Genomic comparison of novel Staphylococcus aureus bacteriophage and their anti-biofilm properties against MRSA sequence type 22 and 36

Staphylococcus aureus (including methicillin-resistant S. aureus - MRSA) remains a leading cause of both nosocomial and community acquired infections globally and despite constant improvement efforts to patient safety within healthcare, it still remains associated with considerable rates of morbidity and mortality. S. aureus is a common cause of biofilm-associated infections observed in chronic wounds, exhibiting a reduced susceptibility to the action of conventional antimicrobial agents and are often difficult to eradicate. The acquisition of resistance to almost any antibiotic with reference to MRSA has greatly reduced the number of alternative antimicrobial agents effective in the treatment of infections. Current development pipeline for new classes of antibiotics are greatly limited, requiring new, alternative approaches for therapeutic and prophylactic intervention in attempt to effectively control and overcome this current global health threat. Bacteriophage therapy exploits the natural killing ability of lytic bacteriophage (phage) as a means of controlling multidrug-resistant pathogenic bacteria. The utility of phage and their derivatives has been shown to effectively reduced the biofilms of major MRSA clones in vitro and in vivo. Global MRSA infections are caused by highly-successful isolates from a small number of epidemic lineages (clones). ST22 and ST36 are two of the most prevalent clones with global impact and largely responsible for the national epidemic of MRSA infections within UK healthcare system throughout the mid-1990s up until the mid-2000s. Understanding the phenotypic and genotypic characteristics of these clones in relation to the ability of bacteriophage to infect and disrupt established biofilms has yet to be explored. In this study, a total of 46 novel obligately lytic phage were isolated from wastewater samples by utilising a modified Staphylococcus carnosus TM300 isolate with expressed S. aureus wall teichoic acids to aid in phage adsorption. The addition of 32 more phage from our current laboratory stocks helped to establish a collection of 78 phage that were screened against a panel of 185 genetically diverse S. aureus, consisting of major clonal groups with high prevalence within the UK and United States, including 43 ST22 and 24 ST36 strains. The majority of the members displayed a wide host range against our panel. Based on this, the four most effective (wide host-range) phage were assessed for their anti-biofilm properties in polystyrene plates biofilm assays produced using four ST22 and four ST36 isolates. Treatment of mature biofilms was shown to significantly reduce biofilm biomass and viable cell counts. However these assays selected for the emergence of phage resistant mutants. Whole genome sequencing was performed on 22 phage isolates and these were found to share a high degree of similarity to genomes of 38 previously classified Twortvirinae phages represented in GenBank. Comparisons of these 60 phage genomes found a surprisingly high level of genetic diversity. Pairwise distances resolved groups of phage in distinct clusters representing individual genera within the Twortvirinae subfamily. Pan-genome analysis identified no single gene present amongst all phage genomes, however phage displayed a core genome amongst other members of the cluster. The structural homology tool HHpred was used to predict the protein structure of genes encoding for lytic enzymes among our phage genomes. We found that all phage encode a protein that shares high structural similarity to the same CHAP domain protein, a catalytic domain of endolysins employed by phage to degrade the bacterial host cell wall in order thus, mediate cell lysis. Suggesting that the all phage most likely share the same catalytic N-terminal endopeptidase domain of endolysins which have a modular domain structure. Interestingly, endolysins have been proposed as possible candidates for the control of antibiotic resistant S. aureus infections

Detection of Highly Pathogenic Avian Influenza Virus H5N1 Clade 2.3.4.4b in Great Skuas:A Species of Conservation Concern in Great Britain

The UK and Europe have seen successive outbreaks of highly pathogenic avian influenza across the 2020/21 and 2021/22 autumn/winter seasons. Understanding both the epidemiology and transmission of these viruses in different species is critical to aid mitigating measures where outbreaks cause extensive mortalities in both land- and waterfowl. Infection of different species can result in mild or asymptomatic outcomes, or acute infections that result in high morbidity and mortality levels. Definition of disease outcome in different species is of great importance to understanding the role different species play in the maintenance and transmission of these pathogens. Further, the infection of species that have conservation value is also important to recognise and characterise to understand the impact on what might be limited wild populations. Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b has been detected in great skuas (Stercorarius skua) across different colonies on islands off the shore of Scotland, Great Britain during summer 2021. A large number of great skuas were observed as developing severe clinical disease and dying during the epizootic and mortalities were estimated to be high where monitored. Of eight skuas submitted for post-mortem examination, seven were confirmed as being infected with this virus using a range of diagnostic assays. Here we overview the outbreak event that occurred in this species, listed as species of conservation concern in Great Britain and outline the importance of this finding with respect to virus transmission and maintenance

Emergence and spread of novel H5N8, H5N5 and H5N1 clade 2.3.4.4 highly pathogenic avian influenza in 2020

Analyses of HPAI H5 viruses from poultry outbreaks across a wide Eurasian region since July 2020 including the Russian Federation, Republics of Iraq and Kazakhstan, and recent detections in migratory waterfowl in the Netherlands, revealed undetected maintenance of H5N8, likely in galliform poultry since 2017/18 and both H5N5 and H5N1. All viruses belong to A/H5 clade 2.3.4.4b with closely related HA genes. Heterogeneity in Eurasian H5Nx HPAI emerging variants threatens poultry production, food security and veterinary public health

First detection of highly pathogenic avian influenza virus in Norway

Abstract Background Several outbreaks of highly pathogenic avian influenza (HPAI) caused by influenza A virus of subtype H5N8 have been reported in wild birds and poultry in Europe during autumn 2020. Norway is one of the few countries in Europe that had not previously detected HPAI virus, despite widespread active monitoring of both domestic and wild birds since 2005. Results We report detection of HPAI virus subtype H5N8 in a wild pink-footed goose (Anser brachyrhynchus), and several other geese, ducks and a gull, from south-western Norway in November and December 2020. Despite previous reports of low pathogenic avian influenza (LPAI), this constitutes the first detections of HPAI in Norway. Conclusions The mode of introduction is unclear, but a northward migration of infected geese or gulls from Denmark or the Netherlands during the autumn of 2020 is currently our main hypothesis for the introduction of HPAI to Norway. The presence of HPAI in wild birds constitutes a new, and ongoing, threat to the Norwegian poultry industry, and compliance with the improved biosecurity measures on poultry farms should therefore be ensured. [MK1]Finally, although HPAI of subtype H5N8 has been reported to have very low zoonotic potential, this is a reminder that HPAI with greater zoonotic potential in wild birds may pose a threat in the future. [MK1]Updated with a sentence emphasizing the risk HPAI pose to poultry farms, both in the Abstract and in the Conclusion-section in main text, as suggested by Reviewer 1 (#7)

First detection of highly pathogenic avian influenza virus in Norway

Background: Several outbreaks of highly pathogenic avian influenza (HPAI) caused by influenza A virus of subtype H5N8 have been reported in wild birds and poultry in Europe during autumn 2020. Norway is one of the few countries in Europe that had not previously detected HPAI virus, despite widespread active monitoring of both domestic and wild birds since 2005. Results: We report detection of HPAI virus subtype H5N8 in a wild pink-footed goose (Anser brachyrhynchus), and several other geese, ducks and a gull, from south-western Norway in November and December 2020. Despite previous reports of low pathogenic avian influenza (LPAI), this constitutes the first detections of HPAI in Norway. Conclusions: The mode of introduction is unclear, but a northward migration of infected geese or gulls from Denmark or the Netherlands during the autumn of 2020 is currently our main hypothesis for the introduction of HPAI to Norway. The presence of HPAI in wild birds constitutes a new, and ongoing, threat to the Norwegian poultry industry, and compliance with the improved biosecurity measures on poultry farms should therefore be ensured. [MK1]Finally, although HPAI of subtype H5N8 has been reported to have very low zoonotic potential, this is a reminder that HPAI with greater zoonotic potential in wild birds may pose a threat in the future. [MK1]Updated with a sentence emphasizing the risk HPAI pose to poultry farms, both in the Abstract and in the Conclusionsection in main text, as suggested by Reviewer 1 (#7). nseriformes, Charadriiformes, HPAI, H5N8, Surveillance, Measure

First detection of highly pathogenic avian influenza virus in Norway

Background: Several outbreaks of highly pathogenic avian influenza (HPAI) caused by influenza A virus of subtype H5N8 have been reported in wild birds and poultry in Europe during autumn 2020. Norway is one of the few countries in Europe that had not previously detected HPAI virus, despite widespread active monitoring of both domestic and wild birds since 2005. Results: We report detection of HPAI virus subtype H5N8 in a wild pink-footed goose (Anser brachyrhynchus), and several other geese, ducks and a gull, from south-western Norway in November and December 2020. Despite previous reports of low pathogenic avian influenza (LPAI), this constitutes the first detections of HPAI in Norway. Conclusions: The mode of introduction is unclear, but a northward migration of infected geese or gulls from Denmark or the Netherlands during the autumn of 2020 is currently our main hypothesis for the introduction of HPAI to Norway. The presence of HPAI in wild birds constitutes a new, and ongoing, threat to the Norwegian poultry industry, and compliance with the improved biosecurity measures on poultry farms should therefore be ensured. [MK1]Finally, although HPAI of subtype H5N8 has been reported to have very low zoonotic potential, this is a reminder that HPAI with greater zoonotic potential in wild birds may pose a threat in the future. [MK1]Updated with a sentence emphasizing the risk HPAI pose to poultry farms, both in the Abstract and in the Conclusionsection in main text, as suggested by Reviewer 1 (#7). nseriformes, Charadriiformes, HPAI, H5N8, Surveillance, Measure