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Antibiotic resistance is currently an extensive medical challenge worldwide, with global numbers increasing steadily. Recent data have highlighted wastewater treatment plants as a reservoir of resistance genes. The impact of these findings for human health can best be summarized using a One Health concept. However, the molecular mechanisms impacting resistance spread have not been carefully evaluated. Bacterial viruses, that is bacteriophages, have recently been shown to be important mediators of bacterial resistance genes in environmental milieus and are transferrable to human pathogens. Herein, we investigated the biogeographical impact on resistance spread through river-borne bacteriophages using amplicon deep sequencing of the microbiota, absolute quantification of resistance genes using ddPCR, and phage induction capacity within wastewater. Microbial biodiversity of the rivers is significantly affected by river site, surrounding milieu and time of sampling. Furthermore, areas of land associated with agriculture had a significantly higher ability to induce bacteriophages carrying antibiotic resistance genes, indicating their impact on resistance spread. It is imperative that we continue to analyse global antibiotic resistance problem from a One Health perspective to gain novel insights into mechanisms of resistance spread.

Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of six representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.

The bacterial species Cutibacterium acnes (formerly known as Propionibacterium acnes) is tightly associated with humans. It is the dominant bacterium in sebaceous regions of the human skin, where it preferentially colonizes the pilosebaceous unit. Multiple strains of C. acnes that belong to phylogenetically distinct types can co-exist. In this review we summarize and discuss the current knowledge of C. acnes regarding bacterial properties and traits that allow host colonization and play major roles in host-bacterium interactions and also regarding the host responses that C. acnes can trigger. These responses can have beneficial or detrimental consequences for the host. In the first part of the review, we highlight and critically review disease associations of C. acnes, in particular acne vulgaris, implant-associated infections and native infections. Here, we also analyse the current evidence for a direct or indirect role of a C. acnes-related dysbiosis in disease development or progression, i.e., reduced C. acnes strain diversity and/or the predominance of a certain phylotype. In the second part of the review, we highlight historical and recent findings demonstrating beneficial aspects of colonization by C. acnes such as colonization resistance, immune system interactions, and oxidant protection, and discuss the molecular mechanisms behind these effects. This new insight led to efforts in skin microbiota manipulation, such as the use of C. acnes strains as probiotic options to treat skin disorders.

Streptococcus pyogenes is known to cause both mucosal and systemic infections in humans. In this study, we used a combination of quantitative and structural mass spectrometry techniques to determine the composition and structure of the interaction network formed between human plasma proteins and the surface of different S. pyogenes serotypes. Quantitative network analysis revealed that S. pyogenes form serotype-specific interaction networks that are highly dependent on the domain arrangement of the surface-attached M protein. Subsequent structural mass spectrometry analysis and computational modelling on one of the M proteins, M28 revealed that the network structure changes across different host microenvironments. We report that M28 binds secretory IgA via two separate binding sites with high affinity in saliva. During vascular leakage mimicked by increasing plasma concentrations in saliva, the binding of secretory IgA was replaced by binding of monomeric IgA and C4BP. This indicates that an upsurge of C4BP in the local microenvironment due to damage of the mucosal membrane drives binding of C4BP and monomeric IgA to M28. The results suggest that S. pyogenes has evolved to form microenvironment-dependent host-pathogen protein complexes to combat the human immune surveillance during both mucosal and systemic infections.

Background Entamoeba gingivalis has been associated with periodontal diseases. Baseline data from the background population, which could help delimit the role of the parasite in health and disease, remain limited. Objective To describe epidemiological features, genetic diversity, and associations with oral microbiome signatures of E. gingivalis colonisation in Tanzanians with non-oral/non-dental diseases. Methods DNAs from 92 oral washings from 52 participants were subject to metabarcoding of ribosomal genes. DNA sequences were identified to genus level and submitted to oral microbiota diversity analyses. Results Sixteen (31%) of the 52 study participants were E. gingivalis-positive, with no difference in positivity rate according to gender or age. Only one subtype (ST1) was found. Individuals testing positive for E. gingivalis had higher oral microbiota alpha diversity than those testing negative (P = 0.03). Eight of the top-ten most common bacterial genera were shared between the two groups (Alloprevotella, Fusobacterium, Gemella, Haemophilus, Neisseria, Porphyromonas, Prevotella, Streptococcus, and Veillonella). Meanwhile, E. gingivalis carriers and non-carriers were more likely to have Aggregatibacter and Rothia, respectively, among the top-ten most common genera. Conclusion About one third of the cohort carried E. gingivalis ST1, and carriers had higher oral microbiome diversity and were more predominantly colonized by Aggregatibacter.

IntroductionStreptococcus dysgalactiae can cause severe recurrent infections. This study aimed to investigate antibody responses following S. dysgalactiae bacteraemia and possible development of protective immunity.Materials and MethodsPatients with S. dysgalactiae bacteraemia in the county of Skåne between 2017 and 2018 were prospectively included. Acute and convalescent sera were obtained. All isolates were emm typed and enzyme-linked immunosorbent assay (ELISA) was utilised to analyse specific antibody responses to bacteria and antigens. Bactericidal- and phagocytosis assays were applied to further establish antibody function.ResultsSixteen patients with S. dysgalactiae bacteraemia were included of whom one had recurrent episodes of bacteraemia. Using ELISA with S. dysgalactiae isolates and mutants, development of IgG antibodies was demonstrated in few patients. Type-specific antibodies were demonstrated in one patient when recombinant M proteins as antigens, were applied. The type-specific serum mediated a small increase in phagocytosis but did not facilitate increased killing of the S. dysgalactiae isolate, carrying that M protein, in blood or by phagocytic cells.ConclusionS. dysgalactiae bacteraemia sometimes results in increased levels of antibodies to the infecting pathogen. We did not find evidence that these antibodies are effectively opsonising. Apparent failure to produce opsonising antibodies might partially explain why S. dysgalactiae can cause recurrent invasive infections in the same host.

Aerococcus urinae is an emerging pathogen that causes urinary tract infections, bacteremia and infective endocarditis. The mechanisms through which A. urinae cause infection are largely unknown. The aims of this study were to describe the surface proteome of A. urinae and to analyse A. urinae genomes in search for genes encoding surface proteins. Two proteins, denoted Aerococcal surface protein (Asp) 1 and 2, were through the use of mass spectrometry based proteomics found to quantitatively dominate the aerococcal surface. The presence of these proteins on the surface was also shown using ELISA with serum from rabbits immunized with the recombinant Asp. These proteins had a signal sequence in the amino-terminal end and a cell wall-sorting region in the carboxy-terminal end, which contained an LPATG-motif, a hydrophobic domain and a positively charged tail. Twenty-three additional A. urinae genomes were sequenced using Illumina HiSeq technology. Six different variants of asp genes were found (denoted asp1-6). All isolates had either one or two of these asp-genes located in a conserved locus, designated Locus encoding Aerococcal Surface Proteins (LASP). The 25 genomes had in median 13 genes encoding LPXTG-proteins (range 6-24). For other Gram-positive bacteria, cell wall-anchored surface proteins with an LPXTG-motif play a key role for virulence. Thus, it will be of great interest to explore the function of the Asp proteins of A. urinae to establish a better understanding of the molecular mechanisms by which A. urinae cause disease.

The increased interest in predatory bacteria due to their ability to kill antibiotic resistant bacteria has also highlighted their inherent plethora of hydrolytic enzymes, and their potential as natural sources of novel therapeutic agents and biotechnological tools. Here, we have identified and characterized a novel protease from the predatory bacterium Bdellovibrio bacteriovorus: BspE (Bdellovibrio elastase-like serine protease). Mapping preferential sites of proteolytic activity showed a single proteolytic cleavage site of native plasma IgA (pIgA) in the Fc-tail; as well as in the secretory component (SC) of secretory IgA (SIgA). Proteolysis of other native immunoglobulins and plasma proteins was either absent (IgG1 and 2, IgM, albumin and orosomucoid) or unspecific with multiple cleavage sites (IgG3 and 4, IgE, IgD). BspE displayed a broad activity against most amino acid bonds in shorter peptides and denatured proteins, with a slight preference for hydrolysis C-terminal of Y, V, F, S, L, R, P, E, and K. BspE autoproteolysis results in numerous cleavage products sustaining activity for more than 6 h. The enzymatic activity remained stable at pH 5.0–9.0 but was drastically reduced in the presence of MnCl2 and completely inhibited by ZnCl2. The hydrolysis of pIgA was subsequently utilized for the specific glycan characterization of the released pIgA Fc-tail (Asn459). Besides contributing to the basic knowledge of Bdellovibrio biology and proteases, we propose that BspE could be used as a potential tool to investigate the importance, and biological function of the pIgA Fc-tail. IMPORTANCE Antibodies are well-established as key components of the immune system, and the importance of antibody glycosylation is steadily gaining recognition. Modifications of antibodies by glycosylation creates a vast repertoire of antibody glycovariants with distinctive and diverse functions in the immune system. Most of the available information regarding antibody glycosylation is based on studies with IgG, which have contributed greatly to the advance of therapeutic antibody treatments. However, much is still unknown regarding the importance of glycosylation and the Fc-structure for the remaining antibody classes. Such research has proven to be technically challenging and demonstrates a need for novel tools to facilitate such investigations. Here we have identified and characterized a novel protease from B. bacteriovorus, facilitating the study of plasma IgA by cleaving the Fc-tail, including the Asn459 N-glycan. This further highlights the potential of B. bacteriovorus as a source to identify potential novel biotechnological tools.

Acinetobacter baumannii is a Gram-negative bacterial pathogen responsible for a range of nosocomial infections. The recent rise and spread of multidrug-resistant A. baumannii clones has fueled a search for alternative therapies, including bacteriophage endolysins with potent antibacterial activities. A common feature of these lysins is the presence of a highly positively charged C-terminal domain with a likely role in promoting outer membrane penetration. In the present study, we show that the C-terminal amino acids 108 to 138 of phage lysin PlyF307, named P307, alone were sufficient to kill A. baumannii (>3 logs). Furthermore, P307 could be engineered for improved activity, the most active derivative being P307 SQ-8C (>5-log kill). Both P307 and P307 SQ-8C showed high in vitro activity against A. baumannii in biofilms. Moreover, P307 SQ-8C exhibited MICs comparable to those of levofloxacin and ceftazidime and acted synergistically with polymyxin B. Although the peptides were shown to kill by disrupting the bacterial cytoplasmic membrane, they did not lyse human red blood cells or B cells; however, serum was found to be inhibitory to lytic activity. In a murine model of A. baumannii skin infection, P307 SQ-8C reduced the bacterial burden by ∼2 logs in 2 h. This study demonstrates the prospect of using peptide derivatives from bacteriophage lysins to treat topical infections and remove biofilms caused by Gram-negative pathogens.

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.

Bacteriophage endolysins have shown great efficacy in killing Gram-positive bacteria. PlyC, a group C streptococcal phage lysin, represents the most efficient lysin characterized to date, with a remarkably high specificity against different streptococcal species, including the important pathogen Streptococcus pyogenes . However, PlyC is a unique lysin, in terms of both its high activity and structure (two distinct subunits). We sought to discover and characterize a phage lysin active against S. pyogenes with an endolysin architecture distinct from that of PlyC to determine if it relies on the same mechanism of action as PlyC. In this study, we identified and characterized an endolysin, termed PlyPy ( p hage ly sin from S. py ogenes ), from a prophage infecting S. pyogenes . By in silico analysis, PlyPy was found to have a molecular mass of 27.8 kDa and a pI of 4.16. It was active against a majority of group A streptococci and displayed high levels of activity as well as binding specificity against group B and C streptococci, while it was less efficient against other streptococcal species. PlyPy showed the highest activity at neutral pH in the presence of calcium and NaCl. Surprisingly, its activity was not affected by the presence of the group A-specific carbohydrate, while the activity of PlyC was partly inhibited. Additionally, PlyPy was active in vivo and could rescue mice from systemic bacteremia. Finally, we developed a novel method to determine the peptidoglycan bond cleaved by lysins and concluded that PlyPy exhibits a rare d -alanyl- l -alanine endopeptidase activity. PlyPy thus represents the first lysin characterized from Streptococcus pyogenes and has a mechanism of action distinct from that of PlyC.

Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of thePropionibacterium acnesbacteriophage research and point out how bacteriophage research has been an important part of the research onP. acnesitself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs). Finally, the potential to useP. acnesbacteriophages as a therapeutic strategy to combatP. acnes-associated diseases will be discussed.

Protein-protein interactions play important roles in bacterial pathogenesis. Surface-bound or secreted bacterial proteins are key in mediating bacterial virulence. Thus, these factors are of high importance to study in order to elucidate the molecular mechanisms behind bacterial pathogenesis. Here, we present a protein-based strategy that can be used to identify and isolate bacterial proteins of importance for bacterial virulence, and allow for identification of both unknown host and bacterial factors. The methods described have among others successfully been used to identify and characterize several IgG-binding proteins, including protein G, protein H, and protein L.

The role of the skin microbiota in human health is poorly understood. Here, we identified and characterized a novel antioxidant enzyme produced by the skin microbiota, designated RoxP for radical oxygenase of Propionibacterium acnes. RoxP is uniquely produced by the predominant skin bacterium P. acnes, with no homologs in other bacteria; it is highly expressed and strongly secreted into culture supernatants. We show that RoxP binds heme, reduces free radicals, and can protect molecules from oxidation. Strikingly, RoxP is crucial for the survival of P. acnes in oxic conditions and for skin colonization of P. acnes ex vivo. Taken together, our study strongly suggests that RoxP facilitates P. acnes’ survival on human skin, and is an important beneficial factor for the host-commensal interaction. Thus, RoxP is the first described skin microbiota-derived mutualistic factor that potentially can be exploited for human skin protection.

Propionibacterium acnes is a common and probably underestimated cause of delayed joint prosthesis infection. Bacterial biofilm formation is central in the pathogenesis of infections related to foreign material, and P. acnes has been shown to form biofilm both in vitro and in vivo. Here, biofilm formation by 93 P. acnes isolates, either from invasive infections (n = 45) or from the skin of healthy people (n = 48), was analysed. The majority of isolates from deep infections produced biofilm in a microtitre model of biofilm formation, whereas the skin isolates were poor biofilm producers (p

Finegoldia magnais a member of the normal human bacterial flora on the skin and other non-sterile body surfaces, but this anaerobic coccus is also an important opportunistic pathogen. SufA was the firstF. magnaproteinase to be isolated and characterized. Many bacterial pathogens interfere with different steps of blood coagulation, and here we describe how purified SufA efficiently and specifically cleaves fibrinogen in human plasma. SufA is both secreted byF. magnaand associated with the bacterial surface. Successful gene targeting has previously not been performed in anaerobic cocci, but in order to study the role of the SufA that is present at the bacterial surface, we constructed anF. magnamutant that expresses a truncated SufA lacking proteolytic activity. In contrast to wild-type bacteria that delayed the coagulation of human plasma, mutant bacteria had no such effect. Wild-type and mutant bacteria adhered to keratinocytes equally well, but in a plasma environment only wild-type bacteria blocked the formation of fibrin networks surrounding adherent bacteria. The effective cleavage of fibrinogen by SufA suggests that the interference with fibrin network formation represents an adaptive mechanism ofF. magnawith potential implications also for pathogenicity.

Bacterial infections are becoming an increasing problem worldwide and there is a need for a deeper understanding of how bacteria turn pathogenic. Here, we suggest that one answer may be found by taking into account the localization of the bacteria, both at an anatomical level and at a microenvironment level. Both commensals and traditional pathogens alter their interaction with the human host depending on the local surroundings – turning either more or less virulent. These localization effects could derive from the characteristics of different anatomical sites but also from local differences within a microenvironment. In order to understand the adaptive functions of bacterial virulence factors, we need to study the bacteria in the environments where they have evolved.

Acinetobacter baumannii , a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killing A. baumannii . The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo . Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found in Acinetobacter phage.

Background Propionibacterium acnes is a Gram positive rod inhabiting the human skin that also infects orthopaedic implants and is associated with acne vulgaris. Previously, one lytic bacteriophage, PA6, from P. acnes has been sequenced and partially characterized. We recently isolated several inducible phages from P. acnes classified as Siphoviruses based on morphology and partial genome sequencing. Results In this study we sequenced the inducible P. acnes phages PAD20 and PAS50, isolated from deep infection and from skin, respectively. The genomes of PAD20 and PAS50 are 29,074 and 29,017 bp, respectively, compared with the 29,739 bp of PA6. The phage genomes have 87.3-88.7% nucleotide sequence identity. The genes are divided into clusters with different levels of similarity between the phages. PAD20 and PAS50 share four genes encoding identical amino acid sequences. Some deletions and insertions in the genomes have occurred, resulting in lack of genes, frame shifts, and possible regulatory differences. No obvious virulence factor gene candidates were found. The phages are inducible, but bacteria can be cured of phages by serial colony isolations and lose their phages during stationary phase, but are still sensitive to new phage infections. Construction of a phylogenetic tree based on more than 459 phage genomes, suggested that P. acnes phages represent a new lineage of Siphoviruses. Conclusions The investigated P. acnes Siphovirus genomes share a high degree of homology to other P. acnes phages sequenced, but not to genomes of other phages isolated from Propionibacteria. The phage genomes are not integrated in the bacterial genome, but instead, most likely have a pseudolysogenic life cycle.

Background Propionibacterium acnes is a commensal of human skin but is also known to be involved in certain diseases, such as acne vulgaris and infections of orthopaedic implants. Treatment of these conditions is complicated by increased resistance to antibiotics and/or biofilm formation of P. acnes bacteria. P. acnes can be infected by bacteriophages, but until recently little has been known about these viruses. The aim of this study was to identify and characterize inducible phages from P. acnes on a genetic and morphological basis. Results More than 70% (65/92) of P. acnes isolates investigated have inducible phages, classified morphologically as Siphoviruses. The phages have a head of 55 nm in diameter and a tail of 145–155 nm in length and 9–10 nm in width. There was no difference in carriage rate of phages between P. acnes isolates from deep infections and isolates from skin. However, there was a significant lower carriage rate of phages in P. acnes biotype IB, mostly attributed to the low carriage rate of inducible phages in biotype IB isolated from deep tissue. Most phages have a strong lytic activity against all P. acnes isolates with inducible phages, but have less lytic activity against isolates that have no prophages. Phages only infected and lysed P. acnes and not other closely related propionibacteria. All phages could infect and lyse their non-induced parental host, indicating that these prophages do not confer superinfection immunity. The phages have identical protein pattern as observed on SDS-PAGE. Finally, sequencing of two phage genes encoding a putative major head protein and an amidase and showed that the phages could be divided into different groups on a genetic basis. Conclusion Our findings indicate that temperate phages are common in P. acnes, and that they are a genetically and functionally homogeneous group of Siphoviruses. The phages are specific for P. acnes and do not seem to confer superinfection immunity.