Saija Kiljunen

Background: Enterococcus faecium and Enterococcus faecalis are opportunistic pathogens that form an increasing concern for hospital-acquired infections due to their natural and acquired antimicrobial resistance (AMR). Although phage therapy has gained global interest as a response to emerging AMR, Enterococcus remains an understudied target for phage therapy. In this context, we aimed to isolate Enterococcus -specific phages and study their in vitro infection efficacy when combined into cocktails, in the presence of antibiotics and human serum and against bacterial biofilms. Results: We isolated one E. faecium -phage fHoEfm07, that did not resemble any known phage genera and four E. faecalis -phages belonging to Saphexavirus (fHoEfa01 and fHoEfa06) and Efquatrovirus (fHoEfa03 and fHoEfa04) genera. The phages from the genus Saphexavirus and fHoEfm07 were suitable for therapeutic applications based on their genome characterization; however, the phages belonging to the Efquatrovirus genus contained a potential AMR-related gene. In vitro studies indicated that interactions between phages, antibiotics, and human serum depended on the specific phage-host pairing or antibiotic concentration. Moreover, only phage fHoEfa03 reduced the biofilm masses after 3 h and 24 h phage treatment. Conclusions: This study provides valuable insights into the potential of Enterococcus phages in conditions mimicking phage therapy. The characterization of novel phages, assessment of their therapeutic suitability, and exploration of synergistic treatment strategies contribute to the foundational knowledge required to advance phage therapy.

The human gut microbiota, particularly during infancy, plays a pivotal role in shaping long-term health outcomes. While research on the bacterial microbiota has advanced rapidly, the infant gut virome—dominated by bacteriophages—remains underexplored due to technical challenges in viral DNA detection and recovery. To address this, we optimized a polyethylene glycol (PEG)-based protocol for phage DNA enrichment tailored to the constraints of infant fecal samples, focusing on maximizing viral yield from minimal input material. We validated the optimized protocol on fecal samples from 41 infants at 1, 6, and 12 months of age and assessed the impact of phage enrichment on the observed gut phageome. The results demonstrate that the optimized protocol improves viral DNA recovery and significantly alters the observed virome composition, especially in older infants. Without appropriate enrichment, key features of the gut virome may be underrepresented or missed entirely. These findings underscore the importance of protocol optimization in virome studies and provide a scalable, cost-effective method for robust infant gut virome profiling. IMPORTANCE: Understanding the viral component of the infant gut microbiome is essential for uncovering its role in early-life health, yet technical limitations have hindered its study. This work presents a systematically optimized and validated protocol for enriching viral DNA from infant stool samples, designed specifically for low-input material typical of early life. By adapting polyethylene glycol-based precipitation methods, we achieved consistent and scalable recovery of viral DNA across infants of different ages. Application of this protocol revealed key age- and delivery mode-specific differences in phage diversity and replication strategies that were undetectable using standard approaches. Our findings demonstrate that careful protocol optimization is critical for accurate virome profiling in infants and offer a practical solution to overcome longstanding methodological challenges in the field.

Enterotoxigenic Escherichia coli (ETEC) is a pathogen responsible for post-weaning diarrhea (PWD) in piglets, which results in economic losses in pig production. The rise of antibiotic-resistant ETEC strains together with restrictions on addition of zinc oxide in pig feed require alternative management approaches. Our research examines bacteriophage therapy as a solution to control ETEC infections in newly weaned piglets. A cocktail of phages targeting a strain of ETEC F4LT1ST2 was identified and subsequently multiplicated in laboratory. We conducted a trial including nine piglets divided into three groups. The negative control group was exposed to the phage cocktail by administration with the bedding material (saw dust) on the floor of their pen. The treatment group was exposed to the ETEC strain and to the phage cocktail, and the positive control group was exposed to the ETEC strain only. Shotgun metagenomic sequencing was performed on fecal samples to characterize bacterial and phage dynamics. Throughout a 10-day period we monitored daily the rectal temperature and the diarrheal score of piglets. Subsequently we evaluated phage and bacterial counts in fecal samples to determine phage therapy effect on gut microbiota dynamics and piglet health. The PHAGE+ETEC group showed 19.2% lower cumulative diarrhea burden ( p = 0.044) and 61.9% higher average daily gain ( p = 0.065). Rectal temperature correlated significantly with diarrhea severity (per-piglet Spearman’s ρ = 0.727, p = 0.027). Alpha diversity did not differ between treatment groups across timepoints, suggesting that phage administration did not cause major shifts in microbial diversity. Metagenomic analyses showed significant reduction of E. coli abundance in PHAGE+ETEC group compared to PHAGE groups ( p = 0.009). Consistent with these observations, plaque assay results confirmed active phage-bacteria interactions: no plaque formation was detected in the feces of the ETEC-only group, whereas the PHAGE+ETEC group showed phage replication, reaching 10 ⁶ PFU/ml. This pilot study highlights the potential of phage therapy as an alternative to antibiotics for ETEC infections in piglets. Additional research with larger pig population and longer duration is required to confirm these findings and develop optimal phage application methods for swine production.

Therapeutically utilized phages should optimally be produced in defined bacterial strains that are free of prophages and virulence factors. However, phage–host interactions in these production strains may be very different from clinical strains. Here, we characterized a lytic Staphylococcus aureus –specific phage vB_SauP_EBHT (EBHT), which had a dramatic change in its host specificity when produced in alternative host 19A2 compared with the original isolation host DSM 104437, even though there were no changes in the phage genome, proteome, structure, or adsorption efficiency. The reason for the altered host range was revealed to be based on different methylation patterns of the EBHT genome by host restriction–modification (R‐M) systems in the two hosts. Even though the alternative host 19A2 produced a higher burst size, the host range of the produced phages was narrower. Together, these results illustrate that the most efficient production host may not necessarily be the most optimal one and that bacterial R‐M systems should be considered when selecting the optimal phage‐production host.

Infections caused by multidrug-resistant Acinetobacter baumannii are an emerging global health threat. Although phages have shown promising results in treating bacterial infections, the mechanisms of the combined effect of phages and innate immunity on clearing A. baumannii remain unclear. Here, we report a synergistic effect of the complement system and phages on clearing multidrug-resistant A. baumannii . We show that A. baumannii rapidly adapts and becomes resistant to phage or serum complement by modifying the expression of capsule and lipooligosaccharides, which can be regulated through reversible transposon mutagenesis in the K locus. Compared to the encapsulated phenotype, the non-encapsulated, phage-resistant A. baumannii showed a higher level of membrane attack complex deposition and were susceptible to killing by complement. In contrast, the encapsulated phenotype escaped the complement system by shedding the membrane attack complex to the environment. Thus, while the complement system targets the non-encapsulated phenotype, the phage infects and eliminates the encapsulated subpopulation. These results suggest means of combatting antibiotic-resistant A. baumannii by a simultaneous treatment with phages and complement, a combination which can be supplemented further with antibacterial antibodies. Graphical abstract

Background: Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections, and belongs to the group of ESKAPE pathogens that are the leading cause of healthcare-associated infections and have high level of antibiotic resistance. The treatment of infections caused by antibiotic-resistant P. aeruginosa is challenging, which makes it a common target for phage therapy. The successful utilization of phage therapy requires a collection of well characterized phages. Methods: Phage fMGyn-Pae01 was isolated from a commercial phage therapy cocktail. The phage morphology was studied by transmission electron microscopy and the host range was analyzed with a liquid culture method. The phage genome was sequenced and characterized, and the genome was compared to closest phage genomes. Phage resistant bacterial mutants were isolated and whole genome sequencing and motility, phage adsorption and biofilm formation assays were performed to the mutants and host bacterium. Results: The genomic analysis revealed that fMGyn-Pae01 is a lytic, phiKZ-like jumbo phage with genome size of 277.8 kb. No genes associated with lysogeny, bacterial virulence, or antibiotic resistance were identified. Phage fMGyn-Pae01 did not reduce biofilm formation of P. aeruginosa, suggesting that it may not be an optimal phage to be used in monophage therapy in conditions where biofilm formation is expected. Host range screening revealed that fMGyn-Pae01 has a wide host range among P. aeruginosa strains and its infection was not dependent on O-serotype. Whole genome sequencing of the host bacterium and phage resistant mutants revealed that the mutations had inactivated either a flagellar or rpoN gene, thereby preventing the biosynthesis of a functional flagellum. The lack of functional flagella was confirmed in motility assays. Additionally, fMGyn-Pae01 failed to adsorb on non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor. Conclusion: fMGyn-Pae01 is a phiKZ-like jumbo phage infecting P. aeruginosa. fMGyn-Pae01 uses the flagellum as its phage-binding receptor, supporting earlier suggestions that flagellum might be utilized by phiKZ but differs from some other previous findings showing that phiKZ-like phages use the type-IV pili as the phage-binding receptor.

Background: Intravascular stent infections are often associated with high risks of morbidity and mortality. We report here a case of a patient with an arterial stent and recurrent Pseudomonas septicaemias successfully treated with phage–meropenem combination therapy. Methods: A 75-year-old female with arteriosclerosis and comorbidities went through a femoropopliteal bypass with prosthesis in the right inguinal area. After the bypass, she developed a recurring Pseudomonas aeruginosa infection and also neutropenia during different antibiotics. A rapidly growing pseudoaneurysm in the right inguinal area led to an emergency intra-arterial stent placement during blood stream infection, later suspected to host a P. aeruginosa biofilm. Removing the stent was deemed precarious, and phage therapy was considered as a compassionate treatment option. A three-phage cocktail infecting the P. aeruginosa strain was prepared and administered intravenously together with meropenem for two weeks, after which, a ten-month follow-up was carried out. Results: No adverse reactions occurred during the phage therapy treatment, while infection markers were normalized. In addition, recovery was seen in a PET-CT scan. During the 10-month follow-up, no further P. aeruginosa septicaemias occurred. Conclusions: Phage–meropenem combination therapy was thus found safe and effective in the treatment of recurrent Pseudomonas septicaemia in a patient with an arterial stent.

Infections caused by multidrug-resistant (MDR) bacteria are a growing global concern. Enterobacter cloacae complex (ECC) species are particularly adept at developing antibiotic resistance. Phage therapy is proposed as an alternative treatment for pathogens that no longer respond to antibiotics. Unfortunately, ECC phages are understudied when compared to phages of many other bacterial species. In this Ghanaian-Finnish study, we isolated two ECC strains from ready-to-eat food samples and three novel phages from natural waters against these strains. We sequenced the genomic DNA of the novel Enterobacter phages, fGh-Ecl01, fGh-Ecl02, and fGh-Ecl04, and assessed their therapeutic potential. All of the phages were found to be lytic, easy to propagate, and lacking any toxic, integrase, or antibiotic resistance genes and were thus considered suitable for therapy purposes. They all were found to be related to T4-type viruses: fGh-Ecl01 and fGh-Ecl04 to karamviruses and fGh-Ecl02 to agtreviruses. Testing of Finnish clinical ECC strains showed promising susceptibility to these novel phages. As many as 61.1% of the strains were susceptible to fGh-Ecl01 and fGh-Ecl04, and 7.4% were susceptible to fGh-Ecl02. Finally, we investigated the susceptibility of the newly isolated ECC strains to three antibiotics – meropenem, ciprofloxacin, and cefepime – in combination with the novel phages. The use of phages and antibiotics together had synergistic effects. When using an antibiotic-phage combination, even low concentrations of antibiotics fully inhibited the growth of bacteria.

Background: Klebsiella aerogenes is an opportunistic pathogen that causes a wide variety of infections. Due to the rising problem of antibiotic resistance, novel antibiotics and strategies to combat bacterial infections are needed. Host-specific bacteriophages are natural enemies of bacteria and can be used in phage therapy as an alternative form of treatment against bacterial infections. Jumbo phages are defined as phages with genomes larger than 200 kb. Relatively few studies have been done on jumbo phages compared to smaller phages. Results: A novel phage, fENko-Kae01, was isolated from a commercial phage cocktail. Genomic analysis revealed that fENko-Kae01 is a lytic jumbo phage with a 360 kb genome encoding 578 predicted genes. No highly similar phage genomes were identified and fENko-Kae01 may be a completely new genus representative. No known genes associated with lysogenic life cycle, bacterial virulence, or antibiotic resistance were identified. The phage had myovirus morphology and a narrow host range. Phage resistant bacterial mutants emerged under phage selection. Whole genome sequencing revealed that the biogenesis of the flagellum was affected in four mutants and the lack of functional flagellum was confirmed in motility assays. Furthermore, phage fENKo-Kae01 failed to adsorb on the non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor. Conclusions: fENko-Kae01 is a novel jumbo bacteriophage that is considered safe for phage therapy. fENko-Kae01 uses the flagellum as the phage-binding receptor and may represent a completely novel genus.