A systematic review of the use of bacteriophages for in vitro biofilm control.

Bacteriophages (phages) are very promising biological agents for the prevention and control of bacterial biofilms. However, little is known about the parameters that can influence the efficacy of phages on biofilms. This systematic review provides a summary and analysis of the published data about the use of phages to control pre-formed biofilms in vitro, suggesting recommendations for future experiments in this area. A total of 68 articles, containing data on 605 experiments addressing the efficacy of phages to control biofilms in vitro were included, after a search conducted in Web of Science, Embase, and Medline (PubMed). The data collected from each experiment included information about biofilm growth conditions, phage characteristics, treatment conditions and biofilm reduction. In most cases, biofilms were formed in the surface of microtiter plates (82.5%); the median time for biofilm formation was 24 h, as is the median treatment duration. Quantification of biofilm biomass (52.6%), viable cells (25.5%) and metabolic activity (17.9%) were the most common biofilm assessment methods. Correlation analysis revealed that some phage parameters can influence the treatment outcome: higher phage concentrations were strongly associated with improved biofilm control, leading to higher levels of biofilm reduction, and phages with higher burst sizes and shorter latent periods seem to be the best candidates to control biofilms in vitro. However, the great variability of the methodologies used prompts the need for the development of standardized in vitro methodologies to characterize phage/biofilm interactions and to assess the efficacy of phages to control biofilms.

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.

Helicobacter pylori infection: from standard to alternative treatment strategies.

Helicobacter pylori is the major component of the gastric microbiome of infected individuals and one of the aetiological factors of chronic gastritis, peptic ulcer disease and gastric cancer. The increasing resistance to antibiotics worldwide has made the treatment of H. pylori infection a challenge. As a way to overhaul the efficacy of currently used H. pylori antibiotic-based eradication therapies, alternative treatment strategies are being devised. These include probiotics and prebiotics as adjuvants in H. pylori treatment, antimicrobial peptides as alternatives to antibiotics, photodynamic therapy ingestible devices, microparticles and nanoparticles applied as drug delivery systems, vaccines, natural products, and phage therapy. This review provides an updated synopsis of these emerging H. pylori control strategies and discusses the advantages, hurdles, and challenges associated with their development and implementation. An effective human vaccine would be a major achievement although, until now, projects regarding vaccine development have failed or were discontinued. Numerous natural products have demonstrated anti-H. pylori activity, mostly in vitro, but further clinical studies are needed to fully disclose their role in H. pylori eradication. Finally, phage therapy has the potential to emerge as a valid alternative, but major challenges remain, namely the isolation of more H. pylori strictly virulent bacterio(phages).

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.

Insights into the genome architecture and evolution of Shiga toxin encoding bacteriophages of Escherichia coli.

BACKGROUND: A total of 179 Shiga toxin-producing Escherichia coli (STEC) complete genomes were analyzed in terms of serotypes, prophage coding regions, and stx gene variants and their distribution. We further examined the genetic diversity of Stx-converting phage genomes (Stx phages), focusing on the lysis-lysogeny decision and lytic cassettes. RESULTS: We show that most STEC isolates belong to non-O157 serotypes (73 %), regardless the sources and geographical regions. While the majority of STEC genomes contain a single stx gene (61 %), strains containing two (35 %), three (3 %) and four (1 %) stx genes were also found, being stx2 the most prevalent gene variant. Their location is exclusively found in intact prophage regions, indicating that they are phage-borne. We further demonstrate that Stx phages can be grouped into four clusters (A, B, C and D), three subclusters (A1, A2 and A3) and one singleton, based on their shared gene content. This cluster distribution is in good agreement with their predicted virion morphologies. Stx phage genomes are highly diverse with a vast number of 1,838 gene phamilies (phams) of related sequences (of which 677 are orphams i.e. unique genes) and, although having high mosaicism, they are generally organized into three major transcripts. While the mechanisms that guide lysis-lysogeny decision are complex, there is a strong selective pressure to maintain the stx genes location close to the lytic cassette composed of predicted SAR-endolysin and pin-holin lytic proteins. The evolution of STEC Stx phages seems to be strongly related to acquiring genetic material, probably from horizontal gene transfer events. CONCLUSIONS: This work provides novel insights on the genetic structure of Stx phages, showing a high genetic diversity throughout the genomes, where the various lysis-lysogeny regulatory systems are in contrast with an uncommon, but conserved, lytic system always adjacent to stx genes.

Differential transcription profiling of the phage LUZ19 infection process in different growth media.

RNA sequencing of phage-infected bacterial cultures offers a snapshot of transcriptional events occurring during the infection process, providing insights into the phage transcriptional organization as well as the bacterial response. To better mimic real environmental contexts, we performed RNA-seq of Pseudomonas aeruginosa PAO1 cultures infected with phage LUZ19 in a mammalian cell culture medium to better simulate a phage therapy event and the data were compared to lysogeny broth medium. Regardless of the media, phage LUZ19 induces significant transcriptional changes in the bacterial host over time, particularly during early infection (t = 5 min) and gradually shuts down bacterial transcription. In a common response in both media, 56 P. aeruginosa PAO1 genes are differentially transcribed and clustered into several functional categories such as metabolism, translation and transcription. Our data allowed us to tease apart a medium-specific response during infection from the identified infection-associated responses. This reinforces the concept that phages overtake bacterial transcriptome in a strict manner to gain control of the bacterial machinery and reallocate resources for infection, in this case overcoming the nutritional limitations of the mammalian cell culture medium. From a phage therapy perspective, this study contributes towards a better understanding of phage-host interaction in human physiological conditions and demonstrates the versatility of phage LUZ19 to adapt to different environments.

Functional Analysis and Antivirulence Properties of a New Depolymerase from a Myovirus That Infects Acinetobacter baumannii Capsule K45.

Acinetobacter baumannii is an important pathogen causative of health care-associated infections and is able to rapidly develop resistance to all known antibiotics, including colistin. As an alternative therapeutic agent, we have isolated a novel myovirus (vB_AbaM_B9) which specifically infects and makes lysis from without in strains of the K45 and K30 capsule types, respectively. Phage B9 has a genome of 93,641 bp and encodes 167 predicted proteins, of which 29 were identified by mass spectrometry. This phage holds a capsule depolymerase (B9gp69) able to digest extracted exopolysaccharides of both K30 and K45 strains and remains active in a wide range of pH values (5 to 9), ionic strengths (0 to 500 mM), and temperatures (20 to 80°C). B9gp69 was demonstrated to be nontoxic in a cell line model of the human lung and to make the K45 strain fully susceptible to serum killing in vitro Contrary to the case with phage, no resistance development was observed by bacteria targeted with the B9gp69. Therefore, capsular depolymerases may represent attractive antimicrobial agents against A. baumannii infections.IMPORTANCE Currently, phage therapy has revived interest for controlling hard-to-treat bacterial infections. Acinetobacter baumannii is an emerging Gram-negative pathogen able to cause a variety of nosocomial infections. Additionally, this species is becoming more resistant to several classes of antibiotics. Here we describe the isolation of a novel lytic myophage B9 and its recombinant depolymerase. While the phage can be a promising alternative antibacterial agent, its success in the market will ultimately depend on new regulatory frameworks and general public acceptance. We therefore characterized the phage-encoded depolymerase, which is a natural enzyme that can be more easily managed and used. To our knowledge, the therapeutic potential of phage depolymerase against A. baumannii is still unknown. We show for the first time that the K45 capsule type is an important virulence factor of A. baumannii and that capsule removal via the recombinant depolymerase activity helps the host immune system to combat the bacterial infection.

Effect of progesterone on Candida albicans biofilm formation under acidic conditions: A transcriptomic analysis.

Vulvovaginal candidiasis (VVC) caused by Candida albicans is a common disease worldwide. A very important C. albicans virulence factor is its ability to form biofilms on epithelium and/or on intrauterine devices promoting VVC. It has been shown that VVC has a hormonal dependency and that progesterone affects virulence traits of C. albicans cells. To understand how the acidic environment (pH 4) and progesterone (either alone and in combination) modulate C. albicans response during formation of biofilm, a transcriptomic analysis was performed together with characterization of the biofilm properties. Compared to planktonic cells, acidic biofilm-cells exhibited major changes in their transcriptome, including modifications in the expression of 286 genes that were not previously associated with biofilm formation in C. albicans. The vast majority of the genes up-regulated in the acidic biofilm cells (including those uniquely identified in our study) are known targets of Sfl1, and consistently, Sfl1 deletion is herein shown to impair the formation of acidic biofilms (pH 4). Under the acidic conditions used, the presence of progesterone reduced C. albicans biofilm biomass and structural cohesion. Transcriptomic analysis of biofilms developed in the presence of progesterone led to the identification of 65 down-regulated genes including, among others, the regulator Tec1 and several of its target genes, suggesting that the function of this transcription factor is inhibited by the presence of the hormone. Additionally, progesterone reduced the susceptibility of biofilm cells to fluconazole, consistent with an up-regulation of efflux pumps. Overall, the results of this study show that progesterone modulates C. albicans biofilm formation and genomic expression under acidic conditions, which may have implications for C. albicans pathogenicity in the vaginal environment.

A Tailspike with Exopolysaccharide Depolymerase Activity from a New Providencia stuartii Phage Makes Multidrug-Resistant Bacteria Susceptible to Serum-Mediated Killing.

Providencia stuartii is emerging as a significant drug-resistant nosocomial pathogen, which encourages the search for alternative therapies. Here, we have isolated Providencia stuartii phage Stuart, a novel podovirus infecting multidrug-resistant hospital isolates of this bacterium. Phage Stuart is a proposed member of a new Autographivirinae subfamily genus, with a 41,218-bp genome, direct 345-bp repeats at virion DNA ends, and limited sequence similarity of proteins to proteins in databases. Twelve out of the 52 predicted Stuart proteins are virion components. We found one to be a tailspike with depolymerase activity. The tailspike could form a highly thermostable oligomeric ß-structure migrating close to the expected trimer in a nondenaturing gel. It appeared to be essential for the infection of three out of four P. stuartii hosts infected by phage Stuart. Moreover, it degraded the exopolysaccharide of relevant phage Stuart hosts, making the bacteria susceptible to serum killing. Prolonged exposure of a sensitive host to the tailspike did not cause the emergence of bacteria resistant to the phage or to serum killing, opposite to the prolonged exposure to the phage. This indicates that phage tail-associated depolymerases are attractive antivirulence agents that could complement the immune system in the fight with P. stuartiiIMPORTANCE The pace at which multidrug-resistant strains emerge has been alarming. P. stuartii is an infrequent but relevant drug-resistant nosocomial pathogen causing local to systemic life-threatening infections. We propose an alternative approach to fight this bacterium based on the properties of phage tailspikes with depolymerase activity that degrade the surface bacterial polymers, making the bacteria susceptible to the immune system. Unlike antibiotics, phage tailspikes have narrow and specific substrate spectra, and by acting as antivirulent but not bactericidal agents they do not cause the selection of resistant bacteria.

Phage therapy efficacy: a review of the last 10 years of preclinical studies.

Due to the rise of multidrug-resistant infections in humans, phage therapy is gaining renewed attention in Western medicine. Despite the increasing number of publications focussed on the isolation, characterization and in vitro performance of different phages, there is still a lack of concise pre-clinical information to guide the application of phage therapy in clinical practice. Nevertheless, over the last decade, efforts have been made to conduct more detailed studies of the in vivo efficacy of phages. Here, we review the most relevant in vivo studies performed in the last decade covering phage efficacy in both preclinical and clinical trials. We compare different routes of administration, dosage effect and different animal models of distinct types of infections. Moreover, insights into case studies and results from clinical trials are presented. Challenges and limitations of phage use as evidenced by the current state of research are also discussed in order to improve both the trustworthiness and success of the implementation of phage therapy.

Bacteriophages to control Shiga toxin-producing E. coli - safety and regulatory challenges.

Shiga toxin-producing Escherichia coli (STEC) are usually found on food products due to contamination from the fecal origin, as their main environmental reservoir is considered to be the gut of ruminants. While this pathogen is far from the incidence of other well-known foodborne bacteria, the severity of STEC infections in humans has triggered global concerns as far as its incidence and control are concerned. Major control strategies for foodborne pathogens in food-related settings usually involve traditional sterilization/disinfection techniques. However, there is an increasing need for the development of further strategies to enhance the antimicrobial outcome, either on food-contact surfaces or directly in food matrices. Phages are considered to be a good alternative to control foodborne pathogens, with some phage-based products already cleared by the Food and Drug Administration (FDA) to be used in the food industry. In European countries, phage-based food decontaminants have already been used. Nevertheless, its broad use in the European Union is not yet possible due to the lack of specific guidelines for the approval of these products. Furthermore, some safety concerns remain to be addressed so that the regulatory requirements can be met. In this review, we present an overview of the main virulence factors of STEC and introduce phages as promising biocontrol agents for STEC control. We further present the regulatory constraints on the approval of phages for food applications and discuss safety concerns that are still impairing their use.

Bacteriophage-receptor binding proteins for multiplex detection of Staphylococcus and Enterococcus in blood.

Healthcare-associated infections (HCAIs) affect hundreds of millions of patients, representing a significant burden for public health. They are usually associated to multidrug resistant bacteria, which increases their incidence and severity. Bloodstream infections are among the most frequent and life-threatening HCAIs, with Enterococcus and Staphylococcus among the most common isolated pathogens. The correct and fast identification of the etiological agents is crucial for clinical decision-making, allowing to rapidly select the appropriate antimicrobial and to prevent from overuse and misuse of antibiotics and the consequent increase in antimicrobial resistance. Conventional culture methods are still the gold standard to identify these pathogens, however, are time-consuming and may lead to erroneous diagnosis, which compromises an efficient treatment. (Bacterio)phage receptor binding proteins (RBPs) are the structures responsible for the high specificity conferred to phages against bacteria and thus are very attractive biorecognition elements with high potential for specific detection and identification of pathogens. Taking into account all these facts, we have designed and developed a new, fast, accurate, reliable and unskilled diagnostic method based on newly identified phage RBPs and spectrofluorometric techniques that allows the multiplex detection of Enterococcus and Staphylococcus in blood samples in less than 1.5 hr after an enrichment step.

Effect of sub-lethal chemical disinfection on the biofilm forming ability, resistance to antibiotics and expression of virulence genes of Salmonella Enteritidis biofilm-surviving cells.

Although disinfection procedures are widely implemented in food environments, bacteria can survive and present increased virulence/resistance. Since little is known about these phenomena regarding biofilms, this study aimed to investigate the effect of chemical disinfection on biofilm-derived cells of Salmonella Enteritidis. Using a reference strain (NCTC 13349) and a food isolate (350), biofilm susceptibility to benzalkonium chloride (BAC), sodium hypochlorite (SH) and hydrogen peroxide (HP) was evaluated and biofilms were exposed to sub-lethal concentrations of each disinfectant. Biofilm-derived cells were characterized for their biofilm forming ability, antibiotic resistance and expression of virulence-associated genes. Except for a few instances, disinfectant exposure did not alter antibiotic susceptibility. However, SH and HP exposure enhanced the biofilm forming ability of Salmonella Enteritidis NCTC 13349. After BAC and HP exposure, biofilm-derived cells presented a down-regulation of rpoS. Exposure to BAC also revealed an up-regulation of invA, avrA and csgD on Salmonella Enteritidis NCTC 13349. The results obtained suggest that biofilm-derived cells that survive disinfection may represent an increased health risk.

Encapsulated bacteriophages in alginate-nanohydroxyapatite hydrogel as a novel delivery system to prevent orthopedic implant-associated infections.

An innovative delivery system based on bacteriophages-loaded alginate-nanohydroxyapatite hydrogel was developed as a multifunctional approach for local tissue regeneration and infection prevention and control. Bacteriophages were efficiently encapsulated, without jeopardizing phage viability and functionality, nor affecting hydrogel morphology and chemical composition. Bacteriophage delivery occurred by swelling-disintegration-degradation process of the alginate structure and was influenced by environmental pH. Good tissue response was observed following the implantation of bacteriophages-loaded hydrogels, sustaining their biosafety profile. Bacteriophages-loaded hydrogels did not affect osteoblastic cells' proliferation and morphology. A strong osteogenic and mineralization response was promoted through the implantation of hydrogels system with nanohydroxyapatite. Lastly, bacteriophages-loaded hydrogel showed excellent antimicrobial activity inhibiting the attachment and colonization of multidrug-resistant E. faecalis surrounding and within femoral tissues. This new local delivery approach could be a promising approach to prevent and control bacterial contamination during implantation and bone integration.

Staphylococci phages display vast genomic diversity and evolutionary relationships.

BACKGROUND: Bacteriophages are the most abundant and diverse entities in the biosphere, and this diversity is driven by constant predator-prey evolutionary dynamics and horizontal gene transfer. Phage genome sequences are under-sampled and therefore present an untapped and uncharacterized source of genetic diversity, typically characterized by highly mosaic genomes and no universal genes. To better understand the diversity and relationships among phages infecting human pathogens, we have analysed the complete genome sequences of 205 phages of Staphylococcus sp. RESULTS: These are predicted to encode 20,579 proteins, which can be sorted into 2139 phamilies (phams) of related sequences; 745 of these are orphams and possess only a single gene. Based on shared gene content, these phages were grouped into four clusters (A, B, C and D), 27 subclusters (A1-A2, B1-B17, C1-C6 and D1-D2) and one singleton. However, the genomes have mosaic architectures and individual genes with common ancestors are positioned in distinct genomic contexts in different clusters. The staphylococcal Cluster B siphoviridae are predicted to be temperate, and the integration cassettes are often closely-linked to genes implicated in bacterial virulence determinants. There are four unusual endolysin organization strategies found in Staphylococcus phage genomes, with endolysins predicted to be encoded as single genes, two genes spliced, two genes adjacent and as a single gene with inter-lytic-domain secondary translational start site. Comparison of the endolysins reveals multi-domain modularity, with conservation of the SH3 cell wall binding domain. CONCLUSIONS: This study provides a high-resolution view of staphylococcal viral genetic diversity, and insights into their gene flux patterns within and across different phage groups (cluster and subclusters) providing insights into their evolution.

K2 Capsule Depolymerase Is Highly Stable, Is Refractory to Resistance, and Protects Larvae and Mice from Acinetobacter baumannii Sepsis.

Acinetobacter baumannii is emerging as a major nosocomial pathogen in intensive care units. The bacterial capsules are considered major virulence factors, and the particular A. baumannii capsular type K2 has been associated with high antibiotic resistance. In this study, we identified a K2 capsule-specific depolymerase in a bacteriophage tail spike C terminus, a fragment that was heterologously expressed, and its antivirulence properties were assessed by in vivo experiments. The K2 depolymerase is active under a broad range of environmental conditions and is highly thermostable, with a melting point (Tm ) at 67°C. In the caterpillar larva model, the K2 depolymerase protects larvae from bacterial infections, using either pretreatments or with single-enzyme injection after bacterial challenge, in a dose-dependent manner. In a mouse sepsis model, a single K2 depolymerase intraperitoneal injection of 50 µg is able to protect 60% of mice from an otherwise deadly infection, with a significant reduction in the proinflammatory cytokine profile. We showed that the enzyme makes bacterial cells fully susceptible to the host complement system killing effect. Moreover, the K2 depolymerase is highly refractory to resistance development, which makes these bacteriophage-derived capsular depolymerases useful antivirulence agents against multidrug-resistant A. baumannii infections.IMPORTANCEAcinetobacter baumannii is an important nosocomial pathogen resistant to many, and sometimes all, antibiotics. The A. baumannii K2 capsular type has been associated with elevated antibiotic resistance. The capsular depolymerase characterized here fits the new trend of alternative antibacterial agents needed against multidrug-resistant pathogens. They are highly specific, stable, and refractory to resistance, as they do not kill bacteria per se; instead, they remove bacterial surface polysaccharides, which diminish the bacterial virulence and expose them to the host immune system.

Control of Salmonella Enteritidis on food contact surfaces with bacteriophage PVP-SE2.

Salmonella is one of the worldwide leading foodborne pathogens responsible for illnesses and hospitalizations, and its capacity to form biofilms is one of its many virulence factors. This work evaluated (bacterio)phage control of adhered and biofilm cells of Salmonella Enteritidis on three different substrata at refrigerated and room temperatures, and also a preventive approach in poultry skin. PVP-SE2 phage was efficient in reducing both 24- and 48-h old Salmonella biofilms from polystyrene and stainless steel causing 2 to 5 log CFU cm-2 reductions with a higher killing efficiency at room temperature. PVP-SE2 phage application on poultry skins reduced levels of Salmonella. Freezing phage-pretreated poultry skin samples had no influence on the viability of phage PVP-SE2 and their in vitro contamination with S. Enteritidis provided evidence that phages prevented their further growth. Although not all conditions favor phage treatment, this study endorses their use to prevent and control foodborne pathogen colonization of surfaces.

Ability of phages to infect Acinetobacter calcoaceticus-Acinetobacter baumannii complex species through acquisition of different pectate lyase depolymerase domains.

Bacteriophages are ubiquitous in nature and represent a vast repository of genetic diversity, which is driven by the endless coevolution cycle with a diversified group of bacterial hosts. Studying phage-host interactions is important to gain novel insights into their dynamic adaptation. In this study, we isolated 12 phages infecting species of the Acinetobacter baumannii-Acinetobacter calcoaceticus complex which exhibited a narrow host range and similar morphological features (podoviruses with short tails of 9-12 nm and isometric heads of 50-60 nm). Notably, the alignment of the newly sequenced phage genomes (40-41 kb of DNA length) and all Acinetobacter podoviruses deposited in Genbank has shown high synteny, regardless of the date and source of isolation that spans from America to Europe and Asia. Interestingly, the C-terminal pectate lyase domain of these phage tail fibres is often the only difference found among these viral genomes, demonstrating a very specific genomic variation during the course of their evolution. We proved that the pectate lyase domain is responsible for phage depolymerase activity and binding to specific Acinetobacter bacterial capsules. We discuss how this mechanism of phage-host co-evolution impacts the tail specificity apparatus of Acinetobacter podoviruses.

A Lytic Providencia rettgeri Virus of Potential Therapeutic Value Is a Deep-Branching Member of the T5virus Genus.

Providencia rettgeri is emerging as a new opportunistic pathogen with high antibiotic resistance. The need to find alternative methods to control antibiotic-resistant bacteria and the recent advances in phage therapy motivate the search for new phages able to infect Providencia spp. This study describes the isolation and characterization of an obligatory lytic phage, vB_PreS_PR1 (PR1), with therapeutic potential against drug-resistant P. rettgeri PR1 is a siphovirus. Its virion DNA size (118,537 bp), transcriptional organization, terminal repeats (10,461 bp), and nicks in the 3'-to-5' strand are similar to those of phage T5. However, sequence similarities of PR1 to phages of the T5virus genus at the DNA and protein levels are limited, suggesting that it belongs to a new species within the Siphoviridae family. PR1 exhibits the ability to kill P. rettgeri antibiotic-resistant strains, is highly specific to the species, and did not present known genomic markers indicating a temperate lifestyle. The lack of homologies between its proteins and proteins of the only other sequenced Providencia prophage, Redjac, suggests that these two phages evolved separately and may target different host proteins.IMPORTANCE The alarming increase in the number of bacteria resistant to antibiotics has been observed worldwide. This is particularly true for Gram-negative bacteria. For certain of their strains, no effective antibiotics are available. Providencia sp. has been a neglected pathogen but is emerging as a multidrug-resistant bacterium. This has revived interest in bacteriophages as alternative therapeutic agents against this bacterium. We describe the morphological, physiological, and genomic characterization of a novel lytic virus, PR1, which is able to kill drug-resistant P. rettgeri clinical isolates. Genomic and phylogenetic analyses indicate that PR1 is a distant relative of T5virus genus representatives. The lack of known virulence- or temperate lifestyle-associated genes in the genome of PR1 makes this phage a potential candidate for therapeutic use. Analysis of its genome also improves our knowledge of the ecology and diversity of T5-like siphoviruses, providing a new link for evolutionary studies of this phage group.