Lytic activity of phages against bacterial pathogens infecting diabetic foot ulcers.

Complications of diabetes, such as diabetic foot ulcers (DFUs), are common, multifactorial in origin, and costly to treat. DFUs are the cause of nearly 90% of limb amputations among persons with diabetes. In most chronic infections such as DFU, biofilms are involved. Bacteria in biofilms are 100-1000 times more resistant to antibiotics than their planktonic counterparts. Multidrug-resistant (MDR) Staphylococcus aureus and Pseudomonas aeruginosa infections in DFUs may require alternative therapeutic agents such as bacteriophages ("phages"). This study describes the lytic activity of phage cocktails AB-SA01 (3-phage cocktail) and AB-PA01 (4-phage cocktail), which target S. aureus and P. aeruginosa, respectively. The host range and lytic effect of AB-SA01 and AB-PA01 on a planktonic culture, single-species biofilm, and mixed-species biofilm were evaluated. In vitro testing showed that 88.7% of S. aureus and 92.7% of P. aeruginosa isolates were susceptible to AB-SA01 and AB-PA01, respectively, in the planktonic state. The component phages of AB-SA01 and AB-PA01 infected 66% to 94.3% of the bacterial isolates tested. Furthermore, AB-SA01 and AB-PA01 treatment significantly (p < 0.05) reduced the biofilm biomass of their hosts, regardless of the antibiotic-resistant characteristics of the isolates and the presence of a non-susceptible host. In conclusion, the strong lytic activity, broad host range, and significant biofilm biomass reduction of AB-SA01 and AB-PA01 suggest the considerable potential of phages in treating antibiotic-resistant S. aureus and P. aeruginosa infections alone or as coinfections in DFUs.

Inactivation of human coronaviruses using an automated room disinfection device.

The emergence of more virulent and epidemic strains of viruses, especially in the context of COVID-19, makes it more important than ever to improve methods of decontamination. The objective of this study was to evaluate the potential of on-demand production of chlorine species to inactivate human coronaviruses. The commercial prototype disinfection unit was provided by Unipolar Water Technologies. The Unipolar device generates active chlorine species using an electrochemical reaction and dispenses the disinfectant vapour onto surfaces with an aspirator. The minimum effective concentration and exposure time of disinfectant were evaluated on human hepatoma (Huh7) cells using 50% tissue culture infectious dose (TCID50) assay and human coronavirus 229E (HCoV-229E), a surrogate for pathogenic human coronaviruses. We showed that chlorine species generated in the Unipolar device inactivate HCoV-229E on glass surfaces at ≥ 400 parts per million active chlorine concentration with a 5 min exposure time. Here, inactivation refers to the inability of the virus to infect the Huh7 cells. Importantly, no toxic effect was observed on Huh7 cells for any of the active chlorine concentrations and contact times tested.

Marine mollusc extracts-Potential source of SARS-CoV-2 antivirals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus and the causative agent of coronavirus disease 2019 (Covid-19). There is an urgent need for effective antivirals to treat current Covid-19 cases and protect those unable to be vaccinated against SARS-CoV-2. Marine molluscs live in an environment containing high virus densities (>107 virus particles per ml), and there are an estimated 100,000 species in the phylum Mollusca, demonstrating the success of their innate immune system. Mollusc-derived antivirals are yet to be used clinically despite the activity of many extracts, including against human viruses, being demonstrated in vitro. Hemolymph of the Pacific oyster (Crassostrea gigas) has in vitro antiviral activity against herpes simplex virus and human adenovirus, while antiviral action against SARS-CoV-2 has been proposed by in silico studies. Such evidence suggests that molluscs, and in particular C. gigas hemolymph, may represent a source of antivirals for human coronaviruses.

Potential for bacteriophage therapy for Staphylococcus aureus pneumonia with influenza A coinfection.

The ability of influenza A virus to evolve, coupled with increasing antimicrobial resistance, could trigger an influenza pandemic with great morbidity and mortality. Much of the 1918 influenza pandemic mortality was likely due to bacterial coinfection, including Staphylococcus aureus pneumonia. S. aureus resists many antibiotics. The lack of new antibiotics suggests alternative antimicrobials, such as bacteriophages, are needed. Potential delivery routes for bacteriophage therapy (BT) include inhalation and intravenous injection. BT has recently been used successfully in compassionate access pulmonary infection cases. Phage lysins, enzymes that hydrolyze bacterial cell walls and which are bactericidal, are efficacious in animal pneumonia models. Clinical trials will be needed to determine whether BT can ameliorate disease in influenza and S. aureus coinfection.

Efficacy of Lytic Phage Cocktails on Staphylococcus aureus and Pseudomonas aeruginosa in Mixed-Species Planktonic Cultures and Biofilms.

The efficacy of phages in multispecies infections has been poorly examined. The in vitro lytic efficacies of phage cocktails AB-SA01, AB-PA01, which target Staphylococcus aureus and Pseudomonas aeruginosa, respectively, and their combination against their hosts were evaluated in S. aureus and P. aeruginosa mixed-species planktonic and biofilm cultures. Green fluorescent protein (GFP)-labelled P. aeruginosa PAO1 and mCherry-labelled S. aureus KUB7 laboratory strains and clinical isolates were used as target bacteria. During real-time monitoring using fluorescence spectrophotometry, the density of mCherry S. aureus KUB7 and GFP P. aeruginosa PAO1 significantly decreased when treated by their respective phage cocktail, a mixture of phage cocktails, and gentamicin. The decrease in bacterial density measured by relative fluorescence strongly associated with the decline in bacterial cell counts. This microplate-based mixed-species culture treatment monitoring through spectrophotometry combine reproducibility, rapidity, and ease of management. It is amenable to high-throughput screening for phage cocktail efficacy evaluation. Each phage cocktail, the combination of the two phage cocktails, and tetracycline produced significant biofilm biomass reduction in mixed-species biofilms. This study result shows that these phage cocktails lyse their hosts in the presence of non-susceptible bacteria. These data support the use of phage cocktails therapy in infections with multiple bacterial species.

Virio- and bacterioplankton microscale distributions at the sediment-water interface.

The marine sediment-water interface is an important location for microbially controlled nutrient and gas exchange processes. While microbial distributions on the sediment side of the interface are well established in many locations, the distributions of microbes on the water side of the interface are less well known. Here, we measured that distribution for marine virio- and bacterioplankton with a new two-dimensional technique. Our results revealed higher heterogeneity in sediment-water interface biomass distributions than previously reported with a greater than 45- and 2500-fold change cm(-1) found within bacterial and viral subpopulations compared to previous maxima of 1.5- and 1.4-fold cm(-1) in bacteria and viruses in the same environments. The 45-fold and 2500-fold changes were due to patches of elevated and patches of reduced viral and bacterial abundance. The bacterial and viral hotspots were found over single and multiple sample points and the two groups often coincided whilst the coldspots only occurred over single sample points and the bacterial and viral abundances showed no correlation. The total mean abundances of viruses strongly correlated with bacteria (r = 0.90, p<0.0001, n = 12) for all three microplates (n = 1350). Spatial autocorrelation analysis via Moran's I and Geary's C revealed non-random distributions in bacterial subpopulations and random distributions in viral subpopulations. The variable distributions of viral and bacterial abundance over centimetre-scale distances suggest that competition and the likelihood of viral infection are higher in the small volumes important for individual cell encounters than bulk measurements indicate. We conclude that large scale measurements are not an accurate measurement of the conditions under which microbial dynamics exist. The high variability we report indicates that few microbes experience the 'average' concentrations that are frequently measured.

Confined aquifers as viral reservoirs.

Knowledge about viral diversity and abundance in deep groundwater reserves is limited. We found that the viral community inhabiting a deep confined aquifer in South Australia was more similar to reclaimed water communities than to the viral communities in the overlying unconfined aquifer community. This similarity was driven by high relative occurrence of the single-stranded DNA viral groups Circoviridae, Geminiviridae and Microviridae, which include many known plant and animal pathogens. These groups were present in a 1500-year-old water situated 80 m below the surface, which suggests the potential for long-term survival and spread of potentially pathogenic viruses in deep, confined groundwater. Obtaining a broader understanding of potentially pathogenic viral communities within aquifers is particularly important given the ability of viruses to spread within groundwater ecosystems.

Hide and seek: the immunology of HSV persistence.

Herpes simplex virus (HSV) has been thought to persist as a latent infection by completely extinguishing antigen expression. In this issue of Immunity, Khanna et al. challenge this view by showing that CTL specific for antigens normally associated with active viral replication are crucial participants in regulating virus latency.

Impaired clearance of herpes simplex virus type 1 from mice lacking CD1d or NKT cells expressing the semivariant V alpha 14-J alpha 281 TCR.

Ag-presenting molecule CD1 and CD1-restricted NKT cells are known to contribute to defense against a range of infectious pathogens, including some viruses. CD1-restricted NKT cells, a distinct subpopulation of T cells, have striking and rapid effector functions that contribute to host defense, including rapid production of IFN-gamma and IL-4, and activation of NK cells. Consideration of the important contributions of innate and adaptive immunity to clearance of HSV prompted us to investigate the role of CD1 and of NKT cells expressing the V alpha 14-J alpha 281 TCR in the pathogenesis of HSV infection. To address this issue, we compared infection in wild-type mice with that in CD1 gene knockout (GKO) and J alpha 281 GKO mice. In this study, we report impaired clearance of virus and viral Ags, and more florid acute infection in mice lacking CD1 (and by inference, CD1-restricted T cells), in comparison with parental C57BL6 mice. In J alpha 281 GKO mice there was also impairment of virus clearance, resembling that seen in CD1 GKO mice. These results imply roles for the V alpha 14-J alpha 281 subset of NKT cells and for CD1d in control of HSV infection.