Assessing the synergistic potential of bacteriophage endolysins and antimicrobial peptides for eradicating bacterial biofilms.

Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.

Low-Voltage Bacterial and Viral Killing Using Laser-Induced Graphene-Coated Non-woven Air Filters.

Laser-induced graphene (LIG) is uniquely positioned to advance applications in which electrically conductive carbon coatings are required. Recently, the antifouling, antiviral, and antibacterial properties of LIG have been proven in both air and water filtration applications. For example, an unsupported LIG based filter (pore size: ∼0.3 µm) demonstrated exceptional air filtration properties, while its joule heating effects successfully sterilized and removed unwanted biological components in air despite persisting challenges such as pressure drop, energy consumption, and lack of mechanical robustness. Here, we developed a polyimide (PI) non-woven supported LIG air filter with negligible pressure drop changes compared to the non-woven support material and showed that low electrical current density inactivates aerosolized bacteria. A current density of 4.5 mA/cm2 did not cause significant joule heating, and 97.2% bacterial removal was obtained. The low-voltage antibacterial mechanism was elucidated using bacterial inhibition experiments on a titanium surface and on an LIG surface fabricated on dense PI films. Complete sterilization was obtained using current densities of ∼8 mA/cm2 applied for 2 min or ∼ 6 mA/cm2 for 10 min upon the dense PI-LIG surface. Lastly, >98% bacterial removal was observed using a low-resistance LIG-coated non-woven polyimide air filter at 5 V. However, only very low voltages (∼0.3 V) were needed to remove ∼99% Pseudomonas aeruginosa bacteria and 100% of T4 virus when the LIG-coated filters were hybridized with a stainless steel mesh. Our results show that low current density levels at very low voltages are sufficient for substantial bacterial and viral inactivation, and that these principles might be effectively used in a wide number of air filtration applications such as air conditioners or other ventilation systems, which might limit the spread of infectious particles in hospitals, homes, workplaces, and the transportation industry.

Biomolecular Simulations of Halogen Bonds with a GROMOS Force Field.

Halogen bonds (XBs) are non-covalent interactions in which halogens (X), acting as electrophiles, interact with Lewis bases. XBs are able to mediate protein-ligand recognition and therefore play an important role in rational drug design. In this context, the development of molecular modeling tools that can tackle XBs is paramount. XBs are predominantly explained by the existence of a positive region on the electrostatic potential of X named the σ-hole. Typically, with molecular mechanics force fields, this region is modeled using a charged extra point (EP) linked to X along the R-X covalent bond axis. In this work, we developed the first EP-based strategy for GROMOS force fields (specifically GROMOS 54A7) using bacteriophage T4 lysozyme in complex with both iodobenzene and iodopentafluorobenzene as a prototype system. Several EP parametrization schemes were tested by adding a virtual interaction site to ligand topologies retrieved from the Automated Topology Builder (ATB) and Repository. Contrary to previous approaches using other force fields, our analysis is based on the capability of each parametrization scheme to sample XBs during MD simulations. Our results indicate that the implementation of an EP at a distance from iodine corresponding to Rmin provides a good qualitative description of XBs in MD simulations, supporting the compatibility of our approach with the GROMOS 54A7 force field.

Mechanism of Virus Inactivation by Cold Atmospheric-Pressure Plasma and Plasma-Activated Water.

Viruses cause serious pathogenic contamination that severely affects the environment and human health. Cold atmospheric-pressure plasma efficiently inactivates pathogenic bacteria; however, the mechanism of virus inactivation by plasma is not fully understood. In this study, surface plasma in argon mixed with 1% air and plasma-activated water was used to treat water containing bacteriophages. Both agents efficiently inactivated bacteriophages T4, Φ174, and MS2 in a time-dependent manner. Prolonged storage had marginal effects on the antiviral activity of plasma-activated water. DNA and protein analysis revealed that the reactive species generated by plasma damaged both nucleic acids and proteins, consistent with the morphological examination showing that plasma treatment caused the aggregation of bacteriophages. The inactivation of bacteriophages was alleviated by the singlet oxygen scavengers, demonstrating that singlet oxygen played a primary role in this process. Our findings provide a potentially effective disinfecting strategy to combat the environmental viruses using cold atmospheric-pressure plasma and plasma-activated water.IMPORTANCE Contamination with pathogenic and infectious viruses severely threatens human health and animal husbandry. Current methods for disinfection have different disadvantages, such as inconvenience and contamination of disinfection by-products (e.g., chlorine disinfection). In this study, atmospheric surface plasma in argon mixed with air and plasma-activated water was found to efficiently inactivate bacteriophages, and plasma-activated water still had strong antiviral activity after prolonged storage. Furthermore, it was shown that bacteriophage inactivation was associated with damage to nucleic acids and proteins by singlet oxygen. An understanding of the biological effects of plasma-based treatment is useful to inform the development of plasma into a novel disinfecting strategy with convenience and no by-product.

Sensitive fluorescence sensing of T4 polynucleotide kinase activity and inhibition based on DNA/polydopamine nanospheres platform.

5'-Polynucleotide kinase (PNK) is a crucial enzyme that catalyzes the phosphorylation of nucleic acid with 5'-OH termini and this phosphorylation reaction has been involved in many important cellular activities. The evaluation of PNK activity has received an increasing attention due to the significance of PNK. Here, the polydopamine nanospheres (PDANS) could adsorb single-stranded DNA (ssDNA) through π-π stacking or hydrogen bonding between nucleobases and aromatic groups of PDANS, while the interaction between double-stranded DNA (dsDNA) with PDANS was weakened due to the changed conformation. Hence, a novel DNA/PDANS platform was constructed for the sensitive and selective determination of T4 PNK activity based on the preferential binding properties of PDANS for ssDNA over dsDNA and the excellent fluorescence quenching property of PDANS. The dye-labeled dsDNA was phosphorylated by T4 PNK and then digested by λ exonuclease, yielding dye-labeled ssDNA, which would be adsorbed on the surface of the PDANS and the fluorescence was greatly quenched by PDANS. Because of the preferential binding properties of PDANS for ssDNA over dsDNA and the high quenching property of PDANS, the developed DNA/PDANS platform exhibited good analytical performance for T4 PNK sensing in complex biological matrix and applied to screening inhibitors. The proposed DNA/PDANS based platform is promising in developing high-throughput assays for drug screening and clinical diagnostics.

New Insights into DNA Polymerase Function Revealed by Phosphonoacetic Acid-Sensitive T4 DNA Polymerases.

The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of nonherpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S, and V475W mutations in DNA pol. These amino acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols. We investigated whether the T4 phages expressing the engineered mutant DNA pols were sensitive to the antiviral drug phosphonoacetic acid (PAA) and characterized the in vivo replication fidelity of the phage DNA pols. We found that G466S substitution marginally increased PAA sensitivity, whereas Y460F substitution conferred resistance. The phage expressing a double mutant G466S/Y460F DNA pol was more PAA-sensitive. V475W T4 DNA pol was highly sensitive to PAA, as was the case with V478W RB69 DNA pol. However, DNA replication was severely compromised, which resulted in the selection of phages expressing more robust DNA pols that have strong ability to replicate DNA and contain additional amino acid substitutions that suppress PAA sensitivity. Reduced replication fidelity was observed in all mutant phages expressing PAA-sensitive DNA pols. These observations indicate that PAA sensitivity and fidelity are balanced in DNA pols that can replicate DNA in different environments.

Antiviral effects of polyphenols: development of bio-based cleaning wipes and filters.

Polyphenol molecules play multiple essential roles in plant physiology such as defences against plant-pathogens and micro-organisms. The present study reports a chemical modification of the surface of non-woven cellulosic fibre filters (Kimwipes(®)) by fixing polyphenol in order to confer them antiviral properties. The grafting of the non-woven fibres by the antiviral entity was performed using laccase. T4D bacteriophage virus of Escherichia coli B was used as virus model. Catechin polyphenol was tested as antiviral entity. Proteomic experiments were performed to quantify the potential protein target of catechin on viruses. When the modified filter was in contact with the viral suspension a large improvement in the reduction of the viral concentration was observed (5-log after 1h). Thus, we propose that this material could be used as virucidal wipes for the virus elimination from contaminated surfaces. Virus filtration experiments were performed by spraying an aerial suspension of T4D bacteriophage virus through the designed filter. The best virus capture factor f (ratio of upstream to downstream virus contents) was obtained when using 2 functionalized filters (f=2.9×10(3)). When these 2 layers were placed inside a commercial medical mask in place of its cellulose layer (Kolmi M24001 mask) (f=3.5×10(4)), the f ratio then reached 2.6×10(5) for 2h of filtration. Based on these results, this novel bio-based antiviral mask represents a significant improvement over conventional medical masks.

Reversible inactivation and desiccation tolerance of silicified viruses.

Long-distance host-independent virus dispersal is poorly understood, especially for viruses found in isolated ecosystems. To demonstrate a possible dispersal mechanism, we show that bacteriophage T4, archaeal virus Sulfolobus spindle-shaped virus Kamchatka, and vaccinia virus are reversibly inactivated by mineralization in silica under conditions similar to volcanic hot springs. In contrast, bacteriophage PRD1 is not silicified. Moreover, silicification provides viruses with remarkable desiccation resistance, which could allow extensive aerial dispersal.

Involvement of type I and type II mechanisms on the photoinactivation of non-enveloped DNA and RNA bacteriophages.

Microbial photodynamic inactivation (PDI), involving the use of a photosensitizer (PS), light and molecular oxygen, with the subsequent production of reactive oxygen species (ROS), has been considered a promising and effective technology for viral inactivation. Although singlet oxygen is generally accepted as the main damaging species in PDI, ROS like free radicals may also be involved in the process, inducing damages to proteins, lipids, nucleic acids and other molecular structures. In this study, the relative importance of each mechanism (type I and type II) on the photoinactivation of non-enveloped DNA (T4-like phage) and RNA (Qß phage) viruses was evaluated. For this purpose, two cationic porphyrins (Tri-Py(+)-Me-PF and Tetra-Py(+)-Me) and four different ROS scavengers were used. The scavenging effect of sodium azide and L-histidine (singlet oxygen quenchers) and of D-mannitol and L-cysteine (free radical scavengers) was assessed by exposure of both phages (T4-like and Qß) to each cationic porphyrin (5.0µM for T4-like phage and 0.5µM for Qß phage) and white light (40Wm(-2)) in the presence of different concentrations of the scavengers (5, 10, 50 and 100mM). Sodium azide and L-histidine gave the best protection, reducing the phototoxic effect of Tri-Py(+)-Me-PF on T4-like phage respectively by 80% and 72% and in the presence of Tetra-Py(+)-Me by 90% and 78%. Free radical scavengers D-mannitol and L-cysteine did not significantly reduce the rate of T4-like phage photoinactivation (around 20% protection, for both PS). The sodium azide protection on Qß phage photoinactivation, in the presence of Tri-Py(+)-Me-PF, was lower (39%) when compared with T4-like phage. D-mannitol did not exert on Qß phage any protective effect after 90min of irradiation. The effect of the simultaneous presence of singlet oxygen and free radicals scavengers at 100mM confirmed that singlet oxygen (type II mechanism) is clearly the main ROS involved in T4-like and Qß phages photoinactivation by these two cationic PS. As RNA-type phages are more easily photoinactivated when compared with DNA-type ones, the protection conferred by the scavengers during the PDI process is lower and this should be taken into account when the main mechanism involved in PDI of different viruses is to be studied.

Amber mutants of bacteriophage T4D: their isolation and genetic characterization.

We have isolated a large number of mutants of bacteriophage T4D that are unable to form plaques on strain B of Escherichia coli, but are able to grow (nearly) normally on some other strains of E. coli, in particular strain CR63. These mutants, designated amber (am), have been characterized by complementation tests, by genetic crosses, and by their response to chemical mutagens. It is concluded that a particular subclass of base substitution mutations may give rise to amber mutants and that such mutants occur in many genes, which are widely distributed over the T4 genome.

Highly efficient DNA compaction mediated by an in vivo antitumor-active tetrazolato-bridged dinuclear platinum(II) complex.

We investigated the effects of antitumor-active tetrazolato-bridged dinuclear platinum(II) complexes [{cis-Pt(NH(3))(2)}(2)(µ-OH)(µ-tetrazolato-N(1),N(2))](2+) (1) and [{cis-Pt(NH(3))(2)}(2)(µ-OH)(µ-tetrazolato-N(2),N(3))](2+) (2) on the higher-order structure of a large DNA molecule (T4 phage DNA, 166 kbp) in aqueous solution through single-molecule observation by fluorescence microscopy. Complexes 1 and 2 cause irreversible compaction of DNA through an intermediate state in which coil and compact parts coexist in a single DNA molecule. The potency of compaction is in the order 2 > 1 ≫ cisplatin. Transmission electron microscopic observation showed that both complexes collapsed DNA into an irregularly packed structure. Circular dichroism measurements revealed that the dinuclear platinum(II) complexes change the secondary structure of DNA from the B to C form. These characteristics of platinum(II) complexes are markedly different from those of the usual condensing agents such as spermidine(3+) and [Co(III)(NH(3))(6)](3+). The ability to cause DNA compaction by the platinum(II) complexes is discussed in relation to their potent antitumor activity.

Photocatalytic inactivation of bacteriophages by TiO2-coated glass plates under low-intensity, long-wavelength UV irradiation.

The International Organization for Standardization (ISO) was used to evaluate antibacterial activity by titanium dioxide (TiO(2)) photocatalysis since 2006. We evaluated photocatalytic inactivation of Qß and T4 bacteriophages induced by low-intensity, long-wavelength ultraviolet A (UVA; 0.1 mW cm(-2) and 0.001 mW cm(-2)) irradiation on a TiO(2)-coated glass plate using the ISO methodology. The results indicated that both bacteriophages were inactivated at 0.001 mW cm(-2) UVA. The intensity of UV light, including long-wavelength light (UVA), is very low in an actual indoor environment. Thus, TiO(2) photocatalysis can be beneficial for inactivating viruses in an indoor environment. Experiments using qPCR and bovine serum albumin degradation assume that viral inactivation is caused by outer viral protein disorder and not by viral RNA reduction by reactive oxygen species produced during TiO(2) photocatalysis. Furthermore, we showed that the ISO methodology for standard testing of antibacterial activity by TiO(2) photocatalysis can be applied to assess antiviral activity.

Virus inactivation during coagulation with aluminum coagulants.

We used the bacteriophages Qß and MS2 to determine whether viruses are inactivated by aluminum coagulants during the coagulation process. We performed batch coagulation and filtration experiments with virus-containing solutions. After filtering the supernatant of the coagulated solution through a membrane with a pore size of 50 nm, we measured the virus concentration by both the plaque forming unit (PFU) and polymerase chain reaction (PCR) methods. The virus concentration determined by the PFU method, which determines the infectious virus concentration, was always lower than that determined by the PCR-based method, which determines total virus concentration, regardless of infectivity. This discrepancy can be explained by the formation of aggregates consisting of several virus particles or by the inactivation of viruses in the coagulation process. The former possibility can be discounted because (i) aggregates of several virus particles would not pass through the 50-nm pores of the filtration membrane, and (ii) our particle size measurements revealed that the virus particles in the membrane filtrate were monodispersed. These observations clearly showed that non-infectious Qß particles were present in the membrane filtrate after the coagulation process with aluminum coagulants. We subsequently revealed that the viruses lost their infectivity after being mixed with hydrolyzing aluminum species during the coagulation process.

Cationic phenylene ethynylene polymers and oligomers exhibit efficient antiviral activity.

The antiviral activities of poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPE) and oligo-phenylene ethynylenes (OPE) were investigated using two model viruses, the T4 and MS2 bacteriophages. Under UV/visible light irradiation, significant antiviral activity was observed for all of the CPEs and OPEs; without irradiation, most of these compounds exhibited high inactivation activity against the MS2 phage and moderate inactivation ability against the T4 phage. Transmission electron microscopy (TEM) and SDS polyacrylamide gel electrophoresis (SDS-PAGE) reveal that the CPEs and OPEs exert their antiviral activity by partial disassembly of the phage particle structure in the dark and photochemical damage of the phage capsid protein under UV/visible light irradiation.

Inactivation of Escherichia coli and bacteriophage T4 by high levels of dissolved CO2.

Little information is available regarding the effectiveness of water disinfection by CO(2) at low pressure. The aim of this study was to evaluate the use of high levels of dissolved CO(2) at 0.3-0.6 MPa for the inactivation of microorganisms. Bacteriophage T4 was chosen as the model virus and Escherichia coli was selected as the representative bacterium. The results of the study showed a highly effective log inactivation of E. coli and bacteriophage T4 at low and medium initial concentrations by high levels of dissolved CO(2) at 0.3 MPa with a treatment time of 20 min. When the pressure was increased to 0.6 MPa, inactivation of both microorganisms at high initial concentrations was improved to different extents. Neither pressurized air nor O(2) effectively inactivated both E. coli and bacteriophage T4. The pH was not a key factor affecting the inactivation process by this method. The results of scanning electron microscopy of E. coli and transmission electron microscopy of bacteriophage T4 suggested that "CO(2) uptake at high pressure and bursting of cells by depressurization" were the main reasons for lethal effect on microorganisms. This technology has potential for application in the disinfection of water, wastewater, and liquid food in the future.

Functional cationic nanomagnet-porphyrin hybrids for the photoinactivation of microorganisms.

Cationic nanomagnet-porphyrin hybrids were synthesized and their photodynamic therapy capabilities were investigated against the Gram (-) Escherichia coli bacteria, the Gram (+) Enterococcus faecalis bacteria and T4-like phage. The synthesis, structural characterization, photophysical properties, and antimicrobial activity of these new materials are discussed. The results show that these new multicharged nanomagnet-porphyrin hybrids are very stable in water and highly effective in the photoinactivation of bacteria and phages. Their remarkable antimicrobial activity, associated with their easy recovery, just by applying a magnetic field, makes these materials novel photosensitizers for water or wastewater disinfection.

Regression supports two mechanisms of fork processing in phage T4.

Replication forks routinely encounter damaged DNA and tightly bound proteins, leading to fork stalling and inactivation. To complete DNA synthesis, it is necessary to remove fork-blocking lesions and reactivate stalled fork structures, which can occur by multiple mechanisms. To study the mechanisms of stalled fork reactivation, we used a model fork intermediate, the origin fork, which is formed during replication from the bacteriophage T4 origin, ori(34). The origin fork accumulates within the T4 chromosome in a site-specific manner without the need for replication inhibitors or DNA damage. We report here that the origin fork is processed in vivo to generate a regressed fork structure. Furthermore, origin fork regression supports two mechanisms of fork resolution that can potentially lead to fork reactivation. Fork regression generates both a site-specific double-stranded end (DSE) and a Holliday junction. Each of these DNA elements serves as a target for processing by the T4 ATPase/exonuclease complex [gene product (gp) 46/47] and Holliday junction-cleaving enzyme (EndoVII), respectively. In the absence of both gp46 and EndoVII, regressed origin forks are stabilized and persist throughout infection. In the presence of EndoVII, but not gp46, there is significantly less regressed origin fork accumulation apparently due to cleavage of the regressed fork Holliday junction. In the presence of gp46, but not EndoVII, regressed origin fork DSEs are processed by degradation of the DSE and a pathway that includes recombination proteins. Although both mechanisms can occur independently, they may normally function together as a single fork reactivation pathway.

Photocatalytic antimicrobial activity of thin surface films of TiO(2), CuO and TiO (2)/CuO dual layers on Escherichia coli and bacteriophage T4.

TiO(2)-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with thin films of TiO(2), CuO and hybrid CuO/TiO(2) prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO(2) prepared by a sol-gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of viruses. The chemical oxidising activity was also determined by measuring stearic acid oxidation. The results showed that the rate of inactivation of bacteriophage T4 increased with increasing chemical oxidising activity with the maximum rate obtained on highly active sol-gel preparations. However, these were delicate and easily damaged unlike the Ap-CVD coatings. Inactivation rates were highest on CuO and CuO/TiO(2) which had the lowest chemical oxidising activities. The inactivation of T4 was higher than that of Escherichia coli on low activity surfaces. The combination of photocatalysis and toxicity of copper acted synergistically to inactivate bacteriophage T4 and retained some self-cleaning activity. The presence of phosphate ions slowed inactivation but NaCl had no effect. The results show that TiO(2)/CuO coated surfaces are highly antiviral and may have applications in the food and healthcare industries.

Purification and concentration of bacteriophage T4 using monolithic chromatographic supports.

Phages are gaining importance due to their wide usage. In this work strong anion exchange monolithic chromatographic column was used for single step phage purification. Most of the proteins and DNA were removed and recovery of approximately 70% of infective virus was reproducibly achieved. 30 ml of phage sample was purified in around 10 min.