Phage-host co-evolution has led to distinct generalized transduction strategies.

Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuity or directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction.

FLAMS: Find Lysine Acylations and other Modification Sites.

SUMMARY: Today, hundreds of post-translational modification (PTM) sites are routinely identified at once, but the comparison of new experimental datasets to already existing ones is hampered by the current inability to search most PTM databases at the protein residue level. We present FLAMS (Find Lysine Acylations and other Modification Sites), a Python3-based command line and web-tool that enables researchers to compare their PTM sites to the contents of the CPLM, the largest dedicated protein lysine modification database, and dbPTM, the most comprehensive general PTM database, at the residue level. FLAMS can be integrated into PTM analysis pipelines, allowing researchers to quickly assess the novelty and conservation of PTM sites across species in newly generated datasets, aiding in the functional assessment of sites and the prioritization of sites for further experimental characterization. AVAILABILITY AND IMPLEMENTATION: FLAMS is implemented in Python3, and freely available under an MIT license. It can be found as a command line tool at https://github.com/hannelorelongin/FLAMS, pip and conda; and as a web service at https://www.biw.kuleuven.be/m2s/cmpg/research/CSB/tools/flams/.

Endolysin NC5 improves early cloxacillin treatment in a mouse model of Streptococcus uberis mastitis.

Streptococcus uberis frequently causes bovine mastitis, an infectious udder disease with significant economic implications for dairy cows. Conventional antibiotics, such as cloxacillin, sometimes have limited success in eliminating S. uberis as a stand-alone therapy. To address this challenge, the study objective was to investigate the VersaTile engineered endolysin NC5 as a supplemental therapy to cloxacillin in a mouse model of bovine S. uberis mastitis. NC5 was previously selected based on its intracellular killing and biofilm eradicating activity. To deliver preclinical proof-of-concept of this supplemental strategy, lactating mice were intramammarily infected with a bovine S. uberis field isolate and subsequently treated with cloxacillin (30.0 µg) combined with either a low (23.5 µg) or high (235.0 µg) dose of NC5. An antibiotic monotherapy group, as well as placebo treatment, was included as controls. Two types of responders were identified: fast (n = 17), showing response after 4-h treatment, and slow (n = 10), exhibiting no clear response at 4 h post-treatment across all groups. The high-dose combination therapy in comparison with placebo treatment impacted the hallmarks of mastitis in the fast responders by reducing (i) the bacterial load 13,000-fold (4.11 ± 0.78 Δlog10; p < 0.001), (ii) neutrophil infiltration 5.7-fold (p > 0.05), and (iii) the key pro-inflammatory chemokine IL-8 13-fold (p < 0.01). These mastitis hallmarks typically followed a dose response dependent on the amount of endolysin added. The current in vivo study complements our in vitro data and provides preclinical proof-of-concept of NC5 as an adjunct to intramammary cloxacillin treatment. KEY POINTS: • Engineered endolysin NC5 was preclinically evaluated as add-on to cloxacillin treatment. • Two types of mice (slow and fast responding) were observed. • The add-on treatment decreased bacterial load, neutrophil influx, and pro-inflammatory mediators.

Isolation and characterization of novel Staphylococcus aureus bacteriophage Hesat from dairy origin.

A novel temperate phage, named Hesat, was isolated by the incubation of a dairy strain of Staphylococcus aureus belonging to spa-type t127 with either bovine or ovine milk. Hesat represents a new species of temperate phage within the Phietavirus genus of the Azeredovirinae subfamily. Its genome has a length of 43,129 bp and a GC content of 35.11% and contains 75 predicted ORFs, some of which linked to virulence. This includes (i) a pathogenicity island (SaPln2), homologous to the type II toxin-antitoxin system PemK/MazF family toxin; (ii) a DUF3113 protein (gp30) that is putatively involved in the derepression of the global repressor Stl; and (iii) a cluster coding for a PVL. Genomic analysis of the host strain indicates Hesat is a resident prophage. Interestingly, its induction was obtained by exposing the bacterium to milk, while the conventional mitomycin C-based approach failed. The host range of phage Hesat appears to be broad, as it was able to lyse 24 out of 30 tested S. aureus isolates. Furthermore, when tested at high titer (108 PFU/ml), Hesat phage was also able to lyse a Staphylococcus muscae isolate, a coagulase-negative staphylococcal strain. KEY POINTS: • A new phage species was isolated from a Staphylococcus aureus bovine strain. • Pathogenicity island and PVL genes are encoded within phage genome. • The phage is active against most of S. aureus strains from both animal and human origins.

Characterization of mycophage endolysin cell wall binding domains targeting Mycobacterium bovis peptidoglycan.

Mycophage endolysins are highly diverse and modular enzymes composed of domains involved in peptidoglycan binding and degradation. Mostly, they are characterized by a three-module design: an N-terminal peptidase domain, a central catalytic domain and a C-terminal peptidoglycan binding domain. Previously, the affinity of cell wall binding domains (CBDs) to the mycobacterial peptidoglycan layer was shown for some of these endolysins. In this study, an in depth screening was performed on twelve mycophage endolysins. The discovered CBDs were characterized for their binding affinity to Mycobacterium (M.) bovis bacille Calmette-Guérin (BCG), a largely unexplored target and an attenuated strain of M. bovis, responsible for bovine tuberculosis. Using homology-based annotation, only four endolysins showed the presence of a known peptidoglycan binding domain, the previously characterized pfam 01471 domain. However, analysis of the secondary structure aided by AlphaFold predictions revealed the presence of a C-terminal domain in the other endolysins. These were hypothesized as new, uncharacterized CBDs. Fusion proteins composed of these domains linked to GFP were constructed and positively assayed for their affinity to M. bovis BCG in a peptidoglycan binding assay. Moreover, two CBDs were able to fluorescently label M. bovis BCG in milk samples, highlighting the potential to further explore their possibility to function as CBD-based diagnostics.

SASpector: analysis of missing genomic regions in draft genomes of prokaryotes.

SUMMARY: Missing regions in short-read assemblies of prokaryote genomes are often attributed to biases in sequencing technologies and to repetitive elements, the former resulting in low sequencing coverage of certain loci and the latter to unresolved loops in the de novo assembly graph. We developed SASpector, a command-line tool that compares short-read assemblies (draft genomes) to their corresponding closed assemblies and extracts missing regions to analyze them at the sequence and functional level. SASpector allows to benchmark the need for resolved genomes, can be integrated into pipelines to control the quality of assemblies, and could be used for comparative investigations of missingness in assemblies for which both short-read and long-read data are available in the public databases. AVAILABILITY AND IMPLEMENTATION: SASpector is available at https://github.com/LoGT-KULeuven/SASpector. The tool is implemented in Python3 and available through pip and Docker (0mician/saspector). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity.

Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung.

Isolation and molecular characterization of the Salmonella Typhimurium orphan phage Arash.

The current threat of multidrug resistant strains necessitates development of alternatives to antibiotics such as bacteriophages. This study describes the isolation and characterization of a novel Salmonella Typhimurium phage 'Arash' from hospital wastewater in Leuven, Belgium. Arash has a myovirus morphology with a 95 nm capsid and a 140 nm tail. The host range of Arash is restricted to its isolation host. Approximately 86% of the phage particles are adsorbed to a host cell within 10 min. Arash has latent period of 65 min and burst size of 425 PFU/cell. Arash has a dsDNA genome of 180,819 bp with GC content of 53.02% with no similarities to any characterized phages, suggesting Arash as a novel species in the novel 'Arashvirus' genus. Arash carries no apparent lysogeny-, antibiotic resistance- nor virulence-related genes. Proteome analysis revealed 116 proteins as part of the mature phage particles of which 27 could be assigned a function. Therefore, the present findings shed light on the morphological, microbiological and genomic characteristics of Arash and suggest its potential application as therapeutic and/or biocontrol agent.

Thermocaproicibacter melissae gen. nov., sp. nov., a thermophilic chain-elongating bacterium, producing n-caproate from polymeric carbohydrates.

Strain MDTJ8T is a chain-elongating thermophilic bacterium isolated from a thermophilic acidogenic anaerobic digestor treating human waste while producing the high commodity chemical n-caproate. The strain grows and produces formate, acetate, n-butyrate, n-caproate and lactate from mono-, di- and polymeric saccharides at 37-60 °C (optimum, 50-55 °C) and at pH 5.0-7.0 (optimum, pH 6.5). The organism is an obligate anaerobe, is motile and its cells form rods (0.3-0.5×1.0-3.0 µm) that stain Gram-positive and occur primarily as chains. Phylogenetic analysis of both the 16S rRNA gene and full genome sequence shows that strain MDTJ8T belongs to a group that consists of mesophylic chain-elongating bacteria within the family Oscillospiraceae, being nearest to Caproicibacter fermentans EA1T (94.8 %) and Caproiciproducens galactitolivorans BS-1T (93.7 %). Its genome (1.96 Mbp) with a G+C content of 49.6 mol% is remarkably smaller than those of other chain-elongating bacteria of the family Oscillospiraceae. Pairwise average nucleotide identity and DNA-DNA hybridization values between strain MDJT8T and its mesophilic family members are less than 70 and 35 %, respectively, while pairwise average amino acid identity values are less than 68 %. In addition, strain MDJT8T uses far less carbohydrate and non-carbohydrate substrates compared to its nearest family members. The predominant cellular fatty acids of strain MDTJ8T are C14 : 0, C14 : 0 DMA (dimethyl acetal) and C16 : 0, while its polar lipid profile shows three unidentified glycophospholipids, 11 glycolipids, 13 phospholipids and six unidentified lipids. No respiratory quinones and polyamines are detected. Based on its phylogenetic, genotypic, morphological, physiological, biochemical and chemotaxonomic characteristics, strain MDTJ8T represents a novel species and novel genus of the family Oscillospiraceae and Thermocaproicibacter melissae gen. nov., sp. nov. is proposed as its name. The type strain is MDTJ8T (=DSM 114174T=LMG 32615T=NCCB 100883T).

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Impact of phage predation on P. aeruginosa adhered to human airway epithelium: major transcriptomic changes in metabolism and virulence-associated genes.

Phage therapy is a promising adjunct therapeutic approach against bacterial multidrug-resistant infections, including Pseudomonas aeruginosa-derived infections. Nevertheless, the current knowledge about the phage-bacteria interaction within a human environment is limited. In this work, we performed a transcriptome analysis of phage-infected P. aeruginosa adhered to a human epithelium (Nuli-1 ATCC® CRL-4011™). To this end, we performed RNA-sequencing from a complex mixture comprising phage-bacteria-human cells at early, middle, and late infection and compared it to uninfected adhered bacteria. Overall, we demonstrated that phage genome transcription is unaltered by bacterial growth and phage employs a core strategy of predation through upregulation of prophage-associated genes, a shutdown of bacterial surface receptors, and motility inhibition. In addition, specific responses were captured under lung-simulating conditions, with the expression of genes related to spermidine syntheses, sulphate acquisition, biofilm formation (both alginate and polysaccharide syntheses), lipopolysaccharide (LPS) modification, pyochelin expression, and downregulation of virulence regulators. These responses should be carefully studied in detail to better discern phage-induced changes from bacterial responses against phage. Our results establish the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, being obvious the phage versatility on bacterial cell invasion.

CkP1 bacteriophage, a S16-like myovirus that recognizes Citrobacter koseri lipopolysaccharide through its long tail fibers.

Citrobacter koseri is an emerging Gram-negative bacterial pathogen, which causes urinary tract infections. We isolated and characterized a novel S16-like myovirus CKP1 (vB_CkoM_CkP1), infecting C. koseri. CkP1 has a host range covering the whole C. koseri species, i.e., all strains that were tested, but does not infect other species. Its linear 168,463-bp genome contains 291 coding sequences, sharing sequence similarity with the Salmonella phage S16. Based on surface plasmon resonance and recombinant green florescence protein fusions, the tail fiber (gp267) was shown to decorate C. koseri cells, binding with a nanomolar affinity, without the need of accessory proteins. Both phage and the tail fiber specifically bind to bacterial cells by the lipopolysaccharide polymer. We further demonstrate that CkP1 is highly stable towards different environmental conditions of pH and temperatures and is able to control C. koseri cells in urine samples. Altogether, CkP1 features optimal in vitro characteristics to be used both as a control and detection agent towards drug-resistant C. koseri infections. KEY POINTS: • CkP1 infects all C. koseri strains tested • CkP1 recognizes C. koseri lipopolysaccharide through its long tail fiber • Both phage CkP1 and its tail fiber can be used to treat or detect C. koseri pathogens.

Phage therapy for hidradenitis suppurativa: a unique challenge and possible opportunity for personalized treatment of a complex, inflammatory disease.

Phage therapy is an emerging antimicrobial treatment for critical multidrug-resistant pathogens. In this review, the specific potential and challenges of phage therapy for patients with hidradenitis suppurativa (HS) are discussed. This represents a unique challenge as HS is a chronic inflammatory disease, but presenting with acute exacerbations, which have an enormous negative impact on patient's quality of life. The therapeutic arsenal for HS has expanded in the past decade, for example, with adalimumab and several other biologicals that are currently under investigation. However, treatment of HS remains challenging for dermatologists because there are individuals who do not respond to any classes of the current treatment options when used for a first or second time. Furthermore, after several courses of treatment, a patient may lose their response to therapy, meaning long-term use is not always an option. Culturing studies and 16S ribosomal RNA profiling highlight the complex polymicrobial nature of HS lesions. Despite the detection of various bacterial species in lesion samples, several key pathogens, including Staphylococcus, Corynebacterium and Streptococcus, may be potential targets for phage therapy. Using phage therapy for the treatment of a chronic inflammatory disease could potentially provide new insights into the role of bacteria and the immune system in HS development. In addition, it is possible more details on the immunomodulatory effects of phages may come to light.

Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications in Pseudomonas Species.

The phage T7 RNA polymerase (RNAP) and lysozyme form the basis of the widely used pET expression system for recombinant expression in the biotechnology field and as a tool in microbial synthetic biology. Attempts to transfer this genetic circuitry from Escherichia coli to non-model bacterial organisms with high potential have been restricted by the cytotoxicity of the T7 RNAP in the receiving hosts. We here explore the diversity of T7-like RNAPs mined directly from Pseudomonas phages for implementation in Pseudomonas species, thus relying on the co-evolution and natural adaptation of the system towards its host. By screening and characterizing different viral transcription machinery using a vector-based system in P. putida., we identified a set of four non-toxic phage RNAPs from phages phi15, PPPL-1, Pf-10, and 67PfluR64PP, showing a broad activity range and orthogonality to each other and the T7 RNAP. In addition, we confirmed the transcription start sites of their predicted promoters and improved the stringency of the phage RNAP expression systems by introducing and optimizing phage lysozymes for RNAP inhibition. This set of viral RNAPs expands the adaption of T7-inspired circuitry towards Pseudomonas species and highlights the potential of mining tailored genetic parts and tools from phages for their non-model host.

Long-Range PCR Reveals the Genetic Cargo of IncP-1 Plasmids in the Complex Microbial Community of an On-Farm Biopurification System Treating Pesticide-Contaminated Wastewater.

Promiscuous plasmids like IncP-1 plasmids play an important role in the bacterial adaptation to pollution by acquiring and distributing xenobiotic catabolic genes. However, most information comes from isolates and the role of plasmids in governing community-wide bacterial adaptation to xenobiotics and other adaptive forces is not fully understood. Current information on the contribution of IncP-1 plasmids in community adaptation is limited because methods are lacking that directly isolate and identify the plasmid borne adaptive functions in whole-community DNA. In this study, we optimized long-range PCR to directly access and identify the cargo carried by IncP-1 plasmids in environmental DNA. The DNA between the IncP-1 backbone genes trbP and traC, a main insertion site of adaptive trait determinants, is amplified and its content analyzed by high-throughput sequencing. The method was applied to DNA of an on-farm biopurification system (BPS), treating pesticide contaminated wastewater, to examine whether horizontal gene exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. The cargo recovered from BPS community DNA encoded catabolic but also resistance traits and various other (un)known functions. Unexpectedly, genes with catabolic traits composed only a minor fraction of the cargo, indicating that the IncP-1 region between trbP and traC is not a major contributor to catabolic adaptation of the BPS microbiome. Instead, it contains a functionally diverse set of genes which either may assist biodegradation functions, be remnants of random gene recruitment, or confer other crucial functions for proliferation in the BPS environment. IMPORTANCE This study presents a long-range PCR for direct and cultivation-independent access to the identity of the cargo of a major insertion hot spot of adaptive genes in IncP-1 plasmids and hence a new mobilome tool for understanding the role of IncP-1 plasmids in complex communities. The method was applied to DNA of an on-farm biopurification system (BPS) treating pesticide-contaminated wastewater, aiming at new insights on whether horizontal exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. Unexpectedly, catabolic functions represented a small fraction of the cargo genes while multiple other gene functions were recovered. These results show that the cargo of the target insertion hot spot in IncP-1 plasmids in a community, not necessarily relates to the main obvious selective trait imposed on that community. Instead, these functions might contribute to adaptation to unknown selective forces or represent remnants of random gene recruitment.

'Drc', a structurally novel ssDNA-binding transcription regulator of N4-related bacterial viruses.

Bacterial viruses encode a vast number of ORFan genes that lack similarity to any other known proteins. Here, we present a 2.20 Å crystal structure of N4-related Pseudomonas virus LUZ7 ORFan gp14, and elucidate its function. We demonstrate that gp14, termed here as Drc (ssDNA-binding RNA Polymerase Cofactor), preferentially binds single-stranded DNA, yet contains a structural fold distinct from other ssDNA-binding proteins (SSBs). By comparison with other SSB folds and creation of truncation and amino acid substitution mutants, we provide the first evidence for the binding mechanism of this unique fold. From a biological perspective, Drc interacts with the phage-encoded RNA Polymerase complex (RNAPII), implying a functional role as an SSB required for the transition from early to middle gene transcription during phage infection. Similar to the coliphage N4 gp2 protein, Drc likely binds locally unwound middle promoters and recruits the phage RNA polymerase. However, unlike gp2, Drc does not seem to need an additional cofactor for promoter melting. A comparison among N4-related phage genera highlights the evolutionary diversity of SSB proteins in an otherwise conserved transcription regulation mechanism.

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori.

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.

Transcriptional Organization of the Salmonella Typhimurium Phage P22 pid ORFan Locus.

Many phage genes lack sequence similarity to any other open reading frame (ORF) in current databases. These enigmatic ORFan genes can have a tremendous impact on phage propagation and host interactions but often remain experimentally unexplored. We previously revealed a novel interaction between phage P22 and its Salmonella Typhimurium host, instigated by the ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon. The pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. Here, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5' untranslated region that is instructive for a secondary pid mRNA species, and has a 3' Rho-independent termination loop that is responsible for stability of the pid transcript.

The evolutionary trade-offs in phage-resistant Klebsiella pneumoniae entail cross-phage sensitization and loss of multidrug resistance.

Bacteriophage therapy is currently being evaluated as a critical complement to traditional antibiotic treatment. However, the emergence of phage resistance is perceived as a major hurdle to the sustainable implementation of this antimicrobial strategy. By combining comprehensive genomics and microbiological assessment, we show that the receptor-modification resistance to capsule-targeting phages involves either escape mutation(s) in the capsule biosynthesis cluster or qualitative changes in exopolysaccharides, converting clones to mucoid variants. These variants introduce cross-resistance to phages specific to the same receptor yet sensitize to phages utilizing alternative ones. The loss/modification of capsule, the main Klebsiella pneumoniae virulence factor, did not dramatically impact population fitness, nor the ability to protect bacteria against the innate immune response. Nevertheless, the introduction of phage drives bacteria to expel multidrug resistance clusters, as observed by the large deletion in K. pneumoniae 77 plasmid containing blaCTX-M , ant(3″), sul2, folA, mph(E)/mph(G) genes. The emerging bacterial resistance to viral infection steers evolution towards desired population attributes and highlights the synergistic potential for combined antibiotic-phage therapy against K. pneumoniae.

qDNase assay: A quantitative method for real-time assessment of DNase activity on coated surfaces.

DNase coatings show great potential to prevent biofilm formation in various applications of the medical implant, food and marine industry. However, straightforward and quantitative methods to characterize the enzymatic activity of these coatings are currently not available. We here introduce the qDNase assay, a quantitative, real-time method to characterize the activity of DNase coatings. The assay combines (1) the use of an oligonucleotide probe, which fluoresces upon cleavage by coated DNases, and (2) the continuous read-out of the fluorescent signal within a microplate fluorometer format. The combination of these two properties results in a real-time fluorescent signal that is used to directly quantify the activity of DNase coatings. As a proof of concept, bovine DNase I coatings were immobilized on titanium by means of chemical grafting and their activity was estimated at 3.87 × 10-4 U. To our knowledge, the qDNase assay provides the first approach to report the activity of a DNase coating in absolute DNase activity units. This assay will not only serve to compare existing DNase coating methods more accurately, but will also enable the rational design of new DNase coating methods in the future.