Overcoming antibiotic resistance: non-thermal plasma and antibiotics combination inhibits important pathogens.

Antibiotic resistance (ATBR) is increasing every year as the overuse of antibiotics (ATBs) and the lack of newly emerging antimicrobial agents lead to an efficient pathogen escape from ATBs action. This trend is alarming and the World Health Organization warned in 2021 that ATBR could become the leading cause of death worldwide by 2050. The development of novel ATBs is not fast enough considering the situation, and alternative strategies are therefore urgently required. One such alternative may be the use of non-thermal plasma (NTP), a well-established antimicrobial agent actively used in a growing number of medical fields. Despite its efficiency, NTP alone is not always sufficient to completely eliminate pathogens. However, NTP combined with ATBs is more potent and evidence has been emerging over the last few years proving this is a robust and highly effective strategy to fight resistant pathogens. This minireview summarizes experimental research addressing the potential of the NTP-ATBs combination, particularly for inhibiting planktonic and biofilm growth and treating infections in mouse models caused by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa. The published studies highlight this combination as a promising solution to emerging ATBR, and further research is therefore highly desirable.

Silver nanoparticles with plasma-polymerized hexamethyldisiloxane coating on 3D printed substrates are non-cytotoxic and effective against respiratory pathogens.

Due to the emerging resistance of microorganisms and viruses to conventional treatments, the importance of self-disinfecting materials is highly increasing. Such materials could be silver or its nanoparticles (AgNPs), both of which have been studied for their antimicrobial effect. In this study, we compared the biological effects of AgNP coatings with and without a plasma-polymerized hexamethyldisiloxane (ppHMDSO) protective film to smooth silver or copper coatings under three ambient conditions that mimic their potential medical use (dry or wet environments and an environment simulating the human body). The coatings were deposited on 3D printed polylactic acid substrates by DC magnetron sputtering, and their surface morphology was visualized using scanning electron microscopy. Cytotoxicity of the samples was evaluated using human lung epithelial cells A549. Furthermore, antibacterial activity was determined against the Gram-negative pathogenic bacterium Pseudomonas aeruginosa PAO1 and antiviral activity was assessed using human rhinovirus species A/type 2. The obtained results showed that overcoating of AgNPs with ppHMDSO creates the material with antibacterial and antiviral activity and at the same time without a cytotoxic effect for the surrounding tissue cells. These findings suggest that the production of 3D printed substrates coated with a layer of AgNPs-ppHMDSO could have potential applications in the medical field as functional materials.

Decontamination of High-Efficiency Mask Filters From Respiratory Pathogens Including SARS-CoV-2 by Non-thermal Plasma.

The current pandemic resulted in a rapidly increasing demand for personal protective equipment (PPE) initially leading to severe shortages of these items. Hence, during an unexpected and fast virus spread, the possibility of reusing highly efficient protective equipment could provide a viable solution for keeping both healthcare professionals and the general public equipped and protected. This requires an efficient decontamination technique that preserves functionality of the sensitive materials used for PPE production. Non-thermal plasma (NTP) is a decontamination technique with documented efficiency against select bacterial and fungal pathogens combined with low damage to exposed materials. We have investigated NTP for decontamination of high-efficiency P3 R filters from viral respiratory pathogens in comparison to other commonly used techniques. We show that NTP treatment completely inactivates SARS-CoV-2 and three other common human respiratory viruses including Influenza A, Rhinovirus and Adenovirus, revealing an efficiency comparable to 90°C dry heat or UVC light. Unlike some of the tested techniques (e.g., autoclaving), NTP neither influenced the filtering efficiency nor the microstructure of the filter. We demonstrate that NTP is a powerful and economic technology for efficient decontamination of protective filters and other sensitive materials from different respiratory pathogens.

Intestinal Shedding of SARS-CoV-2 in Children: No Evidence for Infectious Potential.

The clinical courses of COVID-19 in children are often mild and may remain undiagnosed, but prolonged intestinal virus shedding has been documented, thus potentially enabling fecal-oral transmission. However, the infectious potential of SARS-CoV-2 viruses excreted with feces has remained unclear. Here, we investigated 247 stool specimens from 213 pediatric patients to assess the prevalence of intestinal SARS-CoV-2 shedding in hospitalized children without or with COVID-19 and determined the infectious capacity of stool-borne viruses. Upon RT-qPCR screening, the infectivity of virus-positive samples was tested in cell culture using the Vero-E6 permissive cell line. SARS-CoV-2 RNA was detected by RT-qPCR in 32 (13%) stool specimens, but the analysis of virus-positive samples in cell culture revealed no cytopathic effects attributable to SARS-CoV-2-related cell damage. Our findings do not support the notion of potential fecal-oral SARS-CoV-2 spreading, thus questioning the role of hygienic measures designed to prevent this mode of viral transmission.

Presence of viremia during febrile neutropenic episodes in patients undergoing chemotherapy for malignant neoplasms.

The importance of viral infections as a leading cause of morbidity and mortality is well documented in severely immunosuppressed patients undergoing allogeneic stem cell transplantation. By contrast, viral infections generally receive less attention in patients with malignant disorders undergoing chemotherapy, where the onset of neutropenic fever is mostly associated with bacterial or fungal infections, and screening for viral infections is not routinely performed. To address the occurrence of invasive viral infections in a clinical setting commonly associated with less pronounced immunosuppression, we have prospectively screened 237 febrile neutropenic episodes in pediatric (n = 77) and adult (n = 69) patients undergoing intensive chemotherapy, primarily for treatment of acute leukemia. Serial peripheral blood specimens were tested by RQ-PCR assays for the presence and quantity of the clinically relevant viruses CMV, EBV, HHV6 and HAdV, commonly reactivated in highly immunocompromised patients. Viremia was documented in 36 (15%) episodes investigated, including the detection of HHV6 (n = 14), EBV (n = 15), CMV (n = 6), or HAdV (n = 1). While low or intermediate levels of viremia (<104 virus copies/mL) were commonly associated with bacterial or fungal co-infection, viremia at higher levels (>104 copies/mL) was documented in patients without evidence for other infections, raising the possibility that at least in some instances the onset of fever may have been attributable to the virus detected. The observations suggest that viral infections, potentially resulting from reactivation, might also play a clinically relevant role in patients receiving chemotherapy for treatment of malignant neoplasms, and routine screening for viremia in this clinical setting might be warranted.

Polylactic acid as a suitable material for 3D printing of protective masks in times of COVID-19 pandemic.

A critical lack of personal protective equipment has occurred during the COVID-19 pandemic. Polylactic acid (PLA), a polyester made from renewable natural resources, can be exploited for 3D printing of protective face masks using the Fused Deposition Modelling technique. Since the possible high porosity of this material raised questions regarding its suitability for protection against viruses, we have investigated its microstructure using scanning electron microscopy and aerosol generator and photometer certified as the test system according to the standards EN 143 and EN 149. Moreover, the efficiency of decontaminating PLA surfaces by conventional chemical disinfectants including 96% ethanol, 70% isopropanol, and a commercial disinfectant containing 0.85% sodium hypochlorite has been determined. We confirmed that the structure of PLA protective masks is compact and can be considered a sufficient barrier protection against particles of a size corresponding to microorganisms including viruses. Complete decontamination of PLA surfaces from externally applied Staphylococcus epidermidis, Escherichia coli, Candida albicans and SARS-CoV-2 was achieved using all disinfectants tested, and human adenovirus was completely inactivated by sodium hypochlorite-containing disinfectant. Natural contamination of PLA masks worn by test persons was decontaminated easily and efficiently by ethanol. No disinfectant caused major changes to the PLA surface properties, and the pore size did not change despite severe mechanical damage of the surface. Therefore, PLA may be regarded as a suitable material for 3D printing of protective masks during the current or future pandemic crises.

No evidence for Ago2 translocation from the host erythrocyte into the Plasmodium parasite.

Background: Plasmodium parasites rely on various host factors to grow and replicate within red blood cells (RBC). While many host proteins are known that mediate parasite adhesion and invasion, few examples of host enzymes co-opted by the parasite during intracellular development have been described. Recent studies suggested that the host protein Argonaute 2 (Ago2), which is involved in RNA interference, can translocate into the parasite and affect its development. Here, we investigated this hypothesis. Methods: We used several different monoclonal antibodies to test for Ago2 localisation in the human malaria parasite, P. falciparum and rodent P. berghei parasites. In addition, we biochemically fractionated infected red blood cells to localize Ago2. We also quantified parasite growth and sexual commitment in the presence of the Ago2 inhibitor BCI-137. Results: Ago2 localization by fluorescence microscopy produced inconclusive results across the three different antibodies, suggesting cross-reactivity with parasite targets. Biochemical separation of parasite and RBC cytoplasm detected Ago2 only in the RBC cytoplasm and not in the parasite. Inhibition of Ago2 using BCl-137 did not result in altered parasite development. Conclusion: Ago2 localization in infected RBCs by microscopy is confounded by non-specific binding of antibodies. Complementary results using biochemical fractionation and Ago2 detection by western blot did not detect the protein in the parasite cytosol, and growth assays using a specific inhibitor demonstrated that its catalytical activity is not required for parasite development. We therefore conclude that previous data localising Ago2 to parasite ring stages are due to antibody cross reactivity, and that Ago2 is not required for intracellular Plasmodium development.

CDK4/CDK6 inhibition as a novel strategy to suppress the growth and survival of BCR-ABL1T315I+ clones in TKI-resistant CML.

PURPOSE: Ponatinib is the only approved tyrosine kinase inhibitor (TKI) suppressing BCR-ABL1T315I-mutated cells in chronic myeloid leukemia (CML). However, due to side effects and resistance, BCR-ABL1T315I-mutated CML remains a clinical challenge. Hydroxyurea (HU) has been used for cytoreduction in CML for decades. We found that HU suppresses or even eliminates BCR-ABL1T315I+ sub-clones in heavily pretreated CML patients. Based on this observation, we investigated the effects of HU on TKI-resistant CML cells in vitro. METHODS: Viability, apoptosis and proliferation of drug-exposed primary CML cells and BCR-ABL1+ cell lines were examined by flow cytometry and 3H-thymidine-uptake. Expression of drug targets was analyzed by qPCR and Western blotting. FINDINGS: HU was more effective in inhibiting the proliferation of leukemic cells harboring BCR-ABL1T315I or T315I-including compound-mutations compared to cells expressing wildtype BCR-ABL1. Moreover, HU synergized with ponatinib and ABL001 in inducing growth inhibition in CML cells. Furthermore, HU blocked cell cycle progression in leukemic cells, which was accompanied by decreased expression of CDK4 and CDK6. Palbociclib, a more specific CDK4/CDK6-inhibitor, was also found to suppress proliferation in primary CML cells and to synergize with ponatinib in producing growth inhibition in BCR-ABL1T315I+ cells, suggesting that suppression of CDK4/CDK6 may be a promising concept to overcome BCR-ABL1T315I-associated TKI resistance. INTERPRETATION: HU and the CDK4/CDK6-blocker palbociclib inhibit growth of CML clones expressing BCR-ABL1T315I or complex T315I-including compound-mutations. Clinical studies are required to confirm single drug effects and the efficacy of `ponatinib+HU´ and ´ponatinib+palbociclib´ combinations in advanced CML. FUNDING: This project was supported by the Austrian Science Funds (FWF) projects F4701-B20, F4704-B20 and P30625.

Transmission of the malaria parasite requires ferlin for gamete egress from the red blood cell.

Ferlins mediate calcium-dependent vesicular fusion. Although conserved throughout eukaryotic evolution, their function in unicellular organisms including apicomplexan parasites is largely unknown. Here, we define a crucial role for a ferlin-like protein (FLP) in host-to-vector transmission of the rodent malaria parasite Plasmodium berghei. Infection of the mosquito vectors requires the formation of free gametes and their fertilisation in the mosquito midgut. Mature gametes will only emerge upon secretion of factors that stimulate the disruption of the red blood cell membrane and the parasitophorous vacuole membrane. Genetic depletion of FLP in sexual stages leads to a complete life cycle arrest in the mosquito. Although mature gametes form normally, mutants lacking FLP remain trapped in the red blood cell. The egress defect is rescued by detergent-mediated membrane lysis. In agreement with ferlin vesicular localisation, HA-tagged FLP labels intracellular speckles, which relocalise to the cell periphery during gamete maturation. Our data define FLP as a novel critical factor for Plasmodium fertilisation and transmission and suggest an evolutionarily conserved example of ferlin-mediated exocytosis.

A Plasmodium Cross-Stage Antigen Contributes to the Development of Experimental Cerebral Malaria.

Cerebral malaria is a complex neurological syndrome caused by an infection with Plasmodium falciparum parasites and is exclusively attributed to a series of host-parasite interactions at the pathological blood-stage of infection. In contrast, the preceding intra-hepatic phase of replication is generally considered clinically silent and thereby excluded from playing any role in the development of neurological symptoms. In this study, however, we present an antigen PbmaLS_05 that is presented to the host immune system by both pre-erythrocytic and intra-erythrocytic stages and contributes to the development of cerebral malaria in mice. Although deletion of the endogenous PbmaLS_05 prevented the development of experimental cerebral malaria (ECM) in susceptible mice after both sporozoite and infected red blood cell (iRBC) infections, we observed significant differences in contribution of the host immune response between both modes of inoculation. Moreover, PbmaLS_05-specific CD8+ T cells contributed to the development of ECM after sporozoite but not iRBC-infection, suggesting that pre-erythrocytic antigens like PbmaLS_05 can also contribute to the development of cerebral symptoms. Our data thus highlight the importance of the natural route of infection in the study of ECM, with potential implications for vaccine and therapeutic strategies against malaria.