust and adaptable for use on natural phytoplankton.

Invasion and initial replication of ultraviolet irradiated waterborne infective stages of Myxobolus cerebralis results in immunity to whirling disease in rainbow trout.

Myxobolus cerebralis is a microscopic metazoan parasite (Phylum Myxozoa: Myxosporea) associated with salmonid whirling disease. There are currently no vaccines to minimise the serious negative economical and ecological impacts of whirling disease among populations of salmonid fish worldwide. UV irradiation has been shown to effectively inactivate the waterborne infective stages or triactinomyxons of M. cerbralis in experimental and hatchery settings but the mechanisms by which the parasite is compromised are unknown. Treatments of triactinomyxons with UV irradiation at doses from 10 to 80 mJ/cm(2) either prevented (20-80 mJ/cm(2)) or significantly inhibited (10 mJ/cm(2)) completion of the parasite life cycle in experimentally exposed juvenile rainbow trout (Oncorhynchus mykiss). However, even the highest doses of UV irradiation examined (80 mJ/cm(2)) did not prevent key steps in the initiation of parasite infection, including attachment and penetration of the epidermis of juvenile rainbow trout as demonstrated by scanning electron and light microscopy. Furthermore, replication of UV-treated parasites within the first 24h following invasion of the caudal fin was suggested by the detection of concentrations of parasite DNA by quantitative PCR comparable to that among fish exposed to an equal concentration of untreated triactinomyxons. Subsequent development of parasites treated with an 80 mJ/cm(2) dose of UV irradiation however, was impaired as demonstrated by the decline and then lack of detection of parasite DNA; a trend beginning at 10 days and continuing thereafter until the end of the study at 46 days post parasite exposure. Treatments of triactinomyxons with a lower dose of UV irradiation (20 mJ/cm(2)) resulted in a more prolonged survival with parasite DNA detected, although at very low concentrations, in fish up to 49 days post parasite exposure. The successful invasion but only short-term survival of parasites treated with UV in rainbow trout resulted in a protective response to challenges with fully infective triactinomyxons. Prior treatments of juvenile rainbow trout with UV-treated triactinomyxons (10 and 20 mJ/cm(2)) resulted in a reduced prevalence of infection and significantly lower concentrations of cranial myxospores (two direct measures of the severity of whirling disease) compared with trout receiving no prior treatments when assessed 5 months post parasite exposure to fully infective triactinomyxons.

[Impact of particle on UV disinfection for wastewater].

Water samples with similar particle distribution were irradiated by low-pressure UV. Experiment results were input into the two-kinetic model for calculation. The approach applied in this experiment allowed the assessment of the effect of particles on UV inactivation of FC in their natural state. The results showed that FC concentration increased with the particle number. Particle significantly lowered the inactivation rate of the indigenous FC in the secondary water with elevated particle content when the particle number was less than 10(5) C x mL(-1). On the other hand, the impact was not very significant when the particle number exceeded 10(5) C x mL(-1). Based on the calculation results of a two-kinetic model, for the different number of particles, the ratio of the FC which was difficult to inactivate was stable, while most of the FC which were holed in the particles were easy to inactivate.

Effects of freezing, drying, ultraviolet irradiation, chlorine, and quaternary ammonium treatments on the infectivity of myxospores of Myxobolus cerebralis for Tubifex tubifex.

The effects of freezing, drying, ultraviolet irradiation (UV), chlorine, and a quaternary ammonium compound on the infectivity of the myxospore stage of Myxobolus cerebralis (the causative agent of whirling disease) for Tubifex tubifex were examined in a series of laboratory trials. Freezing at either -20 degrees C or -80 degrees C for a period of 7 d or 2 months eliminated infectivity as assessed by the absence of production of the actinospore stage (triactinomyxons [TAMs]) from T. tubifex cultures inoculated with treated myxospores over a 4-5-month period. Myxospores retained infectivity when held in well water at 5 degrees C or 22 degrees C for 7 d and when held at 4 degrees C or 10 degrees C d for 2 months. In contrast, no TAMs were produced from T. tubifex cultures inoculated with myxospores held at 20 degrees C for 2 months. Drying of myxospores eliminated any evidence of infectivity for T. tubifex. Doses of UV from 40 to 480 mJ/cm2 were all effective for inactivating myxospores of M. cerebralis, although a few TAMs were detected in one replicate T. tubifex culture at 240 mJ/cm2 and in one replicate culture at 480 mJ/cm2. Treatments of myxospores with chlorine bleach at active concentrations of at least 500 mg/L for 15 min largely inactivated myxospore infectivity for T. tubifex. Likewise, there was no evidence of TAMs produced by T. tubifex inoculated with myxospores treated with alkyl dimethyl benzyl ammonium chloride (ADBAC) at 1,500 mg/L for 10 min. Treatments of myxospores with 1,000-mg/L ADBAC for 10 min reduced TAM production in T. tubifex cultures sevenfold relative to that in cultures inoculated with an equal number of untreated myxospores. These results indicate that myxospores of M. cerebralis demonstrate a selective rather than broad resistance to selected physical and chemical treatments, and this selective resistance is consistent with conditions that myxospores are likely to experience in nature.