Alcohol-free hand sanitizer and other quaternary ammonium disinfectants quickly and effectively inactivate SARS-CoV-2.

BACKGROUND: SARS-CoV-2 is the virus responsible for the current global pandemic, COVID-19. Because this virus is novel, little is known about its sensitivity to disinfection. METHODS: We performed suspension tests against SARS-CoV-2 using three commercially available quaternary ammonium compound (Quat) disinfectants and one laboratory-made 0.2% benzalkonium chloride solution. FINDINGS: Three of the four formulations completely inactivated the virus within 15 s of contact, even in the presence of a soil load or when diluted in hard water. CONCLUSION: Quats rapidly inactivate SARS-CoV-2, making them potentially useful for controlling SARS-CoV-2 spread in hospitals and the community.

3× multiplexed detection of antibiotic resistant plasmids with single molecule sensitivity.

Bacterial pathogens resistant to antibiotics have become a serious health threat. Those species which have developed resistance against multiple drugs such as the carbapenems, are more lethal as these are last line therapy antibiotics. Current diagnostic tests for these resistance traits are based on singleplex target amplification techniques which can be time consuming and prone to errors. Here, we demonstrate a chip based optofluidic system with single molecule sensitivity for amplification-free, multiplexed detection of plasmids with genes corresponding to antibiotic resistance, within one hour. Rotating disks and microfluidic chips with functionalized polymer monoliths provided the upstream sample preparation steps to selectively extract these plasmids from blood spiked with E. coli DH5α cells. Waveguide-based spatial multiplexing using a multi-mode interference waveguide on an optofluidic chip was used for parallel detection of three different carbapenem resistance genes. These results point the way towards rapid, amplification-free, multiplex analysis of antibiotic-resistant pathogens.

Gas chromatography-mass spectrometry method for rapid identification and differentiation of Burkholderia pseudomallei and Burkholderia mallei from each other, Burkholderia thailandensis and several members of the Burkholderia cepacia complex.

AIMS: To develop a simple gas chromatography-mass spectrometry (GC-MS) method for the detection and differentiation of Burkholderia pseudomallei and Burkholderia mallei from each other, Burkholderia thailandensis and several members of the Burkholderia cepacia complex. METHODS AND RESULTS: Biomarkers were generated by one-step thermochemolysis (TCM) and analysed using a GC-MS system. Fragments of poly-3-hydroxybutyrate-co-hydroxyvalerate [poly(3HBA-co-3HVA)] produced by TCM were useful biomarkers. Several cellular fatty acid methyl esters were important in differentiating the various Burkholderia species. A statistical discrimination algorithm was constructed using a combination of biomarkers. The identities of four B. pseudomallei strains, four B. mallei strains and one strain of each near neighbour were confirmed in a statistically designed test using the algorithm. The detection limit for this method was found to be approximately 4000 cells. CONCLUSIONS: The method is fast, accurate and easy to use. The algorithm is robust against different growth conditions (medium and temperature). SIGNIFICANCE AND IMPACT OF THE STUDY: This assay may prove beneficial in a clinical diagnostic setting, where the rapid identification of B. pseudomallei is essential to effective treatment. This method could also be easily employed after a biological attack to confirm the presence of either B. pseudomallei or B. mallei.

Investigating the role of macrophages in tumor formation using a MaFIA mouse model.

Tumor-associated macrophages (TAMs) interact with tumors in their development, growth and metastatic activities. Using a transgenic mouse model that allows for the selective depletion of macrophages we were able to access the macrophage's potential to facilitate metastasis. In the MaFIA (Macrophage Fas-Induced Apoptosis) mouse, transgene-expressing cells of the myeloid lineage undergo death by apoptosis in the presence of the drug AP20187. Enhanced green fluorescent protein (EGFP) was fused to the suicide gene to allow identification of transgene-expressing cells. Tumor induction was accomplished by subdermal and intravenous injections of B16-F10 melanoma cells. Metastasis in mice with depleted macrophages was compared to metastasis in normal control mice. The lungs and kidneys were examined for metastatic cells. The macrophage-depleted groups showed significantly less metastasis (P>0.001) compared to the control groups. We theorize that macrophages may aid the metastatic process by fusing with melanoma cells. Using appropriate cell markers and fluorescence-activated cell sorting, we were able to detect a small population of double-positive cells. We confirmed cell fusion by microscopic analysis, visualizing the cell's morphology by both immunohistochemistry and immunofluorescence. The presence of double-positive cells suggests macrophage/cancer cell fusion could be a possible mechanism for metastasis.

Image-guided, direct convective delivery of glucocerebrosidase for neuronopathic Gaucher disease.

OBJECTIVE: To determine if convection-enhanced delivery (CED) of glucocerebrosidase could be used to treat targeted sites of disease progression in the brain and brainstem of a patient with neuronopathic Gaucher disease while monitoring enzyme distribution using MRI. METHODS: A CED paradigm in rodents (n = 8) and primates (n = 5) that employs co-infusion of a surrogate MRI tracer (gadolinium diethylenetriamine penta-acetic acid [Gd-DTPA]) with glucocerebrosidase to permit real-time monitoring of distribution was developed. The safety and feasibility of this delivery and monitoring paradigm were evaluated in a patient with type 2 Gaucher disease. RESULTS: Animal studies revealed that real-time, T1-weighted, MRI of Gd-DTPA accurately tracked enzyme distribution during CED. Targeted perfusion of clinically affected anatomic sites in a patient with neuronopathic Gaucher disease (frontal lobe and brainstem) with glucocerebrosidase was successfully performed. Real-time MRI revealed progressive and complete filling of the targeted region with enzyme and Gd-DTPA infusate. The patient tolerated the infusions without evidence of toxicity. CONCLUSIONS: Convection-enhanced delivery can be used to safely perfuse large regions of the brain and brainstem with therapeutic levels of glucocerebrosidase. Co-infused imaging surrogate tracers can be used to monitor and control the distribution of therapeutic agents in vivo. Patients with neuronopathic Gaucher disease and other intrinsic CNS disorders may benefit from a similar treatment paradigm.

Ultrasonically enhanced vancomycin activity against Staphylococcus epidermidis biofilms in vivo.

Infection of implanted medical devices by Gram-positive organisms such as Staphylococcus ssp. is a serious concern in the biomaterial community. In this research the application of low frequency ultrasound to enhance the activity of vancomycin against implanted Staphylococcus epidermidis biofilms was examined. Polyethylene disks covered with a biofilm of S. epidermidis were implanted subcutaneously in rabbits on both sides of their spine. The rabbits received systemic vancomycin for the duration of the experiment. Following 24 h of recovery, one disk was insonated for 24 or 48 h while the other was a control. Disks were removed and viable bacteria counted. At 24 h of insonation, there was no difference in viable counts between control and insonated biofilms, while at 48 h of insonation there were statistically fewer viable bacteria in the insonated biofilm. The S. epidermidis biofilms responded favorably to combinations of ultrasound and vancomycin, but longer treatment times are required for this Gram-positive organism than was observed previously for a Gram-negative species.

Pulsed ultrasound enhances the killing of Escherichia coli biofilms by aminoglycoside antibiotics in vivo.

Escherichia coli biofilms on two polyethylene disks were implanted subcutaneously into rabbits receiving systemic gentamicin. Ultrasound was applied for 24 h to one disk. Both disks were removed, and viable bacteria were counted. Pulsed ultrasound significantly reduced bacterial viability below that of nontreated biofilms without damage to the skin.

The mycobactericidal efficacy of ortho-phthalaldehyde and the comparative resistances of Mycobacterium bovis, Mycobacterium terrae, and Mycobacterium chelonae.

OBJECTIVES: To assess the mycobactericidal efficacy of an agent relatively new to disinfection, ortho-phthalaldehyde (OPA) and to compare the resistances of three Mycobacterium species. Mycobacterium bovis (strain BCG) was compared with Mycobacterium chelonae and Mycobacterium terrae to investigate the feasibility of using either of the latter two species in tuberculocidal testing. M. chelonae (a rapid grower) and M. terrae (an intermediate grower) both grow faster and are less virulent than M. bovis (a slow grower). DESIGN: The quantitative suspension protocol specified by the Environmental Protection Agency (EPA), the Tuberculocidal Activity Test Method (EPA test), was used throughout this study. Standard suspensions of all three species were prepared in a similar manner. Two suspensions of M. bovis, created in different laboratories, were used. These were tested against two concentrations of alkaline glutaraldehyde to provide reference data. Two concentrations of OPA were evaluated against all mycobacterial test suspensions. Four replicates of each organism-disinfectant combination were performed. RESULTS: Results were assessed by analysis of variance. M. terrae was significantly more resistant to 0.05% OPA than either M. bovis or M. chelonae. At 0.21% OPA, M. terrae was slightly more susceptible than one test suspension of M. bovis, but not significantly different from the other. M. chelonae was significantly less resistant than the other species at both OPA concentrations. At their respective minimum effective concentration, OPA achieved a 6-log10 reduction of M. bovis in nearly one sixth the time required by glutaraldehyde (5.5 minutes vs. 32 minutes). CONCLUSIONS: These data, along with other recent studies, lend support to the idea that M. terrae may be a suitable test organism for use in the tuberculocidal efficacy testing of disinfectants. They also confirm the relatively rapid tuberculocidal activity of OPA.

Ultrasonic enhancement of antibiotic action on Escherichia coli biofilms: an in vivo model.

Biofilm infections are a common complication of prosthetic devices in humans. Previous in vitro research has determined that low-frequency ultrasound combined with aminoglycoside antibiotics is an effective method of killing biofilms. We report the development of an in vivo model to determine if ultrasound enhances antibiotic action. Two 24-h-old Escherichia coli (ATCC 10798) biofilms grown on polyethylene disks were implanted subcutaneously on the backs of New Zealand White female rabbits, one on each side of the spine. Low-frequency (28.48-kHz) and low-power-density (100- and 300-mW/cm2) continuous ultrasound treatment was applied for 24 h with and without systemic administration of gentamicin. The disks were then removed, and the number of viable bacteria on each disk was determined. At the low ultrasonic power used in this study, exposure to ultrasound only (no gentamicin) caused no significant difference in bacterial viability. In the presence of antibiotic, there was a significant reduction due to 300-mW/cm2 ultrasound (P = 0.0485) but no significant reduction due to 100-mW/cm2 ultrasound. Tissue damage to the skin was noted at the 300-mW/cm2 treatment level. Further development of this technique has promise in treatment of clinical implant infections.

The inhibitory effects of bryostatin 1 administration on the growth of rabbit papillomas.

Bryostatin 1 is a protein kinase C modulator that shows antineoplastic activity in a variety of tumor systems. This study examined the effects of bryostatin 1 administration on papilloma growth in rabbits. Investigations of optimal route, dose, and schedule were performed. Several groups of rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) DNA. Bryostatin 1 was administered i.v., both daily and weekly, and intralesionally both weekly and bi-weekly. Intralesionally dosed papillomas were examined histologically for immune cell infiltration. In weekly and daily i.v. trials, 2.5 and 1.0 microg/kg, respectively, showed the greatest overall reduction in tumor size. Bryostatin 1 administered intralesionally also slowed papilloma growth. Treated lesions had significantly higher numbers of heterophils and eosinophils.

Culture variability associated with the U.S. Environmental Protection Agency Tuberculocidal Activity Test Method.

Tuberculosis continues to be a major world health threat. The etiologic agent is among the vegetative organisms most resistant to chemical disinfection. Tuberculocidal efficacy testing for regulatory approval of chemical germicides has evolved considerably over the past decade. A method currently in use is the Environmental Protection Agency Tuberculocidal Activity Test Method, a suspension test using a Mycobacterium bovis culture grown under specific conditions and stored frozen until used. Differing tuberculocidal label claims on products with similar formulations have raised questions concerning the equivalence of test suspensions prepared by different laboratories. Five M. bovis suspensions from laboratories currently performing this test were compared against a battery of three disinfectants at a single test site. A significant difference between test cultures was found, with two of the five exhibiting a significant difference from the other three and also from each other. There was a significant culture-by-disinfectant interaction, indicating that the five cultures did not respond in a consistent manner across the different disinfectants used. However, these differences were due to cultures that were not prepared in accordance with the standard procedure or otherwise did not meet the test suspension criteria. In addition, a 0.55% sodium hypochlorite solution was found to be a sensitive indicator of culture variability. These data reinforce the need to adhere to published procedures and guidelines when growing and preparing a tuberculocidal test suspension and shed light on the variables associated with this type of testing.

Antimicrobial activity of environmental surface disinfectants in the absence and presence of bioburden.

Thirty-nine products representing six categories of disinfectants (alcohols, chlorines, dilute glutaraldehydes, iodophors, phenolics, and quaternary ammonium compounds) were first tested in the absence of bioburden, using four test methods with five test organisms. Products that performed best were retested with the same methods and organisms in the presence of both serum and whole blood, using 3- and 10-minute contact times. Only products containing high ethyl alcohol had consistently high antimicrobial activity regardless of the test method, test organism, or contact time used both in the absence and presence of bioburden. Although these specific formulations demonstrated ability to penetrate and inactivate high concentrations of microorganisms within heavy bioburden, optimum disinfection of environmental surfaces is highly formulation dependent. Other products tested showed deficiencies that contraindicate their use as environmental surface disinfectants in clinical dental settings.

A suspension method to determine reuse life of chemical disinfectants during clinical use.

In-use testing of disinfectants is necessary to ensure efficacy over time. The current official procedure for testing disinfectants, the Association of Official Analytical Chemists (AOAC) use-dilution method, cannot be adapted to repeated sampling techniques of use-life testing. It is therefore necessary to use an alternative method when evaluating the activity of a disinfectant under actual use. The Clinical Research Associates (CRA) suspension method was developed to fill this need. It consists of adding 0.5 ml of a standard culture to 5.0 ml of test disinfectant and sampling the mixture after 10 min for surviving bacteria. When this test was compared with the AOAC use-dilution method under a simulated use situation, the two methods were generally equivalent in identifying disinfectant inactivation. In addition, the CRA method was less time consuming, easier to perform, and less variable than the AOAC method. Use of the CRA method in a clinical study demonstrated the need for reuse claims to be based on clinical use studies rather than on laboratory testing only.

Use of an enzyme-linked immunosorbent assay to measure antibody responses in turkeys against Pasteurella multocida.

AN enzyme-linked immunosorbent assay (ELISA) and a microtiter agglutination (MA) test were used in a comparative study to measure the humoral antibody responses of turkeys receiving oral vaccination against fowl cholera. The ELISA was sensitive and measured antibody titers as high as 1:4,409, whereas the highest titers the MA test measured were 1:128. A close correlation was seen between ELISA antibody titers and protection against oral challenge with virulent Pasteurella multocida, whereas a poor correlation was seen between antibody titers measured by MA tests and protection. ELISA substrate reactions from a single serum dilution, measured with a spectrophotometer, could be converted directly from absorbance to antibody titers using a linear regression plot.

Ctenocystoidea: new class of primitive echinoderms.

The oldest known "carpoids" of the echinoderm subphylum Homalozoa have been discovered in lower Middle Cambrian rocks of northern Utah. They were free-living benthonic animals characterized by a unique ctenoid feeding apparatus and flattened flexible theca with near bilateral symmetry. A new class, Ctenocystoidea, is proposed to include the new genus and new species, Ctenocystis utahensis.

Paleozoic sedimentary rocks in oaxaca, Mexico.

Fossiliferous Cambrian, Ordovician, Mississippian, and Pennsylvanian rocks, never before found in southern Mexico, have been discovered in the Nochixtlán region. Superjacent unfossiliferous sedimentary rocks may be Permian in age. Early Paleozoic and late Paleozoic intervals of marine sedimentation were bounded by intervals of positive tectonism and erosion.