The increase of antibiotic resistance calls for alternatives to control Helicobacter pylori, a Gram-negative bacterium associated with various gastric diseases. Bacteriophages (phages) can be highly effective in the treatment of pathogenic bacteria. Here, we developed a method to identify prophages in H. pylori genomes aiming at their future use in therapy. A polymerase chain reaction (PCR)-based technique tested five primer pairs on 74 clinical H. pylori strains. After the PCR screening, 14 strains most likely to carry prophages were fully sequenced. After that, a more holistic approach was taken by studying the complete genome of the strains. This study allowed us to identify 12 intact prophage sequences, which were then characterized concerning their morphology, virulence, and antibiotic-resistance genes. To understand the variability of prophages, a phylogenetic analysis using the sequences of all H. pylori phages reported to date was performed. Overall, we increased the efficiency of identifying complete prophages to 54.1 %. Genes with homology to potential virulence factors were identified in some new prophages. Phylogenetic analysis revealed a close relationship among H. pylori-phages, although there are phages with different geographical origins. This study provides a deeper understanding of H. pylori-phages, providing valuable insights into their potential use in therapy.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found in biofilm-associated polymicrobial respiratory infections that are challenging to control. Studies performed in laboratory standard conditions suggest that bacterio(phages) and antibiotic combinations are more active against bacterial communities and biofilms than each agent alone. The purpose of this work was to study the antibacterial efficacy of phage-antibiotic combinations using an in vivo-like three-dimensional lung epithelial model that mimics aspects of the parental tissue, colonized by a mixed bacterial community of P. aeruginosa and S. aureus. The bacterial population was targeted by phages specific to P. aeruginosa and/or gentamicin and ciprofloxacin. The results showed that P. aeruginosa was eradicated from the dual-species community when phage treatment was followed by gentamicin and was significantly reduced when followed by ciprofloxacin. Moreover, applying phages first followed by antibiotics demonstrated superior antibacterial activity compared to simultaneous treatment or treatments with the reverse order of application. This approach also reduced the S. aureus population but not as significant as the P. aeruginosa population. Using an in vivo-like model we demonstrated that phages and antibiotics are effective against dual-species bacterial communities, particularly targeting the P. aeruginosa population. However, but the sequence in which these antimicrobials are applied significantly in fluences the effectiveness of bacterial killing.

ABSTRACT In nature, bacteria often survive in a stationary state with low metabolic activity. Phages use the metabolic machinery of the host cell to replicate, and, therefore, their efficacy against non-dividing cells is usually limited. Nevertheless, it was previously shown that the Staphylococcus epidermidis phage SEP1 has the remarkable capacity to actively replicate in stationary-phase cells, reducing their numbers. Here, we studied for the first time the transcriptomic profiles of both exponential and stationary cells infected with SEP1 phage using RNA-seq to gain a better understanding of this rare phenomenon. We showed that SEP1 successfully takes over the transcriptional apparatus of both exponential and stationary cells. Infection was, however, delayed in stationary cells, with genes within the gp142-gp154 module putatively implicated in host takeover. S. epidermidis responded to SEP1 infection by upregulating three genes involved in a DNA modification system, with this being observed already 5 min after infection in exponential cells and later in stationary cells. In stationary cells, a significant number of genes involved in translation and RNA metabolic and biosynthetic processes were upregulated after 15 and 30 min of SEP1 infection in comparison with the uninfected control, showing that SEP1 activates metabolic and biosynthetic pathways necessary to its successful replication. IMPORTANCE Most phage-host interaction studies are performed with exponentially growing cells. However, this cell state is not representative of what happens in natural environments. Additionally, most phages fail to replicate in stationary cells. The Staphylococcus epidermidis phage SEP1 is one of the few phages reported to date to be able to infect stationary cells. Here, we unveiled the interaction of SEP1 with its host in both exponential and stationary states of growth at the transcriptomic level. The findings of this study provide valuable insights for a better implementation of phage therapy since phages able to infect stationary cells could be more efficient in the treatment of recalcitrant infections.

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.

The development of advanced facemasks stands out as a paramount priority in enhancing healthcare preparedness. In this work, different polypropylene non-woven fabrics (NWF) were characterised regarding their structural, physicochemical and comfort-related properties. The selected NWF for the intermediate layer was functionalised with zinc oxide nanoparticles (ZnO NPs) 0.3 and 1.2wt% using three different methods: electrospinning, dip-pad-dry and exhaustion. After the confirmation of ZnO NP content and distribution within the textile fibres by morphological and chemical analysis, the samples were evaluated regarding their antimicrobial properties. The functionalised fabrics obtained via dip-pad-dry unveiled the most promising data, with 0.017 ± 0.013wt% ZnO NPs being mostly located at the fibre’s surface and capable of total eradication of Staphylococcus aureus and Escherichia coli colonies within the tested 24 h (ISO 22196 standard), as well as significantly contributing (**** p < 0.0001) to the growth inhibition of the bacteriophage MS2, a surrogate of the SARS-CoV-2 virus (ISO 18184 standard). A three-layered structure was assembled and thermoformed to obtain facemasks combining the previously chosen NWF, and its resulting antimicrobial capacity, filtration efficiency and breathability (NP EN ISO 149) were assessed. The developed three-layered and multiscaled fibrous structures with antimicrobial capacities hold immense potential as active individual protection facemasks.

Lytic bacteriophages are considered safe for human consumption as biocontrol agents against foodborne pathogens, in particular in ready-to-eat foodstuffs. Phages could, however, evolve to infect different hosts when passing through the gastrointestinal tract (GIT). This underlines the importance of understanding the impact of phages towards colonic microbiota, particularly towards bacterial families usually found in the colon such as the Enterobacteriaceae . Here we propose in vitro batch fermentation as model for initial safety screening of lytic phages targeting Shiga toxin-producing Escherichia coli (STEC). As inoculum we used faecal material of three healthy donors. To assess phage safety, we monitored fermentation parameters, including short chain fatty acid production and gas production/intake by colonic microbiota. We performed shotgun metagenomic analysis to evaluate the outcome of phage interference with colonic microbiota composition and functional potential. During the 24 h incubation, concentrations of phage and its host were also evaluated. We found the phage used in this study, named E. coli phage vB_EcoS_Ace (Ace), to be safe towards human colonic microbiota, independently of the donors’ faecal content used. This suggests that individuality of donor faecal microbiota did not interfere with phage effect on the fermentations. However, the model revealed that the attenuated STEC strain used as phage host perturbed the faecal microbiota as based on metagenomic analysis, with potential differences in metabolic output. We conclude that the in vitro batch fermentation model used in this study is a reliable safety screening for lytic phages intended to be used as biocontrol agents.

This work was co-funded by UIDB/04469/2020 unit and the project PhageSTEC PTDC/CVT-CVT/29628/2017 [POCI-01-0145-FEDER-029628] funded by FEDER through COMPETE2020 by National Funds thought FCT. GP acknowledges the FCT grant SFRH/BD/117365/2016. C.J.H and S.A.M. were funded by the University of Idaho Agricultural Experiment Station Hatch projects IDA01467 and IDA01406, respectively, and the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103408.

Strictly lytic phages are considered powerful tools for biocontrol of foodborne pathogens. Safety issues needed to be addressed for the biocontrol of Shiga toxin-producing Escherichia coli (STEC) include: lysogenic conversion, Shiga toxin production through phage induction, and emergence/proliferation of bacteriophage insensitive mutants (BIMs). To address these issues, two new lytic phages, vB_EcoS_Ace (Ace) and vB_EcoM_Shy (Shy), were isolated and characterized for life cycle, genome sequence and annotation, pH stability, and efficacy at controlling STEC growth. Ace was efficient in controlling host planktonic cells and did not stimulate the production of the Stx prophage or Shiga toxin. A single dose of phage did not lead to the selection of BIMs. However, when reintroduced, BIMs were detected after 24h of incubation. The gain of resistance was associated with lower virulence, as a subset of BIMs failed to agglutinate with O157-specific antibody and were more sensitive to human serum complement. BIM's biofilm formation capacity and susceptibility to disinfectants was equal to that of the wild-type strain. Overall, this work demonstrated that phage Ace is a safe biocontrol agent against STEC contamination and that the burden of BIM emergence did not represent a greater risk in environmental persistence and human pathogenicity.

Staphylococcus epidermidis is a major causative agent of nosocomial infections, mainly associated with the use of indwelling devices, on which this bacterium forms structures known as biofilms. Due to biofilms’ high tolerance to antibiotics, virulent bacteriophages were previously tested as novel therapeutic agents. However, several staphylococcal bacteriophages were shown to be inefficient against biofilms. In this study, the previously characterized S. epidermidis-specific Sepunavirus phiIBB-SEP1 (SEP1), which has a broad spectrum and high activity against planktonic cells, was evaluated concerning its efficacy against S. epidermidis biofilms. The in vitro biofilm killing assays demonstrated a reduced activity of the phage. To understand the underlying factors impairing SEP1 inefficacy against biofilms, this phage was tested against distinct planktonic and biofilm-derived bacterial populations. Interestingly, SEP1 was able to lyse planktonic cells in different physiological states, suggesting that the inefficacy for biofilm control resulted from the biofilm 3D structure and the protective effect of the matrix. To assess the impact of the biofilm architecture on phage predation, SEP1 was tested in disrupted biofilms resulting in a 2 orders-of-magnitude reduction in the number of viable cells after 6 h of infection. The interaction between SEP1 and the biofilm matrix was further assessed by the addition of matrix to phage particles. Results showed that the matrix did not inactivate phages nor affected phage adsorption. Moreover, confocal laser scanning microscopy data demonstrated that phage infected cells were less predominant in the biofilm regions where the matrix was more abundant. Our results provide compelling evidence indicating that the biofilm matrix can work as a barrier, allowing the bacteria to be hindered from phage infection.

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.

Antibiotic resistance is a major public health challenge worldwide, whose implications for global health might be devastating if novel antibacterial strategies are not quickly developed. As natural predators of bacteria, (bacterio)phages may play an essential role in escaping such a dreadful future. The rising problem of antibiotic resistance has revived the interest in phage therapy and important developments have been achieved over the last years. But where do we stand today and what can we expect from phage therapy in the future? This is the question we set to answer in this review. Here, we scour the outcomes of human phage therapy clinical trials and case reports, and address the major barriers that stand in the way of using phages in clinical settings. We particularly address the potential of phage resistance to hinder phage therapy and discuss future avenues to explore the full capacity of phage therapy.

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.

Morganella morganii is a common but frequent neglected environmental opportunistic pathogen which can cause deadly nosocomial infections. The increased number of multidrug-resistant M. morganii isolates motivates the search for alternative and effective antibacterials. We have isolated two novel obligatorily lytic M. morganii bacteriophages (vB_MmoM_MP1, vB_MmoP_MP2) and characterized them with respect to specificity, morphology, genome organization and phylogenetic relationships. MP1’s dsDNA genome consists of 163,095 bp and encodes 271 proteins, exhibiting low DNA (<40%) and protein (<70%) homology to other members of the Tevenvirinae . Its unique property is a >10 kb chromosomal inversion that encompass the baseplate assembly and head outer capsid synthesis genes when compared to other T-even bacteriophages. MP2 has a dsDNA molecule with 39,394 bp and encodes 55 proteins, presenting significant genomic (70%) and proteomic identity (86%) but only to Morganella bacteriophage MmP1. MP1 and MP2 are then novel members of Tevenvirinae and Autographivirinae , respectively, but differ significantly from other tailed bacteriophages of these subfamilies to warrant proposing new genera. Both bacteriophages together could propagate in 23 of 27 M. morganii clinical isolates of different origin and antibiotic resistance profiles, making them suitable for further studies on a development of bacteriophage cocktail for potential therapeutic applications.

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.

The human periodontium health is commonly compromised by chronic inflammatory conditions and has become a major public health concern. Dental plaque, the precursor of periodontal disease, is a complex biofilm consisting mainly of bacteria, but also archaea, protozoa, fungi and viruses. Viruses that specifically infect bacteria - bacteriophages - are most common in the oral cavity. Despite this, their role in the progression of periodontal disease remains poorly explored. This review aims to summarize how bacteriophages interact with the oral microbiota, their ability to increase bacterial virulence and mediate the transfer of resistance genes and suggests how bacteriophages can be used as an alternative to the current periodontal disease therapies.

Citrobacter spp., although frequently ignored, is emerging as an important nosocomial bacterium able to cause various superficial and systemic life-threatening infections. Considered to be hard-to-treat bacterium due to its pattern of high antibiotic resistance, it is important to develop effective measures for early and efficient therapy. In this study, the first myovirus (vB_CfrM_CfP1) lytic for Citrobacter freundii was microbiologically and genomically characterized. Its morphology, activity spectrum, burst size, and biophysical stability spectrum were determined. CfP1 specifically infects C. freundii, has broad host range (>85 %; 21 strains tested), a burst size of 45 PFU/cell, and is very stable under different temperatures (−20 to 50 °C) and pH (3 to 11) values. CfP1 demonstrated to be highly virulent against multidrug-resistant clinical isolates up to 12 antibiotics, including penicillins, cephalosporins, carbapenems, and fluroquinoles. Genomically, CfP1 has a dsDNA molecule with 180,219 bp with average GC content of 43.1 % and codes for 273 CDSs. The genome architecture is organized into function-specific gene clusters typical for tailed phages, sharing 46 to 94 % nucleotide identity to other Citrobacter phages. The lysin gene encoding a predicted D-Ala-D-Ala carboxypeptidase was also cloned and expressed in Escherichia coli and its activity evaluated in terms of pH, ionic strength, and temperature. The lysine optimum activity was reached at 20 mM HEPES, pH 7 at 37 °C, and was able to significantly reduce all C. freundii (>2 logs) as well as Citrobacter koseri (>4 logs) strains tested. Interestingly, the antimicrobial activity of this enzyme was performed without the need of pretreatment with outer membrane-destabilizing agents. These results indicate that CfP1 lysin is a good candidate to control problematic Citrobacter infections, for which current antibiotics are no longer effective.

Bacteriophages or phages are viruses that only infect bacteria. The International Committee on Taxonomy of Viruses classified these viruses in accordance with the morphology of their free virion particles and type and size of their genome. This system fails on the classification of several phages, which have their genome already sequenced. It also requires a morphological analysis by transmission electron microscopy, which is very expensive and time consuming [1]. In 2002 Rohwer and Edward proposed the only sequence-based system existing up to this moment. Thus, it is of utmost importance to develop new systems for bacteriophage classification that take into consideration the genomic and proteomic information already available [2]. The purpose of this study is to establish a new method for the classification of phage based on the genetic information available. The principal objective is to cluster the bacteriophages in different family and types. To create a new phylogenetic tree we analysed all 670 available genome sequences deposited in the GenBank database. Sequences were aligned using the T-coffee program [3]. A genetic marker for the construction of the phylogenetic tree was designed by creating a concatenate of different gene products that presented the highest similarity. In other words, the most conserved gene products were used to form a broader genetic marker. The method allows the use of a single, created genetic marker to classify unknown phages with existing phage types and families. A comparison to existing methods is discussed.