To estimate resource use and the costs of eye injuries in 2011–2012 in the Helsinki University Eye Hospital (HUEH), which covers 1.6 million people in Southern Finland.This population-based study consisted of all new patients  with eye injuries in one year. The data were from hospital records, internal HUEH accountancy, and prospectively from questionnaires. The costs of direct health care, transportation, and lost productivity were obtained and estimated for the follow-up period of three months. The estimated future costs were discussed.During the follow-up, the total cost was 2,899,000 Euros (EUR) , including lost productivity , direct health care , and transportation . The resources used included 6,902 days of lost productivity, 2,436 admissions and transportations, 314 minor procedures, 313 inpatient days, 248 major surgeries, and 86 radiological images. One open globe injury was the costliest , but contusions had the highest overall cost , due to their high occurrence and number of follow-ups.Eye injuries cause a major burden through high costs of direct health care and lost productivity: the imminent costs were EUR 1,870,000/one million population, and the future costs were estimated to EUR 3,741,400/one million population. Prevention remains the main factor to consider for better cost-efficiency.The online version contains supplementary material available at 10.1007/s00417-021-05399-3. Eye injuries occur universally in everyday activities and are among the leading causes of monocular blindness in the world . They arIt is estimated that 55 million  eye injuries occur in the world each year and that 750,000  injuries require hospitalization [To evaluate the influence of eye injuries, and to set policies for priorities, it is crucial to have information on not only the prevalence and causes, but also on the costs incurred by eye injuries.Existing reports on the costs incurred by eye injuries from different countries are mostly outdated. Moreover, the comparability of these studies is limited due to different insurance and compensation policies, or developmental diversity. Many studies have a narrow focus; for example, they report the expenses of preventable and minor , or seriFew studies have reported the cost of all eye injuries. Mönestam estimated a total cost of eye injuries of SEK 1,300,000  in a hoMany studies have addressed inpatient days and lost productivity days –17. MediIn Finland in 1980–1986, perforating eye injuries caused 5% of permanent disability and inability to work [To our knowledge, no recent studies have described direct and indirect costs, the use of resources, or the costs incurred by serious and minor eye injuries in European countries.The strength of our study is that it is a population-based study. The aim of this study was to estimate the economic burden and resources used for eye injuries in Southern Finland from a societal perspective, including direct health care costs, direct non-health care costs, and indirect costs.In this population-based study, the participants consisted of all new patients with an eye injury admitted to the Helsinki University Eye Hospital (HUEH) emergency department (ED) over one year (1 May 2011 to 30 April 2012).Data was obtained prospectively from patient questionnaires and retrospectively from hospital records. These included information on patient demographics, symptoms, detailed physical eye examinations, treatments, use of resources, and the cause of and events leading to the eye injury. The follow-up period was three months. We divided the cases into three age groups: children aged under 17 years, adults aged 17–60 years, and the elderly aged over 60 years. We classified the ICD-10-coded  cases as BETTs (Birmingham Eye Trauma Terminology system) , 19 and The costs were grouped into direct and indirect costs. Direct costs included health care costs and non-health care costs. Direct health care costs included outpatient visits, hospitalizations, major surgeries, minor procedures, and medication and radiology costs. Direct non-health care costs included transportation costs. Indirect costs included the costs of lost productivity.We derived the unit cost for outpatient visits, hospitalizations, major surgeries, and minor procedures from the internal HUEH cost accounting data .Outpatient admissions were calculated for the three age groups as well as for all diagnostic groups. This included number of admissions of the first visits in the Emergency Department (ED) + the first control visits in ED clinic + the following possible follow-up visits at an eye sub-specialty clinic during three months of follow-up.The cost of hospitalizations was based on the number of inpatient days.The major surgeries included the services of an anesthesiologist . The unit costs were obtained from resourced-based internal HUEH cost accounting data. The unit cost was different for an emergent and for an elective operation Table . In the The number of minor procedures was calculated for the three age groups as well as for all diagnostic groups. To calculate the costs of each case, we used the internal resourced-based HUEH cost accounting data Table .The unit cost of medication was obtained from the Finnish Medical Society’s national health portal .The unit costs for transportation were obtained from the Social Insurance Institution of Finland, KELA, and the Department of Health and Welfare –27. The Lost productivity was estimated by multiplying the number of days absent from work by the employee’s average daily cost of lost productivity, for patients aged 17–64 and for one parent of the injured children needing physical restriction Table . The numOn the last visit, the data obtained included main abnormal status findings, outpatient admissions, days of hospitalization, major surgeries, minor procedures, medication, and computed tomography (CT) or magnetic resonance imaging (MRI). The unit and total costs by age group and the mean direct, indirect, and total costs during follow-up were estimated, in different diagnostic groups. The total cost per one million population was obtained. The costs were represented in euros, aligned with the Finnish health care producer price index 2020 .The estimations of the future costs after follow-up are presented in supplementary Tables This study was approved by the ethics committee of the Helsinki–Uusimaa Hospital District and followed the tenets of the Declaration of Helsinki.Of the 1151 patients, 202 were children aged under 17, 831 were adults aged 17–60, and 118 patients were over 60 years old.The number of outpatient visits was 2,436. Children needed 444 (mean 2.2), adults 1,722 (mean 4.1), and seniors 270 visits (mean 2.3).The number of major operations was 248 for 149 patients. Fourteen percent (28) of children, 12% (97) of adults, and 20% (24) of seniors underwent surgery. The number of minor procedures was 314 for 301 patients.Inpatient days were 313 for 90 patients. Eight percent (17) of children, 7% (57) of adults, and 14% (16) of seniors needed inpatient care.Table Medication was used by 1,024 patients for 13,512 days .We estimated that 84 patients had to undergo 86 imaging of the head .Based on the location of the patients living in HUEH area, 54.8–76% received a travel allowance of EUR 51.98/trip, after paying a deductible of EUR 9.25/trip. The transportation cost for patients not receiving the travel allowance was estimated to be EUR 9.25/trip .A total of 6,902 days of lost productivity were needed for 825 people . From this amount of lost productivity, major operations caused 2470 days of lost productivity .The total cost was EUR 2,899,000 for 1151 patients and EUR 1,870,000 per one million population. This included lost productivity (49%), direct health care (43%), and transportation costs (8%)  of lost productivity were incurred by all new eye injuries that occurred during the 1-year study period in a population of 1.55 million. Contusions  and superficial minor injuries  were the largest diagnostic groups that caused lost productivity.An annual direct and indirect cost of USD 5 million  and a lIn the present study, outpatient visits incurred the greatest cost among the direct health care costs, in the follow-up period.The number of days as inpatient and as lost productivity caused by OGI has varied in previous studies. The mean length of hospital stay for perforating eye injuries in HUEH in Finland was 26 days in the 1950s , 20 daysThe health care cost level is lower in Finland than in the other Nordic countries and the USA , 34. ThiA few other previous studies have reported data on the costs of hospitalization. According to Iftikhar et al., the median inpatient costs for eye injury in the USA was USD 11,000 (EUR 8000) in 2014  , after contusions (EUR 1.02 million), due to their highest occurrence among all diagnostic groups. In Croatia, the costs of minor eye injuries were EUR 135,500 in 2002–2003 [09,600, aWe estimated the future costs incurred by the studied population  derived mainly from the high number of life-long follow-ups. After the follow-up, the estimated total mean cost per patient varied between EUR 470 for a minor superficial injury and EUR 20,110 for a contusion. The cost by a contusion was mainly due to the required high number of life-long follow-up visits . Third, we lacked data on the far-reaching future economic burden caused by permanently impaired patients. As we reported previously –22, 107 The future costs were presented in their nominal value; hence, discount rate was set to zero as the Finland Government Bond 10-year reference has been continuously negative starting from 24.4.2020 except a short period in May 2021 . DiscounOne of the strengths of the present study is that it is a population-based study, as HUEH is practically the only ophthalmology acute care trauma unit in the Helsinki and Uusimaa Hospital District in urban and rural Southern Finland and covers approximately 29% of the population of Finland. Some minor eye injuries may have been treated in private care outpatient clinics, but these operate mainly by appointment and do not offer acute eye injury care or treatment. Another strength of the study is its size which was suitably representative of a sparsely populated country such as Finland. Other strengths are that the study reported the costs of both major and minor eye injuries in the clinical follow-up period as well as life-long costs.In conclusion, eye injuries lead to considerable use of resources and costs in both the short and the long term. Knowledge of the burden and costs that eye injuries cause helps decision-makers set policies for priorities, helps prevent eye injuries, and may improve cost-efficiency.Policies should be set to prevent eye injuries, based on the preventable nature of these injuries, at individual and community levels. Compliance with safety regulations and measures at work and at home should be increased by enhancing people’s general knowledge about the risks and costs of eye injuries. The detailed savings and health-promoting effects of prevention remain to be shown by further studies.Supplementary file1 (DOCX 65.2 KB)Below is the link to the electronic supplementary material.


Cryptococcus gattii (C. gatti) is a rare cause of meningitis in the United States. Outbreaks in new geographic distributions in the past few decades raise concern that climate change may be contributing to a broader distribution of this pathogen. We review a case of C. gattii in a 23-year-old woman in Northern California who was diagnosed via lumbar puncture after six weeks of headache, blurred vision, and tinnitus.C. gattii. The patient was treated with serial lumbar punctures, followed by lumbar drain, as well as amphotericin and flucytosine. The patient had improvement in headache and neurologic symptoms and was discharged to another facility that specializes in management of this disease to undergo further treatment with immunomodulators and steroids.A 23-year-old previously healthy young woman presented to the emergency department (ED) after multiple visits to primary care, other EDs, and neurologists, for several weeks of headache, nausea, tinnitus, and blurred vision. On examination the patient was found to have a cranial nerve VI palsy (impaired abduction of the left eye) and bilateral papilledema on exam. Lumbar puncture had a significantly elevated opening pressure. Cerebrospinal fluid studies were positive for C. gattii may be a new pathogen to consider. This finding raises important questions to the medical community about the way global climate change affects day to day medical care now, and how it may change in the future.Fungal meningitis is uncommon in the US, particularly among immunocompetent patients. Due to climate change,  Cryptococcus gattii (C gattii) is an encapsulated yeast whose global distribution has begun changing, possibly due to climate change. This pathogen lives in the soil and in association with certain trees. It was first isolated from eucalyptus but has also been isolated in other tree species in tropical and subtropical geographic distributions, especially Australia and Papua, New Guinea, and to a lesser degree Africa, Europe, Mexico, and South America.C gattii is not well documented but average symptom onset may be six to seven months from exposure. It is not contagious between people.1Climate change is expanding the landscape of infectious disease and may impact the daily practice of emergency medicine. C gattii has either adapted to a new climate niche or that climate change has opened new areas that C gattii can inhabit.6In the early 2000s, an outbreak was documented in the Pacific Northwest in primarily human immune-deficiency virus (HIV)-negative patients.A 23-year-old previously healthy woman presented to the emergency department (ED) with nausea and vomiting, weight loss, headache, tinnitus, diplopia, photophobia, dizziness, and paresthesia. Six weeks prior to presentation, the patient started having light-headedness, tinnitus and zigzags in her vision. She then developed nausea, vomiting, and headaches, and over several weeks had multiple visits with her primary care provider, a neurologist, and an outside hospital ED. Outpatient labs and a non-contrast computed tomography (CT) of the head were reportedly negative. She was prescribed a succession of antibiotics for presumed sinusitis, migraine prophylaxis and abortive medications, and received botulinum toxin injections to her forehead, temples, and neck.On arrival to our ED, the patient reported progressive headache and emotional lability, as well as new numbness and tingling in her left fourth and fifth fingers and right foot. She denied alcohol, smoking, or drug use. Her only reported travel was to Singapore and Taiwan approximately three months prior to symptoms onset. She denied any preceding illnesses. Intake vital signs were as follows: heart rate of 84 beats per minute, blood pressure 107/83 millimeters of mercury, respiratory rate of 20 breaths per minute, temperature of 36.8° Celsius, and pulse oximetry 99% on room air. Physical exam was notable for a supple neck with a full range of motion without pain. Her neurologic exam was notable for a sixth (VI) cranial nerve palsy of the left eye, and subjective decrease in sensation in her left fourth and fifth digits and in her right ankle and foot. While the patient was alert and oriented, her affect fluctuated between animated and flat. On fundoscopic exam, she had pale optic discs bilaterally and papilledema.20) . Cerebrospinal fluid studies showed 70 white blood cells ; 75% lymphocytes (40–80%); protein 62 milligrams per deciliter (mg/dL) (15–45 mg/dL); glucose 34 mg/dL (40–70 mg/dL), and Gram stain with evidence of fungal elements consistent with C gattii . A non-contrast CT of the head was obtained, which was negative for any acute intracranial process . A lumbaC gattii . CultureWhat do we already know about this clinical entity?is a leading cause of meningitis in immunocompetent hosts in tropical/subtropical regions and has a high mortality rate.Cryptococcus gattii What makes this presentation of disease reportable?The rarity and nonspecific presentation of this pathogen require a high level of suspicion by the emergency clinician to reach the diagnosis.What is the major learning point?is a rare pathogen in the United States, but recent outbreaks suggest climate change may contribute to its broader distribution.C. gattii How might this improve emergency medicine practice?It is crucial that emergency clinicians consider the effect of the planet’s changing climate on the distribution of disease.Magnetic resonance imaging (MRI) showed a 5.6 mm ring-enhancing lesion in the left anterior frontal lobe consistent with a cryptococcoma . AdditioThe patient’s symptoms of headache, diplopia, nausea, vomiting and tinnitus gradually improved after induction therapy with amphotericin and flucytosine and multiple lumbar punctures per week, followed by a lumbar drain. After a one-month admission she was discharged to another facility that specializes in immune modulation and steroids. After treatment at the specialty facility, her headache, nausea, and vomiting resolved. Her exam upon discharge was significant only for mild bilateral cranial nerve VI palsies, and her cerebrospinal fluid cultures remained negative for over two weeks. The patient was ultimately discharged from the specialty facility on indefinite fluconazole prophylaxis.Cryptococcus gattii is an encapsulated yeast that has traditionally been found in tropical and subtropical geographic distributions. In more recent years it has also been cultured from eucalyptus trees exported from Australia to California and the Pacific Northwest.C gattii infection dying directly because of the infection, and 13% dying from complicating factors.9Symptoms may occur months to years before clinical diagnosis, as the onset can be quite insidious. Patients may present with a nonspecific assortment of constitutional, respiratory, gastrointestinal, and central nervous system symptoms over several weeks or months. In addition, a non-contrast CT can be negative in as many as 50% of cases, requiring the clinician to maintain a high level of suspicion to continue pursuing a central nervous system etiology.C gattii. One study in Singapore identified 62 patients with Cryptococcus over eight years at a 1400-bed hospital, and only three of those (4.8%) were infected with C gattii, the majority being Cryptococcus neoformans (C neoformans).C neoformans and 4.1% by C gattii. This is compared to Australia and New Zealand, where 85% of cryptococcal infection are caused by C neoformans and 15% by C gattii.C gattii during her travels to one of these countries or at home in northern California. This is especially concerning given that since the late 1990s there has been an increase in the incidence of this pathogen in the Pacific Northwest and subsequently in other states as well, including California.This patient had traveled to Singapore and Taiwan three months prior to the onset of symptoms, and both countries have rare, reported cases of C gattii infection in the northwestern United States have occurred in Washington and Oregon, and it has been identified in environmental samples in San Francisco, California.C gattii has previously been found. Some hypotheses propose that C gattii has existed in the environment of the Pacific Northwest for the past 35 years but that recent climate changes have allowed for higher concentrations to flourish.Cases of Cryptococcus gattii is a yeast previously found only in specific geographic and climate distributions. Cryptococcal infections are thought to be the most common cause of fungal meningitis in immunocompetent hosts, although most research has focused on HIV-positive patients.C gattii’s potential habitats. It is becoming increasingly crucial that emergency clinicians consider the implications of the planet’s changing climate on the distribution and manifestation of disease, and as a result consider additional neurologic and infectious workup in young, healthy, and immunocompetent persons with intractable headache and no other objective signs of infection.Although still rare, this pathogen is becoming more common in new parts of the world such as the Pacific Northwest and California, which may be related to effects of climate change on broadening


At the same time, the optimized ceramic supports presented a typical mullite phase and excellent resistance to acid and alkali. This work provides a potential route for the preparation of ceramic membrane supports with characteristics of low cost and high performance.Support is a necessary foundation for ceramic membranes to achieve high performance. Finding the optimum balance between high performance and low cost is still a significant challenge in the fabrication of ceramic supports. In this study, low-cost fly ash-based ceramic supports with enhanced performance were prepared by the addition of bauxite. The pore structure, mechanical strength, and shrinkage of fly ash/bauxite supports could be tuned by optimizing the bauxite content and sintering temperature. When the sintering temperature and bauxite content were controlled at 1300 °C and 40 wt%, respectively, the obtained membrane supports exhibited a high pure water permeance of approximately 5.36 m Ceramic membranes have been widely used in water treatment owing to their distinctive characteristics, such as high flux, antifouling properties, long life, and flexibility of operation ,2,3,4,5.2O3 powders and sintered at temperatures above 1700 °C [2O3, SiO2, and other metal oxides. Thus, using fly ash as a raw material to prepare ceramic supports can also reduce the material cost. Zong et al. [Conventional high-performance ceramic membrane supports are usually fabricated from high-purity Al 1700 °C ,9. The h 1700 °C ,11. Thes 1700 °C ,13,14,15 1700 °C ,17, clay 1700 °C ,19, pozz 1700 °C , and fly 1700 °C ,23,24,25 1700 °C , and it 2O3 as the aluminum source, and AlF3 as the mullite whisker promoter. After sintering at 1200 °C, the bending strength of the ceramic supports was 59.1 MPa and the porosity was 26.8%. In our previous work [However, fly ash has a wide particle size distribution and low melting temperature, which hinders the control of the sintering process and the obtainment of a macroporous structure with uniform pore size distribution and high porosity. Moreover, the consistency and composition of fly ash are significantly affected by the source and even the season. In recent years, some researchers have tried to adjust the composition of fly ash and control the porous structure of supports through doping methods. Liu et al.  preparedous work , fly ash2 in fly ash [2 and Al2O3 [2 and produce secondary mullite, thereby enhancing the resistance of the ceramic under harsh conditions [As evidenced above, most of the materials added to fly ash are rich in aluminum. The reason for using these components is that the percentage of mullite in the obtained ceramics can be increased by introducing an additional source of aluminum to combine with the free SiO fly ash . Mullitend Al2O3 ,32. Thernditions .2SO4 and NaOH solutions at 100 °C and the bending strength before and after treatment was measured.In this study, low-cost ceramic membrane supports were fabricated from industrial waste fly ash and bauxite was employed as an aluminum source. The effects of bauxite content and sintering temperature on the structure and performance of the prepared supports were systematically investigated by taking into account porosity, pore size, bending strength, pure water permeance, and micromorphology. In addition, the resistance of the optimized ceramic membrane supports to acid and alkali was characterized using H−1).The fly ash powders used in this study were purchased from a power plant in Hebei Province, China, whereas bauxite powders were purchased from Shijiazhuang, Hebei, China. These materials were used as received, without purification. Glycerol  and polyvinyl alcohol  were used as binders to obtain fly ash supports. The pure water used in this work was deionized water  were added in the respective amounts of 4 and 3 wt% to the total mass of bauxite and fly ash. Glycerol acts as a lubricant to make the powder particles mix more evenly. PVA acts as a binder to improve the strength of the green support. Subsequently, the powder and additives were mixed well in a mortar and then shaped using the dry press molding method to form disk supports (30 mm in diameter and 2.8 mm in thickness) and rectangular column supports (50 mm × 6.1 mm × 5.9 mm). The obtained green samples were sintered at different temperatures varying from 1150 to 1300 °C at an interval temperature of 50 °C. During the sintering process, the holding time was 2 h, and the heating rate was 2 °C/min. Samples were generically denoted as Ax-y, where x indicates the mass of bauxite added to fly ash (100 g), and y is the sintering temperature . The detailed compositions of the samples are presented in φ is the shrinkage of the ceramic support, and V is the volume of liquid on the permeate side, in L; A is the effective filtration area of the ceramic support, in m2; t is the filtration time, in h; ΔP is the transmembrane pressure, in bar; F is the pure water flux of the ceramic support, in L·m−2·h−1; and J is the pure water permeance of the ceramic support, in L·m−2·h−1·bar−1. The three-point bending strength of the rectangular column supports was measured using a universal testing machine  with at least five samples for each test and calculated according to the following equation:σ is the three-point bending strength of the ceramic support, in MPa; a is the length of the ceramic support, in mm; b and c are the width and height of the ceramic support, in mm; and F is the fracture force of the ceramic support, in N.The fly ash and bauxite powders were characterized using field-emission scanning electron microscopy . The particle sizes of fly ash and bauxite were measured using a laser particle sizer , while their elemental compositions were determined via X-ray fluorescence spectroscopy . The surface micromorphology of the ceramic support was characterized using a benchtop scanning electron microscope . The phase compositions of the fly ash and bauxite powders, as well as ceramic supports obtained at different sintering temperatures, were determined by X-ray diffraction . The shrinkage was calculated by measuring the diameters of the disk supports before and after sintering at different temperatures using Vernier calipers . The shrinkage rate was calculated using the following equation:2SO4 (20 wt%) solutions heated to 100 °C in an oil bath, and they exposed for 8 h; a set of samples was taken every 2 h. After 8 h of corrosion, the samples were soaked in 3.65 wt% HCl or 4 wt% NaOH solutions for 30 min to neutralize the residual alkali or acid on the surface of the samples, and then, all samples were rinsed with water; after that, the samples were placed in an oven at 70 °C for 12 h to completely remove the residual moisture. The dried supports were used to test the three-point bending strength and mass loss after corrosion. The mass loss rate was calculated using the following equation:Ceramic supports A40-1200 and A40-1300 were placed in NaOH (10 wt%) and H2O3 and SiO2, with a total proportion of approximately 80 to 90 wt%. Other metal oxides can aid in reducing the sintering temperature because of their low melting points. The morphologies and size distributions of the fly ash and bauxite powders are shown in The detailed compositions of fly ash and bauxite analyzed by XRF are listed in The XRD patterns of fly ash and bauxite are shown in The effects of bauxite addition on the micromorphologies of the ceramic supports sintered at different temperatures were studied, as shown in In general, the resistance of fly ash supports to acid or alkali is determined by their phase composition. The phase of the supports sintered at different temperatures between 1150 and 1300 °C with different bauxite contents is shown in 2O in the fly ash, which react with cristobalite to form a glass phase at high temperatures [When the sintering temperature reached 1250 and 1300 °C c,d, the eratures . In the The effects of different bauxite contents on the shrinkage and porosity of the ceramic supports obtained by sintering at different temperatures are shown in The variation of support porosity as a function of bauxite doping is shown in The effects of bauxite content and sintering temperature on the pore size distributions of fly ash/bauxite supports were investigated at different sintering temperatures between 1150 and 1300 °C; the results are shown in In the A0 supports a, the avFor supports A20 to A100, the pore size increased with increasing sintering temperature. This is consistent with the trend observed in the A0 supports, where a high sintering temperature promoted particle growth. However, the sintering temperature did not have any clear adverse effects on the porosity of the A20 to A100 supports. In addition, when the sintering temperature was 1250 and 1300 °C, the A20 to A100 supports exhibited a narrower pore size distribution than A0. This demonstrates that the addition of bauxite can endow fly ash supports with a narrow pore size distribution while maintaining a high porosity. It is clear that the pore size distributions were determined by the sintering temperature and the amount of bauxite added.F is the pure water flux, In general, high bending strength and high permeance to pure water are desirable properties for ceramic supports. The experimental and calculated pure water permeance of fly ash supports obtained by sintering at different temperatures with different bauxite contents are shown in −2·h−1·bar−1 for an increase in sintering temperature from 1150 to 1300 °C, as shown in The pure water permeance of the A0 supports decreased from 4300 to 0 L·mThe bending strengths of the fly ash supports obtained with different sintering temperatures and bauxite contents are shown in The corrosion resistance of a ceramic membrane determines its range of applications. In this study, the A40-1200 and A40-1300 supports were tested to characterize the corrosion resistance of the fly ash support (see details in The bending strength and mass loss rate of the ceramic supports before and after acid corrosion are shown in A comparison of the properties of the supports prepared in this work with those reported in the literature is shown in 3·m−2·h−1·bar−1 and bending strength of 69.6 MPa. The corrosion tests and comparison with the literature demonstrated the good resistance of this support to acid and alkali. Therefore, the supports prepared in this work can be employed in practical applications where resistance to harsh conditions is necessary.Fly ash and bauxite were used as raw materials to prepare ceramic supports. Bauxite served as the aluminum source to promote mullitization during sintering and increase the corrosion resistance of fly ash supports. The effects of the sintering temperature and amount of bauxite on the performance of the fabricated supports were thoroughly investigated. With an increasing bauxite doping, the shrinkage of the supports decreased while the porosity increased. The pore size of the supports decreased slightly with an increase in bauxite content, but the trend was not definite. In the supports having the same bauxite content, the pore size would increase at higher sintering temperatures. At 1300 °C and 40 wt% bauxite, we manufactured a fly ash support with a pure water permeance of 5.36 m


The microorganisms associated with an organism, the microbiome, have a strong and wide impact in their host biology. In particular, the microbiome modulates both the host defense responses and immunity, thus influencing the fate of infections by pathogens. Indeed, this immune modulation and/or interaction with pathogenic viruses can be essential to define the outcome of viral infections. Understanding the interplay between the microbiome and pathogenic viruses opens future venues to fight viral infections and enhance the efficacy of antiviral therapies. An increasing number of researchers are focusing on microbiome-virus interactions, studying diverse combinations of microbial communities, hosts, and pathogenic viruses. Here, we aim to review these studies, providing an integrative overview of the microbiome impact on viral infection across different pathosystems. In 1676, Antonie van Leeuwenhoek saw and described microbes for the first time ever. Still, the study of microorganisms did not advance much until the late 1800s, when Robert Koch and Louis Pasteur pointed at microbes as the cause of transmissible diseases. Since then, microbes had only been associated with the negative impacts they exerted on their host. This view started to change with the rise of the high-throughput sequencing techniques during the past decades and the study of the metagenome of microbial communities. Nowadays we know that all multicellular organisms have an associated microbiota: a set of microorganisms living within/over them and in their immediate surroundings. The term microbiome can allude to the combined genetic material of the microbiota or refer to the microbiota and their theater of activity: microbial structures, metabolites, and their mobile genetic elements . The microbiome is a factor to consider to successfully achieve a common optimal health  infections. Mice exposed to S. epidermidis suppressed the replication of IAV in the nasal mucosa. This suppression prevented IAV spread to the lung and was caused by the stimulation of interferon (IFN) innate immunity. The same pathway was also activated by S. epidermidis in human cells  that suppressed infection with RSV, protecting the host against the lung disease caused by the virus. This defense response was associated with production of IFN-β in alveolar macrophages and the abundance of Corynebacterium and Lactobacillus in the lungs. Stefan et al. (Bacteroides spp. can induce IFN-β through Toll-like receptor 4 (TLR4) signaling, which enhances resistance to vesicular stomatitis virus Indiana and IAV. The microbiome can also help in restoring host immunity after perturbation with chronic viral infections. Rhesus macaques chronically infected with simian immunodeficiency virus (SIV) showed an increased immune response when their microbiome composition was changed after a fecal microbiota transplant  restored the macrophage activation and type I IFN responses, diminishing virus replication. For chikungunya virus (CHIKV) infections, both germfree and microbiome-depleted mice show an increased viral burden. Providing those mice with Clostridium scindens reduced the viremia due to a restoration of type I IFN responses . Bradley et al.  studied a et al.  studied a et al.  showed tesponses .Drosophila melanogaster flies have a rapid mortality when infected with the RNA viruses cricket paralysis virus, Drosophila C virus, and Flock House virus. However, Hedges et al.  and porcine circovirus type 2 (PCV2)  . Patin e2 (PCV2)  studied n et al.  inoculatAedes aegypti is a vector for multiple viruses, including the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Carlson et al.  to different cultivars, causing catastrophic crop losses. Gong et al.  and TSWV rates of infection and severity of disease.The impact of the plant microbiome on infectious disease has been extensively studied for nonviral diseases , 67. ConThere is a promising future in microbiome-based interventions to improve plants’ defense against virus. This possibility has been shown for other pathogens. Inoculation of germinating plants with a native bacterial consortium significantly attenuates the plants’ mortality against bacterial and fungal pathogens . TransplSpecies face multiple threats that can reduce their population size. One of these threats is infectious disease, which is possibly a main factor in extinction risk . TherefoApis melifera, whose decline is mainly driven by habitat loss, pesticides, and several pathogens  how the microbiome may have different effects on virus infection, depending on the host, and (ii) how the microbiome may not be necessary for an infection to happen, although it can affect the interaction of the virus with the host: the microbiome may regulate important phenotypes of the pathogen in terms of the degree of virus replication or transmission. There are other pathosystems in which an effect of the microbiome on virus replication and transmission was observed. Axenic mice did not transmit the mouse mammary tumor virus (MMTV) to their offspring. Reconstitution of the mouse bacterial community restored MMTV transmission. This seems to happen as MMTV binds bacterial lipopolysaccharides to trigger Toll-like receptor 4, inducing an immune evasion pathway by producing inhibitory cytokine IL-10 (Haemophilus influenzae increases the expression of a receptor used by human rhinoviruses. This results in an enhanced virus replication and inflammatory response to RSV. In the case of poliovirus (PV), the virions’ stability and capacity to attach to host cells are enhanced when the virus binds to bacterial surface polysaccharides. This increase in stability and receptor affinity suggests that the microbiome may also increase PV replication and transmission -induced gastrointestinal symptoms in newborns. Axenic chickens infected with Marek’s disease virus had more severe disease  can cause cervical intraepithelial neoplasia, which could lead to cervical cancer. Oh et al.  found a s et al.  found ani et al.  found an disease . Similar disease .To treat or prevent viral infections, we mainly rely on two interventions: antiviral drugs and vaccines. The effect of antiviral drugs could require the microbiota or be enhanced using microbiome interventions, as the microbiome can affect the degree of efficacy and toxicity of the drug along with drug metabolization –98. Some–Actinobacteria species prevalence may increase the vaccine response, while Enterobacteriales, Pseudomonadales, and Clostridiales were associated with lower response to the aforementioned vaccines. Hagan et al.  the effect of a specific microbiome on one virus might not be the same for other virus strain or species, and (ii) the relationship between the microbiome and virus infections could be unique at the host species level . Furthermunities .Enterococcus faecalis in the host microbiome drove the evolution of a bacterial pathogen toward reduced virulence. Following the idea of redirecting virus evolution toward less pathogenic strains (Finally, the microbiome could be used not only for altering the viral infection outcome, but also for driving the evolution of the virus. Ford et al.  showed t strains , the mic strains  or adver strains . When dr strains . HoweverThe microbiome can play a fundamental role in viral infections. The host’s microbiome may determine the success of a virus infection in an organism and/or the severity of the viral disease. The research on the microbiome-virus interactions is promising: the advances in the near future should give us insights into the nature and mechanisms behind the microbiome’s influence on viral infections. Microbiome-virus research is developing across various fields and organisms. This research will aid the development of interventions that reduce the viruses’ impact, which could be applied to different hosts. These microbiome-based interventions would contribute to the establishment of an integrated health approach to face pathogenic viruses and reduce the impact of diseases.


Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine.EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay.We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.The online version contains supplementary material available at 10.1186/s12985-021-01591-3. Solanum tuberosum L.), the fourth most important crop in the world [Potato , Andean lineage (PVSA), and Phureja lineage (PVSP) [As a high-throughput alternative, one-step real-time mRT-PCR is a desirable candidate. The real-time PCR technique is classified into two methods, one method using fluorescent-labeling probes and the other using double-stranded DNA (dsDNA) binding fluorescent dye. The latter, the intercalator method, is more cost-effective because only two fluorescent dyes, a dsDNA binding fluorescent dye and a reference dye for each well’s standardized fluorescent intensity, need to be compared with fluorescent-labeling probes to each target in the former method. In the intercalator method, the DNA dye is captured to dsDNA, not to single-stranded DNA (ssDNA), in a target non-specific manner, and amplicons can be identified by the melting temperature (Tm), which is measured as the release of the dye during denaturing dsDNA to ssDNA . Among intercalator methods, only one assay, which can identify multiple amplicons of potato viruses with different Tm values by the melt curve analysis, was reported . Their ae (PVSP) . PVY is e (PVSP) . In addie (PVSP) , so theiHere, we developed a new one-step real-time mRT-PCR assay for the four major potato viruses and used a plant gene as the internal control. Combined with the simple RNA preparation method , the detAll viral isolates in the previous report were also used in this study . The virhttps://www.ncbi.nlm.nih.gov/), considering lineages of PVS and strains of PVY. Alignment analysis was performed using the MEGA-X program (https://www.megasoftwa re.net/) to determine the conserved regions. For internal control, which was an indicator of successful RNA preparation and DNA amplification, five sequences of mRNA for potato elongation factor 1 alpha gene (EF1α)  were used and the conserved region was also determined using MEGA-X. The melting temperature (Tm) value of primer was calculated by Oligo calculator , and all primers were designed to be around 60 °C in their Tm values, except degenerated primers for PVS which were resulted in the range of 60–65 °C. The Tm values for predicted amplicons were calculated by uMelt batch software of DNA-UTAH.ORG (https://dna-utah.org/index.html) with the following options: 30 mM mono cation, 1.2 mM free Mg2+, 10% dimethyl sulfoxide (DMSO), “Huguet et al. [Primers for PLRV, PVS, PVX, PVY were designed based on conserved regions among more than six representative sequences  were used. Primer concentrations were optimized by comparing the heights of each specific peak in the melt curve for the four viruses contaminated samples to adjust the amplification efficiency for simultaneous detection of all four viruses. All primer concentrations were initially set at 0.2 µM in the reaction mixture and decreased or increased depending on the height of peaks. For simultaneous detection of the four viruses, the RNA solution was prepared from a potato leaf homogenate containing all four viruses, prepared by mixing an equal volume of four potato leaf homogenates, each containing one of the four viruses.For this experiment, isolates ChLR_2 for PLRV, M for PVSPCR Procedure was as follows. The RNA solution was extracted using a simple filter paper-based method . The RNAR2) for the regression line, which was given by the spreadsheet program, Microsoft Excel 365. The calculation of Tm values for non-Japanese isolates was performed as described above.A non-specific peak was observed in the melt curve for virus-free potato leaf and similar to the PVS-specific peak in the multiple detections. The assay was performed to detect three viruses without PVS genome or PVS primers to reveal whether the peak in multiple detections was a specific or non-specific product. For the assay without PVS genome, RNA solutions were prepared from each singly-virus-infected leaf homogenate, except PVS-infected, and an equal volume of the three solutions was mixed and used as a template. For the assay without PVS primers, the template solution was prepared from the mixture of the four potato leaf homogenates. To validate the calculation, calculated Tm values for Japanese isolates in Table O, O-IC249 for PVX, and Eu-12Jp for PVYNTN. The detection sensitivity was confirmed using tenfold dilution series for each virus, prepared by mixing with singly-virus-infected and virus-free potato leaf homogenate. The RNA preparation, the assay, and the analysis of the result were done as described above.The identical four virus isolates were used in this experiment: ChLR_2 for PLRV, M for PVSA and PVSP , O strain (PVYO), O5 strain (PVYO5), Eu-N strain (PVYEu-N), NA-N strain (PVYNA-N) and E strain (PVYE) [Considering genetic diversity of PVS and PVY, primer sequences reported by Cheng et al.  were comn (PVYE) . The seqEF1α , and the calculated Tm value seemed to be sufficiently reliable to presume Tm values for non-Japanese isolates using their genome sequences. Based on the calculation, Tm values for representatives were not overlapped with other viruses, and the values for PVY were from 77.5 to 79.5 °C, PVS from 81.0 to 82.5 °C, PVX from 84.5 to 85.5 °C, and PLRV from 87.0 to 87.5 °C in ascending order  to 83.1 °C (PVSA) and from 79.4 (PVYNA-N) to 80.5 °C (PVYO or PVYNTN), respectively. This result was consistent with the previous study on real-time mRT-PCR assay with melt curve analysis for four grapevine viruses, and the values for each virus species were also varied about 1.0 °C according to isolates in their report [O and isolate Leona of PVSO. The actual Tm values for these isolates in our assay were unknown, and the Tm values for these isolates might be more closed to each other than our calculation. The shape of the peak for PVY in the melt curve was sharper than that for PVS; this feature would be useful for diagnosing these two viruses. Our primers would be able to identify representatives by Tm values and the shape of the peak.There was no real-time RT-PCR assay with melt curve analysis which would detect all Japanese isolates of the four viruses. All primers for the four potato viruses were newly designed in the conserved region among representatives not to overlap amplicon Tm values with the other species to meet our requirement. Amplicon Tm values for PVS and PVY were within a broad range and varied by lineages of PVS and strains of PVY, from 82.2 (PVSr report . We coulEF1α, so we would suggest the below criterion removed the non-specific peak. The Ct value for RNase-free water as the negative control was over 30.0, so the criterion for successful amplification was also set. Namely, these two criteria were set for our assay.Successful amplification is “Ct value ≤ 30.0.”Virus-specific peak higher than the internal-control peak is evaluated as positiveOur assay could detect the four viruses simultaneously from the mixture of four homogenates of singly-virus-infected potato leaves using newly designed primers. A few real-time mRT-PCR assays with melt curve analysis for plant viruses were reported , 17–19, EF1α-specific primers do not cross exon and detect both mRNA and genomic DNA for EF1α. The peak for EF1α in melt curve analysis will somehow overestimate if crude RNA solution is applied as a template. The result showed that the specific peaks for all four viruses were observed in 1000-fold dilution. However, the 1000 dilution of PVS and PLRV were evaluated as negative by the second criterion set above. Our assay with criteria could detect any of four viruses from 100-fold dilution and seemed to have enough sensitivity to detect each virus in Japanese seed potato quarantine.The two criteria also affect the evaluation of detection sensitivity. The detection sensitivity of real-time RT-PCR assays has been confirmed using purified genome RNA  and plasIn Japan, plants are grown out from tubers, and the leaves of the plants are used for viral detection because the virion of PVY, which is the most common virus in the world, is gradually degrading during the dormancy period of tubers until breaking the dormancy . Our orgOur labor-saving high-throughput detection assay for internationally important potato viruses PLRV, PVS, PVX, and PVY can be used with the simple RNA preparation method we reported previously. This protocol is useful for detecting viruses in many samples for seed potato quarantine and field surveys.Additional file 1. Supplementary Figure 1. The primer binding position in viral genome. The position referred to the representative sequences written in Virus Taxonomy ninth edition: GenBank accession X14600 for PLRV, AJ863509 for PVS, D00344 for PVX, and AJ890348for PVY. Primer binding sequences and their direction were shown under each alignment, and red letters meant the position of mismatches between primer and genome sequences, and degenerated nucleotides were replaced with represented nucleotides. Abbreviations for lineages of PVS; O, PVSO; A, PVSA; P, PVSP, and those for strains of PVY; C, PVYC; NA-N, PVYNA-N; E, PVYE; O5, PVYO5; Eu- N, PVYEu-N; O, PVYO. Supplementary Figure 2. The confirmation of mismatches between viral genome and reported primers. The position referred to the representative sequences written in Virus Taxonomy ninth edition: GenBank accession AJ863509 for PVS and AJ890348 for PVY. Pimers’ binding sequences and their direction were shown under each alignment, and red letters meant the position of mismatches between primer and genome sequences and degenerated nucleotide was replaced with represented nucleotide. Abbreviations for lineages of PVS; O, PVSO; A, PVSA; P, PVSP, and those for strains of PVY; C, PVYC; NA-N, PVYNA-N; E, PVYE; O5, PVYO5; Eu-N, PVYEu- N; O, PVYO.


The ubiquitin system has emerged as a master regulator of many, if not all, cellular functions. With its large repertoire of conjugating and ligating enzymes, the ubiquitin system holds a unique mechanism to provide selectivity and specificity in manipulating protein function. As intracellular parasites viruses have evolved to modulate the cellular environment to facilitate replication and subvert antiviral responses. Poxviruses are a large family of dsDNA viruses with large coding capacity that is used to synthetise proteins and enzymes needed for replication and morphogenesis as well as suppression of host responses. This review summarises our current knowledge on how poxvirus functions rely on the cellular ubiquitin system, and how poxviruses exploit this system to their own advantage, either facilitating uncoating and genome release and replication or rewiring ubiquitin ligases to downregulate critical antiviral factors. Whilst much remains to be known about the intricate interactions established between poxviruses and the host ubiquitin system, our knowledge has revealed crucial viral processes and important restriction factors that open novel avenues for antiviral treatment and provide fundamental insights on the biology of poxviruses and other virus families. Poxviridae family are large DNA viruses that infect a wide variety of species, from insects (classified in the Entomopoxvirinae subfamily) to vertebrates, including fish, reptiles, birds, and mammals (classified in the Chordopoxvirinae subfamily). The best-known poxviruses are variola virus (VARV), the smallpox agent, and vaccinia virus (VACV), its vaccine. In addition, several poxviruses are emerging zoonoses, like monkeypox virus (MPXV) and cowpox virus (CPXV), and others affect economically important animals such as orf virus and capripox viruses. Poxviruses follow a complex replication process in the cell cytosol, producing two types of virions generally known as extracellular virus (EV) and intracellular mature virus (MV) .,79.β-TrCbstrates . WhetherRecent work has shown that some OPXV ANK proteins associate with cellular Cul-2-based CRL (CRL2) complexes . CRL2 utWhilst CRL1 and CRL2 utilise adaptors and substrate receptors, Cul-3-based complexes (CRL3) differ and employ a single protein combining both functions. This type of adaptor therefore contains a domain that mediates interaction with Cul-3 known as bric-a-brac, tramtrack, and broad complex transcription (BTB)  or poxviThe molecular and functional architecture of CRL1, 2, and 3 is generally shared with Cul-4-based CRL (CRL4) and Cul-5-based CRL (CRL5). CRL4 can be formed by Cul-4A or Cul-4B, and in both cases employ the DDB1 adaptor and a large repertoire of substrate receptors known as DCAF (DDB1-Cul-4A-associated factors) ,104. It In addition to CRL1-5, non-canonical Cul such as Cul-7, APC/C, and PARC exist and have been shown to have E3 Ub ligase activity ,109,110.Besides adaptors, E3 Ub ligases have also been identified in poxvirus genomes. This essentially involves two families of proteins: the poxvirus MARCH E3 Ub ligase and p28. The poxvirus MARCH (from membrane-associated RING-CH) consists of a RING-CH domain followed by two transmembrane domains, in an arrangement equivalent to that observed in the Kaposi’s sarcoma associated virus (KSHV) K3 and K5 Ub ligases . It can makorin cDNA [The p28 Ub ligase is a viral protein containing N-terminal KilA-N domains followed by a C-terminal RING domain. It is present in multiple poxvirus genera, including VARV and several OPXV, although it is inactivated or absent in VACV strains widely used for experimentation, such as Western Reserve or Copenhagen ,127. In rin cDNA . Early wrin cDNA . Subsequrin cDNA ,131. Thirin cDNA ,133. Therin cDNA ,133,134.Besides E3 Ub ligases, bioinformatics searches have also identified Ub-like genes in the genomes of certain avipoxvirus species, such as canarypox virus, penguinpox virus, or flamingopox virus, and several entomopoxviruses ,138,139.Besides rewiring cellular E3 Ub ligases and targeting of cellular factors, viral proteins are also ubiquitylated during infection. As indicated above, poxvirus uncoating requires active UPS to mediate the breakdown of the internalised cores, which is consistent with the detection of K48-linked ubiquitylated cores . ConsistSome viral immunomodulators are also known to be directly ubiquitylated. Protein E3 is a dsRNA-binding protein known to prevent activation of protein kinase R and RNAse L and IFN-primed necroptosis ,147,148,Recent proteomics work has shown that VACV infection of human fibroblasts results in the downregulation of 265 cellular proteins . Out of Over the last decades, Ub and ubiquitylation have emerged from being a cryptic little protein modification into a master regulator of cell biology. Our knowledge on how the Ub system interplays with poxviruses has also expanded accordingly. It is now clear that poxviruses encode many different types of molecules interacting with the Ub system. Many of these have been identified as adaptors of cellular E3 Ub ligases due to sequence similarity to their cellular counterparts. Whilst great progress has been made on identifying these adaptors and understanding how they operate, elucidating the substrates that are targeted by these virally assembled ligases remains an important task, as these targets are likely to be important restriction factors and antiviral molecules. Comparative virology and genetic approaches have proven useful, such as in the case of RIPK3 and, likewise, quantitative proteomics has allowed identification of the IFITs and HDAC4/5. Furthermore, complete proteome analyses have shown enormous potential in revealing important classes of cellular proteins downregulated  or modified during infection. These datasets uncover the vast reconfiguration that a cell encounters upon poxvirus infection, and they will shed light onto novel cellular hubs and functions in cell biology and immunity. Whilst ubiquitylation during viral infection is likely to be largely modulated by the many virally encoded Ub adaptors reported so far, it is important to consider the virally encoded E3 Ub ligases and the existence of yet unidentified non-canonical adaptors. Finally, it is now also clear that the UPS plays a major role in allowing uncoating, genome release, and allowing productive infection. Understanding how cellular proteasomes are recruited to the incoming viral cores and how they recognise core proteins are important questions because of the potential to yield new therapeutic approaches for antiviral intervention. Although poxviruses are the only DNA viruses to replicate exclusively in the cytosol, most RNA viruses uncoat and release their RNA genomes in the cytosol. Ubiquitylation is also involved in poxvirus DNA replication, and similar biological processes may be operating in the replication of other large DNA viruses such as African swine fever virus or the Herpesviridae family. Therefore, knowledge obtained using tractable poxvirus models has the potential to enlighten conserved mechanisms used by many viruses to establish infection. With the emergence of induced proximity drugs and related technologies, expanding and deepening our knowledge on virus interactions with the Ub system is a timely and promising avenue to contribute to the fight against transmissible and non-transmissible diseases that continue to be major causes of disability and death.


The efficacy of DPWs to decontaminate carriers spiked with SARS-CoV-2, as well as the transferability of the virus from used DPWs to clean surfaces, is also assessed. Stainless steel carriers inoculated with approximately 6 logs of SARS-CoV-2 prepared in a soil load were disinfected within 5 min through exposure to 66.5% EtOH, 0.5% NaOCl, and 1% KMPS. The addition of mechanical wiping using DPWs impregnated with these biocides rendered the virus inactive almost immediately, with no viral transfer from the used DPW to adjacent surfaces. Carriers treated with 100 ppm of ClO2 showed a significant amount of viable virus remaining after 10 min of biocide exposure, while the virus was only completely inactivated after 10 min of treatment with 500 ppm of ClO2. Wiping SARS-CoV-2-spiked carriers with DPWs containing either concentration of ClO2 for 5 s left significant amounts of viable virus on the carriers. Furthermore, higher titers of infectious virus retained on the ClO2-infused DPWs were transferred to uninoculated carriers immediately after wiping. Overall, 66.5% EtOH, 0.5% NaOCl, and 1% KMPS appear to be highly effective biocidal agents against SARS-CoV-2, while ClO2 formulations are much less efficacious.High-touch environmental surfaces are acknowledged as potential sources of pathogen transmission, particularly in health care settings where infectious agents may be readily abundant. Methods of disinfecting these surfaces often include direct application of a chemical disinfectant or simply wiping the surface with a disinfectant pre-soaked wipe (DPW). In this study, we examine the ability of four disinfectants, ethanol (EtOH), sodium hypochlorite (NaOCl), chlorine dioxide (ClO The coronavirus SARS-CoV-2, first discovered in December 2019 in Wuhan City, Hubei province, China, has caused millions of human deaths since its declaration as a pandemic by the World Health Organization in March 2020. Aerosols in close proximity are believed to be the primary mode of transmission ; however2), and potassium monopersulfate (KMPS), to effectively inactivate SARS-CoV-2 using In this study, we test the ability of DPWs impregnated with one of four microbicidal actives common in the marketplace , namely,v/v penicillin–streptomycin ]. One day prior to testing, cells were trypsinized (Gibco LS25200056) and seeded into appropriate flasks or plates to reach ∼80% confluence the following day. On the day of testing, the CCM was removed and replaced with virus culture medium  for the duration of the experiment.African green monkey Vero E6 cells (ATCC CRL-1586) were propagated in cell culture medium (CCM)  supplemented with 10% fetal bovine serum [FBS; Gibco  and 1% EPI_ISL_425177hCoV-19/Canada/ON-VIDO-01/2020, GISAID accession# ) at an MOI of 0.01. At 3–5 days post infection, the infected supernatant was removed and clarified using low-speed centrifugation at 4,500 × g for 10 min. Supernatant was overlaid onto a 20% w/v sucrose cushion in Tris-NaCl-EDTA buffer (prepared in-house), centrifuged at 28,000 RPM in a Beckman Coulter SW 32 TI rotor for 2 h, and the resulting viral pellet re-suspended in VCM overnight at 4°C. The following day, the pellets were pooled, aliquoted, and quantified as per Virus stocks were prepared as previously described . BrieflyAll four chemical biocides were prepared in accordance with v/v) EtOH , 0.5% (v/v) NaOCl , and 1% (w/v) KMPS  were prepared in sterile hard water . For assays using ClO2 , solutions were prepared 1 day prior to testing and placed at 4°C overnight as per the manufacturer’s instructions. Active chlorine was measured the following morning, and solutions were diluted to 100 ppm  and 500 ppm  in hard water for disinfectant assays. Fresh batches of disinfectant were used for each independent experiment.The disinfectants 66.5%  was recorded . A total2, which was only assessed in two independent experiments.The effects of mechanical wiping were determined in accordance with the Wiperator methodology, 50 assays  decreased by 5.12 logs/carrier to 1.07 after 30 s of exposure. By 60 s, titers fell below the limit of quantitation , althoug0 assays  and safe0 assays .2 at a concentration of 100 ppm had a negligible effect on virus viability. A contact time of 30 s and 1 min with the disinfectant resulted in less than 0.5 log loss of viable virus  was degraded within the first minute of contact with the biocide, with full inactivation by the 5-min mark. KMPS is widely used as a chlorine-free oxidizing agent and is the main active ingredient in the multi-purpose laboratory disinfectant, Virkon . Virkon 2 has been shown to inactivate a variety of viruses, including SARS-CoV-2, in wastewater, albeit in conjunction with several other antimicrobial agents at low concentrations , or iodophor for 5 s removed adenovirus 8 from Goldmann tonometer and pneumotonometer tips, while a 5-min submersion time was required for inactivation by the same compounds in the absence of wiping.The effect of combining microbicidal actives and mechanical wiping action on the inactivation of viruses and bacteria has been evaluated in several studies . The ben2, it was inactivated using 500 ppm (0.05%) ClO2 by the 5-min time point during the QCT-2 assay. Notably, the virus was far more susceptible to both concentrations of the biocide when wiping action was implemented. After 5 s of wiping with 100 ppm (0.01%) and 500 ppm ClO2-infused wipes, a 2.78 and 4.27 log reduction in viable virus was observed on inoculated test carriers, respectively. Considering that both concentrations of the disinfectant had negligible-to-minor effects on the virus during the QCT-2 assay , these findings provide further support for the enhancement of viral inactivation when mechanical wiping is incorporated in the disinfection process.Although SARS-CoV-2 was resistant to degradation by 100 ppm ClO2, considerably high amounts of viable virus were recovered from both test and transfer carriers, indicating that transferring an infectious material from one surface to another via wiping is a legitimate concern. As such, it is imperative that any disinfection strategy involve the use of biocides proven effective against the infectious agent of concern rather than simply relying on commercial products due to ease or availability.Interestingly, in Wiperator tests involving ClO2. Even when dried within a protective organic matrix, roughly 6 logs of virus can be inactivated using the three former biocides within 5 min of exposure, while 500 ppm ClO2 requires a longer duration (at least 10 min). We have also demonstrated that incorporating these biocides into a mechanical wipe greatly enhances their efficacy against the virus, and in agreement with other studies , presence of an organic matrix, disinfectant used , and duration of exposure to the disinfectant . We have studies , infectiAlthough this study was on SARS-CoV-2 isolate Wuhan-Hu-1, we believe that the present findings would also apply to other circulating variants. Others have observed that there was no difference between SARS-CoV-1 and SARS-CoV-2 deposited on surfaces  or in aeHere, we evaluated readily available disinfectants or components of formulated disinfectants that can be used within healthcare facilities or by the general public, either on their own or by incorporating wiping. Following simple application, ethanol was the most effective at reducing 6 logs of dried virus within a soil load in a short amount of time, while sodium hypochlorite (bleach) and KMPS required longer contact times. By incorporating a mechanical wipe, the virucidal effects of ethanol, sodium hypochlorite, and KMPS were immediate, with no detectable virus remaining after only 5 s of wiping the surface.The original contributions presented in the study are included in the article/SK and TC contributed to the conception and design of the study and performed the methods. AS drafted the manuscript. All authors analyzed the results, contributed to the article, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The mutation in farR* alters the activity of the regulator, FarR*, in such a way that not only its own gene, farR*, but also the divergently transcribed farE gene and genes controlled by the global regulator, agr, are highly upregulated. Here, we show that mainly the upregulation of the fatty acid efflux pump FarE causes the RomR phenotype, as farE deletion in either the parent or the RomR strain (RomR ΔfarE) yielded hypersensitivity to Rom. Comparative lipidome analysis of the supernatant (exolipidomics) and the pellet fraction revealed that the RomR strain excreted about 10 times more phospholipids (PGs) than the parent strain or the ΔfarE mutants. Since the PG content in the supernatant  was more than 100-fold higher than that of fatty acids (FA), we assumed that PG interacts with Rom, thereby abrogating its antimicrobial activity. Indeed, by static and dynamic light scattering (SLS and DLS) and isothermal titration calorimetry (ITC) analyses, we could demonstrate that both PG and Rom were vesicular and reacted with each other in milliseconds to form a 1:1.49  complex. The binding is entropically driven and hence hydrophobic and of low specificity in nature. Our results indicate that the cytoplasmic membrane is the actual target of Rom, which is also in agreement with Rom’s induced rapid collapse of the membrane potential and decreased membrane integrity.Rhodomyrtone (Rom) is a plant-derived broad-spectrum antibiotic active against many Gram-positive pathogens. A single point mutation in the regulatory  Rhodomyrtus tomentosa , it has been shown that rhodomyrtosone B prevents skin ulcer formation and reduces the incidence of infection-related morbidity; its activity was comparable to that of vancomycin , originally isolated from plant extracts of omentosa . Rom haspyogenes –6. In a ncomycin . Structuncomycin . The elancomycin , 9.Rom interferes with none of the classical antibiotic targets, such as peptidoglycan biosynthesis, DNA replication, translation, and transcription, but targets the cell membrane by causing a strong dissipation of the membrane potential and release of ATP and cytoplasmic proteins . Rom doeS. aureus HG001 in medium supplemented with Rom (farR (regulator of fatty acid resistance). FarR belongs to the TetR family of regulators (TFRs), which are widely associated with antibiotic resistance and the regulation of genes encoding small-molecule exporters, but they are also involved in controlling many other aspects of prokaryotic physiology  displays an altered regulatory activity. In particular, farR, farE, agr (accessory gene regulator), and the Agr-controlled virulence genes were upregulated in the RomR mutant, the latter explaining the increased pathogenicity of the RomR mutant  became hypersensitive to Rom.The Rom-resistant mutant In this study, we demonstrated by qualitative and quantitative lipidomic analyses that the RomR strain releases large amounts of PGs into the supernatant and cell envelope, suggesting that FarE acts as a PG efflux pump. Rom resistance is mediated by interaction of PG with Rom, thereby abrogating its antimicrobial activity.farE is responsible for Rom resistance in RomR, we constructed markerless farE deletion mutants in the S. aureus HG001 wild-type and RomR mutant strains . Since the promoters of farE and farR are divergent and the corresponding transcripts overlapped as described by the group of Martin McGavin  was added to basic medium (BM) 1 h after starting the incubation, and the growth of S. aureus clones was followed for 20 h. Growth of HG001 was inhibited for the first 12 h but resumed thereafter. Growth of HG001 ΔfarE was inhibited for the whole period. Growth of RomR mutant was unaffected in the presence of Rom (completely resistant to Rom), while growth of RomR ΔfarE was inhibited for about 15 h and resumed thereafter  was followed for 24 h. Growth of HG001 was inhibited for about 12 h but resumed thereafter; growth of HG001 ΔfarE was inhibited for the whole period. Growth of the RomR mutant was unaffected in the presence of Rom (completely resistant to Rom), while the growth of RomR ΔfarE was inhibited for about 15 h and resumed thereafter. The result indicate that FarE is crucial for Rom resistance. The graph is a representative of one of the triplicate growth studies performed. Download FIG S1, PDF file, 0.2 MB.Impact of Rom on the growth of HG001 and RomR and their respective ΔCopyright © 2022 Huang et al.2022Huang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the farE mutants. The killing of the S. aureus clones in BM supplemented with Rom (8 μg/mL) was followed for 8 h. HG001 and HG001 ΔfarE were almost completely killed after 7 to 8 h, while the growth of the RomR mutant continued. With RomR ΔfarE, mutant killing was also observed, but it was markedly delayed compared to the case with HG001 and HG001 ΔfarE  was followed for 8 h. Cells were inoculated to a concentration of about 6 × 105 CFU/mL. HG001 and HG001ΔfarE were almost completely killed after 7 to 8 h, while growth of the RomR mutant continued. With the RomR ΔfarE mutant, killing was also observed, but it was markedly delayed compared to that of HG001 and HG001 ΔfarE. Each bar represents the mean ± SD from three independent biological replicates. Download FIG S2, PDF file, 0.1 MB.Impact of Rom on the killing of HG001 and RomR and their respective ΔCopyright © 2022 Huang et al.2022Huang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the farE mutants.One of our main questions was whether Rom resistance was due to Rom being expelled from the cell by the efflux pump FarE or whether the excreted PGs and fatty acids neutralized Rom. While two-times-concentrated supernatant of RomR strains grown to the end of the exponential phase was already able to protect HG001 from Rom, the supernatant of HG001 needed to be concentrated 20 times to achieve similar protection . This sufarE, and RomR ΔfarE showed no discernible differences. However, their growth in the defined minimal medium was generally decreased and growth of the two ΔfarE mutants was somewhat delayed (578] = 0.9 to 1.0). For the calculation of the sample concentration and the structure, internal standards (ISs) as well as the TripleTOF system and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were used. We analyzed not only the lipid/FAs in the culture supernatant but also those that were loosely bound to the cell wall, referred to as “pellet wash,” by detaching them from the cell wall with 90% isopropanol treatment. Absolute amounts of FAs/lipids were calculated in nanograms/OD578 and adjusted to the corresponding OD578 of 1.0. As described earlier, S. aureus synthesizes mainly saturated FAs , at which the cells were harvested for lipidomic analysis. Download FIG S3, PDF file, 0.1 MB.Comparative growth of HG001 and its mutants in defined minimal medium using a microplate reader. Each point in the graph is the mean ± SD of three independent biological replicates; bar indicates the end of the exponential phase  between HG001 and the mutants. However, the RomR mutant excreted about 2-fold more odd-numbered FAs  than HG001 and the two Δ mutants . If we afarE mutants. On average, the RomR strain released about 8 to 10 times more lipids than HG001 and the ΔfarE mutants. The most abundant lipids ranged from C29:0 to C34:0, with a peak from C30:0 to C32:0  was the most excreted lipid in terms of quantity, and it is in the levels of PG that we observed the greatest difference between RomR and HG001 and the Δto C32:0 . If we afarE, and 16 ng/OD for HG001 ΔfarE. It is reasonable that only a small amount (about 3%) of the PG is lysylated, since expression of MprF (multiple peptide resistance factor) is inducible , which was about 30-fold lower than for PG . If we anducible  and cellfarE mutants (Only trace amounts of diacylglycerol (DG) and monogalactosyldiacylglycerol (MGDG) were released by all strains, and there was no remarkable difference between RomR and HG001 or the Δ mutants . The tot mutants ; the tot mutants . These rDigalactosyldiacylglycerol (DGDG) was found in substantial levels in the supernatant, amounting to about 110 ng/OD for each strain, and there were no significant differences among the four strains, except for HG001 and RomR . Only infarE gene in the RomR mutant reversed the phenotype. This appears to represent direct evidence that overexpression of FarE is responsible for release of PGs.Summarizing the lipidomic results, we can say that the RomR strain excreted multiple amounts of PG and Lys-PG, compared to the other strains . By far S. aureus.To investigate the effects of Rom treatment on the release of lipids by the parent strain HG001 and the mutant strain RomR , Rom was10.1128/mbio.03833-21.4FIG S4S. aureus HG001 and RomR strains in the supernatant (top) and pellet wash (bottom). The bar charts show the amount of fatty acids ranging from C14:0 to C21:0 (A), phosphatidylglycerols (PG) ranging from C28:0 to C36:0 (B), and lysyl phosphatidylglycerols (Lys-PG) ranging from C29:0 to C:34 (C) detected in both strains in the presence of subinhibitory concentration of Rom (0.3 μg/mL). All absolute amounts of FAs and lipids are reported in nanograms/OD578 of the bacterial cultures grown until the end of the exponential phase and adjusted to the corresponding OD578 of 1.0. The supernatant and pellet were separated by centrifugation for lipidomic analyses. The pellet wash was obtained by treating the pellets with 90% isopropanol to remove the fatty acids and lipids from the surface of cells. Each bar shown in the chart represents the mean ± SD from five independent biological replicates. Download FIG S4, PDF file, 0.2 MB.Effect of subinhibitory concentrations of Rom on the release of fatty acids and lipids by Copyright © 2022 Huang et al.2022Huang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Next, we investigated which of the lipid components exported by FarE can most effectively neutralize the antimicrobial activity of Rom. For this, we supplied the medium (BM) with Rom (8 μg/mL ≈ 18 μM) and different lipid components in approximately the same molarity. Rom alone completely inhibited growth . Among t10.1128/mbio.03833-21.5FIG S5mprF. (A and B) Growth of S. aureus HG001 in BM with supplementations of fatty acids at 4 μg/mL of C15:0 (≈16 μM), 4 μg/mL of C18:0 (≈14 μM), 4 μg/mL of C16:0 (≈16 μM), 10 μg/mL of C16:1 (≈39 μM), and 21 μg/mL of C18:2 (≈75 μM) (A) and lipids at 4 μg/mL of PG(18:0) (≈4.6 μM) and 4 μg/mL of Lys-PG(18:1) (≈4 μM) (B). The well containing BM only and bacteria was regarded as a positive control. Growth of the bacteria was measured by OD578 every 4 h for 48 h using a Varioskan Lux microplate reader (Thermo Scientific) in a 48-well plate. Each point in the graph is the mean ± SD from three independent biological replicates. (C) Illustration of RomR ΔmprF deletion mutant. Download FIG S5, PDF file, 0.3 MB.Impact of exogenous supplementation of FAs/lipids on the growth of HG001 and construction of RomR ΔCopyright © 2022 Huang et al.2022Huang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mprF in the RomR strain, generating the strain RomR ΔmprF , suggesting that mprF does not play a role in Rom resistance.MprF encodes a bifunctional membrane protein that synthesizes the positively charged lipid Lys-PG and subsequently translocates it from the inner to the outer membrane leaflet . TherefomR ΔmprF . HoweverIn summary, our results show that essentially only the compounds most abundantly released by the RomR strain (PGs) can neutralize the activity of Rom. To demonstrate the postulated interaction of Rom with PGs, we used two methods: static and dynamic light scattering (SLS/DLS) and isothermal titration calorimetry (ITC).sn-glycero-3-phospho-(1′-rac-glycerol) , as a model PG that is also produced by S. aureus. Both Rom and PG(32:0) are almost insoluble in water. Therefore, we needed to find a common solvent for both compounds. We overcame the problem by dissolving PG(32:0) first in a chloroform/methanol/water mixture, and then we prepared vesicles formed in 10% dimethyl sulfoxide (DMSO)/phosphate-buffered saline (PBS) (pH 7.2), which is the same solvent as used for Rom.DLS is a frequently used technique for measuring the size distribution of dispersed particles in the range of 1 to 3,000 nm. For studies of interaction of Rom with P-lipids, we have chosen PG(32:0), 1,2-dipalmitoyl-DLS analysis with 1 mM PG(32:0) (diluted from a 5 mM sample solution) showed that the mean size at a 90° angle was 90.8 nm . This siH), equilibrium binding constant (DK), and stoichiometry of the reaction (n) involved in the interaction between Rom and PG(32:0) using ITC. The binding of Rom and PG(32:0) was determined titrating PG(32:0) into Rom solution at pH 7.2 in 10% DMSO/PBS buffer at 37°C. Three experiments were conducted with different conditions, all of which resulted in similar heat flows (H > 0). The contribution of the entropy change (ΔS) to the binding was favorable and large . In other words, binding is entropically driven and hence hydrophobic and of low specificity in nature. The n and DK were 1.49 (±0.0478) and 2.30 (±0.747) μM, respectively. The interaction of Rom with PG(32:0) was fast and occurred within milliseconds. The data are summarized in To gain further evidence for neutralization of Rom’s activity by the released PGs into the medium, we determined the enthalpy change (Δat flows : (i) injat flows , (ii) inat flows , and  upregulation of FarE and Rom resistance is correlated, and (ii) deletion of t to Rom . Howevert to Rom , or is FS. aureus strains HG001, RomR, HG001ΔfarE, and RomR ΔfarE by analyzing the FAs, lipids, PG, Lys-PG, DG, MGDG, and DGDG both analytically and quantitatively. Since we know that S. aureus is able to take up and incorporate unsaturated FAs present, for example, on human skin  into PGs and the lipid moiety of lipoproteins   , 22. The bilayer .S. aureus encodes cardiolipin synthases 1 and 2, we did not detect cardiolipin, most likely because we harvested the supernatant at the end of the exponential growth phase and it has been reported that cardiolipin is produced mainly in the stationary phase . Using the methods described, it was finally possible to dissolve both compounds in 10% DMSO/PBS (pH 7.2). Using SLS/DLS analysis, we could demonstrate that in freshly prepared solutions, both components formed vesicles with mean sizes at a 90° angle of 90.8 nm for PG(32:0) and of 120 nm for Rom. When the two components were mixed, the mean size of 100 nm at the beginning increased with time to 200 nm , which i10.1128/mbio.03833-21.6FIG S6farE, and RomR ΔfarE in the absence and presence of Rom. This illustration is similar to the one in FIG S6, PDF file, 0.1 MB.Lipidomic analysis in supernatants only for PGs in HG001, RomR, HG001ΔCopyright © 2022 Huang et al.2022Huang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the aK), reaction stoichiometry (n), the heat capacity (ΔCp) of the reaction, binding free energy (ΔG), entropy (ΔS), and enthalpy (ΔH). We obtained similar heat fluxes in all three experiments, each with slightly different conditions , we used isothermal titration calorimetry (ITC). ITC can measure the association constant (nditions . The resAnother question is how to explain the high Rom resistance (MIC > 128 μg/mL) in the RomR mutant . In the d-alanine racemase gene confers resistance to d-cycloserine in Mycobacterium smegmatis , an autoimmune disease occurring mostly in young women . It woulfarE-farR function is the following. In the wild type, farE expression is low and FarR acts only as a mild activator. In the RomR mutant, the point mutation in farR* causes FarR* to become a strong activator of farE and its own (farR*) gene expression. Overexpression of FarE, which acts most likely as a PG efflux pump, causes massive accumulation of PG in the supernatant and the cell envelope area. PG scavenges Rom, thereby abrogating its antimicrobial activity  containing 1% (wt/vol) soy peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% K2HPO4, and 0.1% glucose at pH 7.2. Bacteria were cultivated aerobically (200 rpm) at 37°C. For lipidomic analysis, we used a defined minimal medium as described by Rudin et al.  of Rom.Bacterial strains and plasmids used in this study are listed in n et al. . To inve10.1128/mbio.03833-21.7TABLE S1Table S1, DOCX file, 0.02 MB.Bacterial strains and plasmids used in this study. Download Copyright © 2022 Huang et al.2022Huang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the farE) and SAOUHSC_01359 (mprF) were amplified from the genomic DNA of S. aureus HG001 and RomR strains, respectively. Then, the fragments were assembled with the EcoRI-linearized pBASE6 using Hi-Fi DNA assembly master mix (New England BioLabs). The ligation mixtures were transformed into E. coli DC10B chemically competent cells. The correct plasmids were confirmed by PCR and sequencing before being transformed via electroporation into HG001 and RomR, respectively. Mutagenesis was performed as previously described  was added to each well. An inoculated broth without Rom was regarded as a positive control, and the well without bacteria was used as a negative control. The 96-well microtiter plates were incubated at 37°C for 24 h. The MIC values were determined as the lowest concentration that completely inhibited the visible growth of bacteria. The experiments were repeated three times.MIC values of Rom were determined in 96-well microtiter plates using BM as previously described . Briefly578 of 0.1, and growth of HG001, HG001 ΔfarE, RomR, and RomR ΔfarE was monitored for 48 h . Bacteria were precultured in defined medium inoculated into 500 μL of fresh defined medium at a starting ODfor 48 h .HG001 was inoculated into BM and incubated at 37°C with shaking overnight, and the impact of Rom (2 μg/mL) and culture supernatant (S) on growth was monitored. Culture supernatants from the HG001 and RomR strains were harvested at the end of the exponential growth phase, sterile filtered (0.2-μm filter), and concentrated 10 times with a speed vacuum concentrator. The supernatant was applied at 1:10 (vol/vol) .farE, RomR, and RomR ΔfarE) were used to inoculate 15 mL of defined minimal medium in 100-mL flasks (OD578 = 0.1) and incubated at 37°C with shaking until the end of the exponential phase was reached. Cells were then centrifuged at 8,000 rpm and 4°C for 10 min, and both the supernatants and pellets were collected. The supernatants were filter sterilized and lyophilized. All samples were prepared in five biological replicates and used for subsequent preparation for lipidomic analyses. We also investigated whether sublethal concentrations of Rom can induce lipid/FA release. In this case, the cultures were grown in the presence of 0.3 μg/mL of Rom.The overnight bacterial cultures , 18:0 phosphatidylglycerol-d70 (18:0 PG-d70), and 18:1 lysyl-phosphatidylglycerol (18:1 Lys-PG) were purchased from Avanti Polar Lipids . Arachidonic acid-d11 (AA-d11) and C18-ceramide-d7 (d18:1-d7/18:0) were obtained from Cayman Chemicals . Isopropanol (IPA), acetonitrile (ACN), and methanol (MeOH) at ultra-LC-MS grade were from Carl Roth . Ammonium formate, formic acid, and IPA at high-performance liquid chromatography (HPLC) grade were purchased from Merck . Purified water was produced by Elga Purelab Ultra .2O to account for a final ratio of MTBE-MeOH-H2O of 10:3:2.5 (vol/vol/vol) and incubation at room temperature for another 10 min to induce phase separation. The upper layer was transferred to a new Falcon tube, and the water phase was reextracted by adding 2 mL of the upper phase from a solution of MTBE-MeOH-H2O (10:3:2.5 [vol/vol/vol]). The upper layer from reextraction was then combined with the phase from first extraction and dried with a Genevac EZ-2 evaporator  with nitrogen protection. Extraction residues after evaporation were reconstituted in 100 μL of MeOH, and after vortexing (10 s), sonication (2 min), and centrifugation , the methanol solutions were transferred to autosampler vials.Lipid extraction from bacterial supernatant was performed by a biphasic extraction method following the protocol of Matyash et al. . First, 2O protocol , 100 μL of CL-d5(18:2) stock solution (1 mg/mL), 100 μL of d18:1-d7/18:0 stock solution (0.25 mg/mL), 100 μL of 18:0 PG-d70 (1 mg/mL), and 100 μL of Lys-PG(18:1) (1 mg/mL) stock solutions. Dry bacterial pellets were suspended in 5 mL of IPA/H2O (9:1 [vol/vol]) with IS, vortexed for 10 s, and sonicated for 2 min. Then the samples were incubated on ice for 1 h with 2 min of sonication every 12 min during the incubation, which means a total of 5 cycles of sonication (2 min). The samples were centrifuged , pellets were kept, and supernatant (lipid extract) was transferred to fresh Falcon tubes and dried with the Genevac EZ-2 evaporator  with nitrogen protection. Afterwards, the extracts were reconstituted in 100 μL of MeOH, vortexed (10 s), sonicated (2 min), centrifuged , and transferred to autosampler vials.For the preparation of bacterial pellet wash, lipid extraction was performed using a monophasic extraction method following the IPA/Hprotocol . An IS m2O (9:1 [vol/vol]) with IS was added to the pellets, which were kept after washing from the previous step. Samples were vortexed and sonicated. After adding beads (1-mm diameter) and enzyme (50 μL of lysostaphin at 0.3 mg/mL) to each sample, the pellets were disrupted in a Fastprep-24  (3 cycles of 30 s each at a speed 6.5 m/s). Samples were then centrifuged , and supernatant was collected for further drying with a Genevac EZ-2 evaporator  with nitrogen protection. The dried extracts were then reconstituted, vortexed, sonicated, and transferred into vials as described above. A pooled quality control (QC) sample was prepared by mixing 15-μL aliquots of each reconstituted sample from three extraction process.Lastly, for the extraction from bacterial pellets, 5 mL of IPA/H18 column  with precolumn . The column temperature was 65°C, with a flow rate 0.6 mL/min. Mobile phase A was composed of H2O/ACN (2:3 [vol/vol]) containing 10 mM ammonium formate and 0.1% (vol/vol) formic acid, while mobile phase B was IPA/ACN/H2O (90:9:1 [vol/vol/vol]) containing 10 mM ammonium formate and 0.1% (vol/vol) formic acid. A gradient elution started from 15% mobile phase B to 30% mobile phase B in 2 min, followed by an increase of mobile phase B to 48% in 0.5 min. Then mobile phase B was further increased to 82% at 11 min and quickly reached 99% in the next 0.5 min, followed by holding this percentage for another 0.5 min. Afterwards, the percentage of mobile phase B was taken back to starting conditions (15% mobile phase B) in 0.1 min to reequilibrate the column for the next injection (2.9 min).The analysis of samples was performed with an Agilent 1290 Infinity ultrahigh-performance liquid chromatography (UHPLC) system  equipped with a binary pump and a PAL-HTX xt DLW autosampler  and coupled to a SCIEX TripleTOF 5600+ quadrupole time of flight (QTOF) mass spectrometer with a DuoSpray source . The chromatographic separation was performed on an Acquity UPLC CSH Cm/z of 50 to 1,250 was selected, while different SWATH windows were acquired for MS/MS experiments  was adjusted to 80 V and −80 V for positive and negative polarity modes, respectively. The cycle time was always 720 ms. The collision energy (CE) and collision energy spread (CES) for each experiment are shown in LC-ESI-MS/MS experiments were operated in both positive and negative modes with injection volumes of 3 μL for positive and 5 μL for negative mode. An MS full-scan experiment with mass range eriments . The ion10.1128/mbio.03833-21.9TABLE S3m/z range, accumulation time (Acc. time), and collision energy. Download Table S3, DOCX file, 0.02 MB.MS/MS experiment of SWATH windows with Copyright © 2022 Huang et al.2022Huang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 578 was determined every 2 h for 24 h. All experiments were conducted in three independent biological replicates.The effects of FAs  and PGs [PG(18:0) and Lys- PG(18:1)] on Rom activity were determined in a 48-well microplate reader in BM at 37°C with shaking overnight. The ODSince PG(32:0) was insoluble in all organic solvents tested, an organic solvent mixture (chloroform- methanol-water at 65:35:8 [vol/vol/vol]) was used to dissolve it. However, this solvent mixture was not suitable to be used for ITC because of the volatilization of chloroform; thus, PG(32:0) was prepared as vesicles formed in 10% DMSO/PBS and used for ITC and DLS. Rom, on the other hand, was soluble in all organic solvents tested but not in water; thus, it was first dissolved in 100% DMSO and subsequently diluted in 10% DMSO/PBS. Thus, both compounds were dissolved in the same solvent, which is a prerequisite for ITC studies.sn-glycero-3-phospho-(1′-rac-glycerol) , was purchased from Avanti Polar Lipids Inc. PG powder was dissolved in organic solvent (chloroform-methanol-water at 65:35:8 [vol/vol/vol]) in a glass tube. The lipid was dried completely by evaporator for 2 h and exicator overnight. The dried lipid film was weighed, dissolved in buffer (10% DMSO/PBS), adjusted to a concentration of 5 mM, and treated in an ultrasonic bath for 2 h. The lipid dispersion was transferred to an Eppendorf tube and subjected to ultrasonication (Titan tip) four times, each for 2 min. Subsequently, the size distribution of the vesicles was determined by DLS.PG(32:0), 1,2-dipalmitoyl-DK), enthalpy (ΔH) and entropy (ΔS) of binding and stoichiometry (n).ITC experiments were performed using a MicroCal PEAQ-ITC calorimeter . PG(32:0) vesicles and Rom were dissolved in 10% DMSO/PBS (1:9 [vol/vol]) and degassed via vacuum before titration. An initial volume of 0.4 μL followed by 18 injections of 2 μL of PG(32:0) vesicles was injected into the cell with Rom solution. Injection of PG(32:0) vesicles into 10% DMSO was used as a control to correct the heat of dilution. The measurements were carried out at 37°C with stirring at 750 rpm with 150-s intervals. The final data were analyzed via the “one-site-binding” model to determine the binding affinity  vesicles and Rom clusters.The growth curves and bar charts were visualized using GraphPad Prism 6.0 software. Mann-Whitney U test was applied to compare significant differences between HG001 and RomR. R was employed for performing the Mann-Whitney U test.The main data supporting the findings of this work are available within the article and the supplemental material files or from the corresponding author upon request.