Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification.Nontuberculous Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid.No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these.Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.Phage treatment of  Mycobacterium infections. We observed no adverse reactions, favorable outcomes in at least 50% of patients, no evidence of phage resistance, and neutralizing immune reactions that do not correlate with treatment success.We describe 20 consecutive cases of bacteriophage treatment of  The therapeutic use of phages for treating drug-resistant bacterial infections has received recent attention, but the types of infections and pathogens deemed suitable; routes, dosage, and frequency of administration; interactions with antibiotics; and pharmacokinetics remain unclear , 2. UnliMycobacterium pathogens, alternative therapies are needed [Mycobacterium abscessus—are particularly challenging, as many are refractory to antibiotics and extended drug therapies are poorly tolerated [M. abscessus infections are particularly challenging and typically preclude lung transplantation. Phages have been proposed for managing CF [Mycobacterium infections, including NTM and tuberculosis [Because of increasing and widespread antibiotic resistance among e needed . Nontubeolerated . NTM infolerated , 13. Peoaging CF , but therculosis , 16, remM. abscessus clinical isolates [M. abscessus isolates have a smooth colony morphotype [Mycobacterium smegmatis; few phages have been isolated directly on any strain of M. abscessus [There is great variation in phage susceptibilities among isolates . Approxirphotype , 18, andrphotype . In contrphotype . Nonethebscessus .M. abscessus infection following bilateral lung transplantation and drug-induced immunosuppression [M. abscessus pulmonary infection [9 plaque-forming units (PFUs) per dose for at least 6 months. Both were also treated with concomitant multidrug antibiotic regimens. The first patient had clinical improvement in lung function, radiographic imaging, and clinical signs and symptoms, although without complete clearance of the infection [M. abscessus colony counts in sputum that was abrogated by emergence of a potent neutralizing antibody response to the phages [Two case reports have described compassionate use of phages for NTM infections , 19. Onepression . The secnfection . The secWe report here therapeutic interventions for a pilot cohort of 20 patients with antibiotic-refractory mycobacterial infections. Phage administration was safe, no resistance was observed, and favorable microbiological or clinical outcomes were observed in a majority of cases.M. abscessus infections, but some had other underlying diseases complicated by NTM  infections. The following criteria determined eligibility for compassionate use phage therapy: age >5 years, microbiologic documentation of mycobacterial infection based on at least 2 positive cultures of relevant tissue or body fluids; drug susceptibility testing (DST) documenting resistance to multiple antimycobacterial drugs; clinical signs, symptoms and radiographic findings involving at least 1 organ system , European Respiratory Society (ERS), European Society of Clinical Microbiology and Infectious Diseases (ESCMID), and Infectious Diseases Society of America (IDSA) diagnostic criteria [Since May 2019, we received approximately 200 requests for adjunctive phage treatment for patients with NTM infections that were either refractory to antibiotic therapies or in which extended drug treatments were not tolerated; antibiotic susceptibilities are shown in 9 PFU intravenously twice daily; some patients also received the same dose by inhalational nebulization  rough colony strains were infected and killed efficiently by at least 1 phage. If an isolate was efficiently infected and killed by 1 or more phage, and the patient’s clinical status indicated eligibility for compassionate use intervention as previously described, regulatory permissions were obtained and purified phages were dispatched to a local dispensing pharmacy. Of the 20 patients offered treatment, 17 had M. abscessus infections; 14 of these had underlying CF, 1 had bronchiectasis, 1 had scleroderma, and 1 had hypersensitivity pneumonitis. One patient had a Mycobacterium chelonae disseminated skin infection, 1 had CF with pulmonary Mycobacterium avium, and 1 had a disseminated BCG infection , 1 had disseminated skin lesions caused by M. chelonae, 1 had scleroderma with M. abscessus infection, and 1 had disseminated BCG infection , 1 with pulmonary M. avium infection (patient 17), and another with M. chelonae skin infection (patient 16); details of patients 15 and 16 were recently reported [M. abscessus cultures and successfully underwent a bilateral lung transplant. Two patients (1 and 13) had substantial clinical improvement, but without clear evidence of culture conversion. One of these (patient 1) was the case reported previously [M. abscessus infection and clinical signs and symptoms greatly improved, but some skin nodules persisted after >1 year of phage treatment. These were only intermittently culture positive, but the patient subsequently died from CF-related health challenges and organ failure 44 months after the start of phage treatment. In the other (patient 13), there was substantial improvement of symptoms and forced expiratory volume in 1 second by spirometry, but the patient remained culture positive.Favorable clinical and microbiological responses were observed in 5 patients  and partial clinical or microbiological responses were noted in 6 patients  ; all wernfection . In 5 pareported , 23. ThrMycobacterium infections. One patient (patient 2) had a severe chest infection requiring sternum resection. Phage treatment resulted in AFB smear-negative chest swabs, but the patient died after failing therapy for multiple bacterial and fungal coinfections. For patient 4, phage treatment resulted in conversion to culture negative tracheal aspirates, but systemic adenovirus infection resulted in death. Patient 7 had M. abscessus infection with both rough and smooth colony morphotypes; the rough strain derived from the smooth strain by a mutation in glycopeptidolipid synthesis [For 4 patients , response to therapy was partial and more difficult to assess largely due to complications from other infections, although there was evidence of improved control of the ynthesis . The phaM. abscessus, and active phage was identified only for the rough strain  had inconclusive responses to therapy or had modest short-lived improvements. Patient 8 developed phage neutralizing antibodies and had little clinical improvement with either aerosolized or IV administered phage, likely due to the phage neutralizing immune response. Like patient 7, patient 11 had a mixed infection with both rough and smooth colony morphotypes of h strain . While clization . PatientM. abscessus infections, showed no overall clinical or microbiologic response , all with pulmonary response . All hadM. smegmatis) for phage growth. Eleven patients were treated with just a single phage (33HTH_HRMGD03), and in the 6 patients from whom rough colony M. abscessus isolates were recovered after the start of phage treatment, all remained fully phage sensitive. Indeed, changes in phage sensitivity were only observed in 1 phage in a cocktail of 3 (Phage administration by either IV or aerosolized routes was well-tolerated with no serious adverse reactions related to the phage in any patient. Phage preparations were highly purified, certified to be sterile, and had undetectable endotoxin levels, an advantage of using a lipopolysaccharide-free bacterial host (Sera from 15 patients were tested for immune reactions, and robust neutralization of at least 1 phage was observed in 8 of the patients following IV treatment . AlthougThis series of 20 patients treated with phages on a compassionate use basis provides support for further evaluation of phages for treatment of mycobacterial infections. Phage administration was well-tolerated, and phage resistance was not observed even when using a single phage. Favorable responses were observed in more than half of the patients, including complete resolution of some infections, and successful lung transplantation in 1 patient. However, some patients saw little clinical benefit, and the basis for this variability in response is unclear. Although phage treatment of mycobacterial infections shows promise, this cohort illustrates some key limitations and lessons.M. abscessus infection was fully resolved, the phages were also deposited bronchoscopically, which may have contributed to more effective phage delivery to the infection. Nebulization may also avoid systemic neutralization. The immune status of the patient is also important; immunocompromised patients may tolerate extended phage administration without antibody-mediated neutralization. However, little is known about intracellular penetration or uptake of phages, particularly by macrophages, where most replicating mycobacteria are found. Third, dosage and regimens warrant optimization. As the treatments are well-tolerated, higher doses could be contemplated, using longer interdose intervals. Further exploration of pharmacodynamics and tissue penetration of phage is critical.First, the repertoire of therapeutically useful phages is small, and expansion requires further phage isolation, developing phages induced from lysogenic strains or using synthetic phages , 24. HowM. abscessus strains, the unpredictable specificity for rough strains, and the limited phage repertoire represent current impediments to broad implementation of phage treatments. However, these limitations are not insurmountable, and these case studies suggest that phage treatments may be valuable tools for clinical control of NTM infections. Successful outcomes for M. chelonae, M. avium, and BCGosis, as well as M. abscessus, suggest a large spectrum of target Mycobacterium diseases.The lack of therapeutically useful phages for smooth Mycobacterium infections and the potential for infection control when none other is effective.Although compassionate use case studies such as these lack the rigor and consistency of treatment and patient monitoring possible in carefully controlled blinded clinical trials, they provide a wealth of insights for designing such trials. Variations in antibiotic regimens, surgical interventions, and management of coinfections can all potentially influence patient status, and direct linkage of phage treatments with outcomes in individual patients is perilous. Nonetheless, this series of case studies strengthens the likelihood of direct benefits from phage treatments of ciac453_Supplementary_DataClick here for additional data file.


Pseudomonas aeruginosa, Acinetobacter sp, and Staphylococci. With investment to develop new antibiotics waning, finding and developing alternative therapeutic strategies to tackle this issue is imperative. One option remerging in popularity is bacteriophage (phage) therapy. This review focuses on Staphylococcus aureus and how it has developed resistance to antibiotics. It also discusses the potential of phage therapy in this setting and its appropriateness in high-risk people, such as those with cystic fibrosis, where it typically forms a biofilm.The production and use of antibiotics increased significantly after the Second World War due to their effectiveness against bacterial infections. However, bacterial resistance also emerged and has now become an important global issue. Those most in need are typically high-risk and include individuals who experience burns and other wounds, as well as those with pulmonary infections caused by antibiotic-resistant bacteria, such as  Staphylococcus aureus (S. aureus), has developed drug resistance mechanisms to the currently available antibiotics, including cloxacillin, vancomycin, daptomycin, and others, and is resultantly responsible for over two million infections and over 23,000 deaths in the United States alone each year . Ba. Ba87]. d others . Severalbiofilms ,94,95. Is et al. . Howevers et al. . Moreoveproteins .Collectively, as described in this review, staphylococci readily form antibiotic resistance, whether in free planktonic forms or biofilms. It can adapt over very short periods when a new antibiotic is introduced to develop resistance, which ultimately increases these infections’ economic and healthcare burden. Thus, an alternative treatment regimen that permanently eliminates infection is desperately required. One postulated solution is bacteriophage therapy.The term “phage” is defined as a type of virus that can enter and destroy bacterial cells whose potential was appreciated early in history ,100. TheTheir bactericidal activity begins with the binding of the virion to specific bacterial cell-surface receptors via phage receptor binding proteins (RBP), followed by the adsorption of phage DNA into a bacterium. This then initiates various molecular mechanisms which promote phage propagation and resultant bactericidal activity . BacteriAs natural predators of bacteria, bacteriophages have several innate mechanisms to destroy biofilms . For exaP. aeruginosa, due to its significant clinical impact as an opportunistic pathogen, in cystic fibrosis (CF), burns, pneumonia, and urinary tract infections [5 PFU direct in the ear as a treatment strategy for chronic otitis media caused by P. aeruginosa, patients were observed for up to 42 days’ post treatment, and encouragingly, there was a reduction in infection by over 50% [Klebsiella pneumoniae. Results showed that a single intraperitoneal dose provided 1 h post-bacterial inoculation resulted in 100% recovery [P. aeruginosa with two phages together or in combination with three antibiotics . Significantly, only moderate activity was observed when each phage/antibiotic treatment was conducted singularly. However, synergistic activity was observed when phages and antibiotics were applied concurrently [P. aeruginosa  for activity against P. aeruginosa strains isolated from children with CF. Excitingly, the authors identified E79 as a possible new therapeutic phage candidate for P. aeruginosa lung infections in CF due to its broad antibacterial activity (91% of the tested strains were sensitive) [Most phage/antibiotic studies have focused on fections . In one over 50% . Similarrecovery . Anotherurrently . These furrently . Trend ensitive) .S. aureus are well known for their resistance to high concentrations of antibiotics [Although much attention has focused on treating Gram-negative bacteria with phage, Estrella and colleagues demonstrated that many strains of staphylococci carried bacteriophages. These phages were able to lyse some but not all strains. However, by exposing staphylococci to several phages, a susceptibility pattern could be recognized, and similarities or differences between strains could be determined . Since tibiotics ,127. S. aureus phage (AB-SA01) in detail. S. aureus phage (AB-SA01) is a bacteriophage cocktail produced by AmpliPhi Biosciences Corporation that consists of three naturally occurring, obligately lytic myoviruses related to Staphylococcus phage K belonging to the Herelleviridae family. Importantly, it did not contain any bacterial virulence or antibiotic resistance genes when sequenced. In addition, they confirmed that this phage’s inherent characteristics met the human use criteria and was predicted to remain active against circulating multidrug-resistant S. aureus strains for long enough to be useful under good manufacturing practices. Overall, results offered great promise, with AB-SA01 killing ~95% of S. aureus isolates [S. aureus strains have been shown [S. aureus ventilator-associated pneumonia (VAP), Prazack and colleagues demonstrated reduced mortality in mice treated with and without teicoplanin compared to the placebo [S. aureus strains [S. aureus biofilms [Lehman et al. were one of the first to characterize an isolates . Furtheren shown . Anotheren shown . Importaen shown . Using a placebo . Rahman  strains . Specifibiofilms . Althougbiofilms , they cabiofilms . Neverthbiofilms .S. aureus using the SATA-8505 phage with vancomycin, cefazolin, linezolid, dicloxacillin, and tetracycline, simultaneously or sequentially. Results demonstrated minimal bactericidal activity when both phage and antibiotic were concurrently added, which was significantly increased when phage treatment preceded antibiotics, particularly with vancomycin and cefazolin [Staphylococcal epidermis, a normal skin microflora with phage saGU1, inhibited the growth of phage-resistant S. aureus in vitro [Kumaran et al. also studied the synergistic activity of phage/antibiotic treatment against biofilm-forming efazolin . Subsequefazolin ,94. Anotefazolin . Identifin vitro . HoweverS. aureus anti-biofilm agents for the treatment of staphylococcal infections over the current time period, as shown in clinicaltrials.gov website about bacteriophage therapy since April 2022. Phage therapy (PT) is used in the majority of the cases that are related to biofilms in S. aureus infections. These infections are mainly attributed to medical devices [S. aureus on these devices. Phage therapy is also included in the management of patients with deep-seated infections such as osteomyelitis [S. aureus infections [S. aureus [S. aureus biofilm-related infections resolved with PT except in only two patients [However, with all the foregoing data on the diversity of phage therapy, both in vitro and in vivo, which greatly outweigh any specific concerns, we summarize some important clinical studies that used phages or phage-related  devices ,138, imp devices ,146,147, devices ,148, whemyelitis ,150,151 myelitis , chronicmyelitis , and bramyelitis ) and chrmyelitis . Howevermyelitis . Exebacafections . Both si. aureus ,154,156 . aureus  detected. aureus ,146,156 . aureus ,153,154 . aureus  The use . aureus ,153,154 . aureus ,151,156.. aureus ,155, witpatients ,145, indS. aureus infections, as well as to address knowledge gaps or issues that need to be addressed. As highlighted, phage offers an extremely promising therapeutic alternative to combat staphylococcal infection found in various human diseases, including septicemia, pneumonia-like infection, venous leg ulcers, etc. [S. aureus infections, it is important to highlight that relatively few phages would be needed and combined with currently available antibiotics to have an effective outcome. Furthermore, different phages can be combined into unique cocktails and administered via different routes, i.e., topically, orally, or both, to facilitate effective penetration into the system/tissue [The knowledge gained from work performed in this research space will identify opportunities to translate phage therapy into practice for rs, etc. . Phages m/tissue .S. aureus isolates has been shown to be significantly associated with the type of the S. aureus clonal complex (CC). Phage V1SA20 is only active against CC80 strains, while all phages exhibit poor activity against CC7, CC59, CC239, and CC398 strains [However, several challenges have also been identified. For example, lysogenic phages are commonly considered unsuitable for treatment due to their high potential for horizontal gene transfer; however, some lytic phages show no positive value as antimicrobial agents . Perform strains . However strains .S. aureus PT failed to completely cure chronic polymicrobial biofilm infection of a bone allograft in a sarcoma patient. The authors explained the possibility of incomplete coverage of anti-S. aureus PT against non-staphylococci bacteria [Phage therapy (PT) should also be carefully assessed in patients with chronic infections caused by polymicrobial agents. For example, anti-bacteria . In addibacteria . Howeverbacteria . Althougbacteria  demonstrbacteria . The uptbacteria . This fibacteria . Tolerabbacteria . On a sibacteria .Work currently being performed is also identifying phage-associated treatment options. For example, phages can produce specific enzymes , which are involved in the rapid degradation and destruction of the bacterial cell wall. The promising findings on phage-encoded endolysins from in vitro studies have been extensively reviewed, and their synergy action with antibiotics could give insight into new therapeutic options . This enS. aureus that must be addressed and understood in order to facilitate the development of phage therapy into standard clinical practice. Overall, several points emerge from this review. Firstly, positive interactions that are observed between phages and antibiotics provide a strong rationale that phage/antibiotic combinations can be successfully used against many multidrug-resistant bacteria [This review summarises the essential aspects of antibiotic resistance observed in bacteria ,176. Secbacteria . Finallybacteria ,133. Nev


Vibrio alginolyticus is an important pathogen of marine animals and has been the target of phage therapy applications in marine aquaculture for many years. Here, we report the isolation and partial characterization of a novel species of the Siphoviridae family, the Vibrio phage Artemius. The novel phage was species-specific and could only infect strains of V. alginolyticus. It could efficiently reduce the growth of the host bacterium at various multiplicities of infection as assessed by an in vitro lysis assay. It had a genome length of 43,349 base pairs. The complete genome has double-stranded DNA with a G + C content of 43.61%. In total, 57 ORFs were identified, of which 19 were assigned a predicted function. A genomic analysis indicated that Vibrio phage Artemius is lytic and does not harbor genes encoding toxins and antibiotic resistance determinants. Vibrio alginolyticus is one of the most important pathogenic bacteria of marine organisms and has become a serious threat to the aquaculture industry [industry . It has industry ,3, crustindustry ,5 and moindustry  all overindustry . In Euroindustry . The manindustry . Therefoindustry . In thisindustry ,12,13. Vibrio alginolyticus isolated from the live feeds of a fish hatchery. This work is part of a broader effort of our research team to isolate and characterize lytic bacteriophages against pathogenic vibrios relevant to marine aquaculture [Here, we describe the isolation and characterization of a novel phage infecting aculture ,14,15,16Artemia salina culture tank of the Institute of Marine Biology, Biotechnology and Aquaculture of the Hellenic Centre for Marine Research in Heraklion, Crete (IMBBC-HCMR). HCMR-2 Art1 was identified as Vibrio alginolyticus using molecular methods  [Table 1). All bacterial strains were maintained in microbeads (MicroBank) at −80 °C; when working, they were grown in a lysogeny broth (LB)  at 25 °C. All strains had been previously identified to a species level using sequencing.The bacterial strain used as a host (HCMR-2 Art1) was isolated from the water of a live feed B, toxR) ,18,19 anArtemia salina culture tank of IMBBC-HCMR following a standard enrichment procedure [Vibrio alginolyticus strain HCMR-2 Art1) and incubated at 25 °C for 24 h. Following filtration through 0.22 µm filters, 10 µL aliquots were plated by a standard double-layer agar method and incubated overnight at 25 °C to detect and enumerate the plaque forming units (PFU). The clearest plaque formed was picked and further purified by re-plating five times to ensure clonal phage stocks. The purified phage was named Vibrio phage Artemius and propagated to reach a titer of 1010 PFU mL−1 and stored at 4 °C.The phage was isolated from the water of a live feed rocedure . Briefly10 PFU mL−1 was negatively stained with 4% w/v uranyl acetate. Vibrio phage Artemius was observed using a JOEL transmission electron microscope operated at 60 kV at the Electron Microscopy Lab of the University of Crete.The phage morphology was observed by transmission electron microscopy (TEM). An aliquot of the phage suspension with a titer of ~107 CFU mL−1) was mixed with 3 mL of warm top agar (culture medium with 0.6% agar) and poured onto bottom LB 1/2 agar plates. When the top agar hardened, spots of 10 µL phage were made and examined after 24 h incubation for lysis. The host range of Vibrio phage Artemius was evaluated by a spot test using the bacterial stains shown in 7 CFU mL−1), it was infected with 20 μL of the phage preparation at 4 different MOIs . Three wells were not infected and were used as the control. The growth curve of the cultures was monitored in real-time over a minimum of 18 h and OD600 measurements were recorded every 10 min.The efficacy of Vibrio phage Artemius on the host strain was assessed by an in vitro lysis assay. Sterile 96-well plates were used and loaded with 180 μL of a freshly prepared culture of the host bacterium. The plate was placed in a TECAN microplate reader (Infinite PRO 200) equipped with a temperature control and incubated at 25 °C with orbital shaking. When the bacterial culture was at the exponential phase (~107 PFU mL−1) to different temperatures . The aliquots were incubated at each temperature for 1 h and then rested at room temperature (RT) for 10 min. Each aliquot was then serially diluted and spotted (10 μL/spot) on a host bacterial lawn (HCMR-2 Art1). After a 24 h incubation of the agar plates, the phage titer was determined for each temperature and 4 °C was used as the control. The thermal stability of the novel phage was examined by exposing 500 μL aliquots of the phage  was infected with the phage at a multiplicity of infection (MOI) of 0.01. The same amount of phage was added to a tube containing only LB and served as the control. Immediately after the infection, 500 μL of the infected culture was placed in tubes containing chloroform and stored in ice (4 °C). The same procedure was repeated for 20 min with a 2 min interval. When all time points were collected, the aliquots were centrifuged at 13,000 rpm for 3 min. They were then serially diluted and spotted onto the host bacterial lawn on LB ½ agar plates. The phage titer was determined after 24 h of incubation of the agar plates at 25 °C.For the adsorption time and one-step growth, we followed the protocol described in Misol et al.  accordin7 CFU mL−1) was centrifuged at 13,000 rpm for 3 min. The supernatant was discarded and the pellet was washed with saline. After two repetitions of this step, the culture was finally resuspended in LB, infected with the phage at a MOI of 0.01 and rested for 15 min at RT. Following centrifugation at 13,000 rpm for 3 min, the supernatant was discarded and the pellet was dissolved in 1 mL saline. The infected culture was then transferred to a new tube containing 25 mL LB and the aliquots were immediately removed and placed in empty Eppendorf vials. The aliquots were then centrifuged at 13,000 rpm for 3 min and the supernatant was transferred to a new 2 mL Eppendorf vial, serially diluted and spotted onto the host bacterial lawn on LB ½ agar plates. This procedure was repeated every 10 min for 120 min. The phage titer was determined after 24 h of incubation of the agar plates at 25 °C.For the one-step growth, 1 mL of freshly grown culture at the exponential phase  was used as a negative control. At least 5 μg of high-purity bacterial DNA, the quality of which was tested using a BioAnalyzer , was used to generate a paired-end 300 PE genomic library with an optimized size selection using magnetic bead purifications based on the standard Illumina protocol and by using a Nextera XT Library Construction Kit . To calculate the average size of the library, we used a Tapestastion 4200 system . The insert size was estimated as the average size of the library minus the Illumina adapter size and was found to be 834 bp. The sequencing was performed using an Illumina NovaSeq 6000 sequencing platform  according to the manufacturer’s protocol at Life Sequencing , which allowed us to obtain at least 1 million paired reads sequences. Possibly contaminated, primer, N-terminus and 3′-, 5′-low quality reads were trimmed off (threshold: 0.05). The raw reads were quality inspected and were assembled in Geneious Prime using the Geneious assembler. RASTk and Glimmer were used as the gene predictors for the structural annotation through the PATRIC webserver [https://phage.ai/, accessed on 1 May 2022). The genome of Vibrio phage Artemius with annotated predicted ORFs was then visualized in a circular representation with Geneious software  and CGview.The DNA extraction of Vibrio phage Artemius was performed using the phenol–chloroform method according to Higuera et al. . The extebserver  and Galaebserver . The preebserver  adjustedebserver , InterPrebserver  and TΜHMebserver ,29 databebserver  and Viruebserver  as well ebserver . The phaebserver . The phaTable 2) [A ViPTree was used to build a viral proteomic tree by comparing the proteome of Vibrio phage Artemius with 4982 dsDNA phage proteomes . The phyTable 2) . A totalTable 2) . The gapTable 2)  with a bTable 2)  was usedphage morphology of the virions as observed with TEM classified the novel phage to the Siphoviridae family. The head was 48.7 ± 0.9 nm wide and the tail was 107.0 ± 2.9 nm long  .The Figure 2), with the highest MOI yielding a more rapid decrease in the bacterial titer. The bacterial titer remained low (close to the detection limit) over a period of approximately 10 h.Vibrio phage Artemius was able to lyse the host bacterial population in vitro using approximate MOIs ranging between 0.1 and 100 . According to the host range assay, Vibrio phage Artemius was able to only lyse strains that belonged to p < 0.05) was observed when the phage was exposed to 70 °C and 80 °C. The phage was not inactivated at high temperatures. We then exposed the phage to acidic and alkaline pH values ranging from pH = 1 to pH = 10 and compared the titer of the phage with the titer at pH = 7, which served as the control (p < 0.001), but remained stable from pH = 3 to pH = 10 with no statistically significant difference compared with the control. We exposed Artemius to different temperatures ranging from 25 °C to 80 °C, with T = 4 °C serving as the control a, to inv control b. The ph−1.The adsorption time assay showed that 90% of the Vibrio phage Artemius virions required 10 min to bind to the bacterial host a. Artemi−3) with an average similarity of 52.67%. Overall, 19 ORFs (10.83%) were assigned a function based on the protein homology. No genes associated with integration, virulence or antibiotic resistance were detected. The genome of Vibrio phage Artemius was not modularly organized . However, several gene encoding proteins required for phage assembly  were arranged in subclusters as well as a few genes encoding for DNA replication and nucleotide metabolism proteins . The genes that were functionally annotated are shown in Table 4.The genome size of Vibrio phage Artemius was 43,349 bp with a GC content of 43.61%. The genome arrangement was dense, as suggested by the 1.31 genes per kbp. The Rapid Annotation using Subsystem Technology (RASTk) server and Glimmer.hmm 3.0 revealed that 57 ORFs were present in the genome. Each individual ORF was manually inspected in order to validate the presence of start codons (ATG/GTG or TTG). There was no presence of tRNA in the genome, as shown by ARAGORN. A total of 50 ORFs used a start codon of ATG, 6 ORFs used GTG and 1 used TTG. A search of the NCBI nr database showed that 45 ORFs (78.95%) had significant hits . The proteins required for phage morphogenesis included major capsid protein (ORF 8), tail-length tape measure protein (ORF 17), tail tube protein (ORF 13), tail tubular protein (ORF 22) and neck protein (ORF 16). In addition, the small terminase subunit (ORF 2), large terminase subunit (ORF 5), stopper protein (ORF 10) and phage portal protein (ORF 6), which are involved in DNA packaging for tailed phages, were identified. Proteins for DNA replication, recombination and repair were also detected: these were DNA polymerase (ORF 23), HNH endonuclease (ORF 51), DNA polymerases, DNA helicase  and other regulatory elements (ORF 54). Finally, several transmembrane proteins were detected  . In addition, it was predicted to infect hosts from the Gammaproteobacteria class, which includes the Vibrionaceae family. A closer view of the tree focusing on the nearest relatives  showed that Vibrio phage Artemius clustered together with Shewanella phage 1/44 (Siphoviridae) and Escherichia phage PTXU04 (Podoviridae).A wide genome proteomic tree analysis validated that Vibrio phage Artemius belonged to the Vibrio alginolyticus phages  showed that Vibrio phage Artemius shared a common ancestor with Vibrio phage phi-St2 and Vibrio phage phi-Grn1. However, the phages were rather distant, resulting in low bootstrap values (<75%); thus, the node was not well-supported. A large terminase subunit gene was used for the phylogeny because it is considered to be a signature, well-conserved gene among the phages and is a strong molecular motor associated with phage packaging [Phylogeny using the large terminase subunits of the ackaging .Vibrio alginolyticus has been used frequently as a target for novel phage isolation. Until today, more than 40 different phages have been isolated against V. alginolyticus, which is suggestive of the importance of this opportunistic pathogen for the aquaculture industry [industry . Severalindustry ,42, but industry .V. alginolyticus), Vibrio phage Artemius was closer to Vibrio phage phi-St2 and Vibrio phage phi-Grn1, which belong to the genus of Schizotequatrovirus [Shewanella sp. Interestingly, this phage was isolated from iced water in the Baltic Sea [Vibrio phage Artemius is a novel phage, as indicated by the BLAST search in the NCBI nr database where the closest phage at the nucleotide level was Vibrio phage 2.044.O._10N.261.51.B8 (MG592661.1) with a 79.6% similarity over a 2% query cover. A phylogenetic analysis of Artemius verified the novelty of this phage. According to the phylogeny obtained with the terminase large subunit using phages infecting the same host (trovirus . Accordiltic Sea ,46. Two of the most crucial characteristics of a phage to be considered for therapeutic purposes are the inability to integrate in the host genome and the lack of unwanted genes such as toxins and antibiotic resistance determinants that could be transferred to its host through lysogenic conversion or recombination . A genomSiphoviridae family [High temperatures usually have a devastating effect on phage integrity, causing tail aggregation, detachment of the phage head and denaturation of the nucleic acid . Interese family .Vibrio alginolyticus as it was not able to infect the other congeneric species; thus, it was a species-specific phage. Broad host-range phages are considered to be ideal for phage therapy, especially in aquaculture where the diversity of the pathogenic strains and species is wide. Phages with a broad host range have been reported against Vibrio alginolyticus [The host range of Vibrio phage Artemius was limited to strains of olyticus ,41. In tSiphoviridae family that infects V. alginolyticus and, based on its biological and genomic characteristics, could be considered for efficient and safe phage therapy applications.In conclusion, Vibrio phage Artemius is a potential new species of the


This cross-sectional study uses Centers for Medicare & Medicaid Services payment data to examine use of short-course radiotherapy from 2015 to 2019 among Medicare beneficiaries with indolent lymphoma. We evaluated SC-RT use with US episode-based payment data from the Centers for Medicare & Medicaid Services.6With increasing medical costs and concerns about COVID-19 exposure, reducing financial and time burdens of cancer treatment is critical.STROBE reporting guideline. Radiotherapy episodes from 2015 to 2019 were analyzed for Medicare beneficiaries 65 years or older who did not receive systemic therapy and lived more than 90 days after RT for lymphoma . Most patients (71%) received LC-RT. Receipt of SC-RT was associated with older age , hospital-affiliated vs free-standing site of care , and conventional RT vs IMRT   used conventional RT; however, IMRT use increased from 17% in 2015 to 25% in 2019 (45-6.02) . Increas45-6.02) .P < .001), conventional RT, younger patients, and hospital-affiliated sites (P < .001), younger patients, conventional RT, and freestanding sites  vs $8484  for LC-RT. For SC-RT, median total spending was $8048  with IMRT vs $4121  with conventional RT. Median total LC-RT spending was $13 085  with IMRT vs $7657  with conventional RT. Professional services–related spending was reduced with SC-RT (adjusted β, $572; 95% CI, $550-$594; ed sites . Technicng sites . In lineMost Medicare beneficiaries treated with radiation monotherapy for lymphoma between 2015 and 2019 received LC-RT. Use of LC-RT has important financial implications given higher total spending. Despite lower health care spending and reduced time, travel, and costs for patients, SC-RT was used in only 29% of patients by 2019. Whereas practice patterns may reflect site-specific case mixes, ensuring differences do not reflect competing financial incentives owing to reimbursement structures or opinions on evidence-based practice is important. Increases in spending were greatest for IMRT; therefore, regimen and technique should be considered for cost savings. Financial alignment with value-based practice irrespective of site of care is critical; future studies may help refine the balance between local control, patient treatment-related burden, and health care spending. Wider adoption of SC-RT may help reduce systemwide costs and optimize personalized RT decision-making. Limitations include lack of available clinical variables; despite efforts to limit the analysis to indolent lymphomas for which SC-RT is considered, more aggressive subtypes may have been included.


Bacteriophages are obligatory parasites propagating in bacterial hosts in a lytic or lysogenic/pseudolysogenic cycle. Phages are the most abundant biological particles in the world, being responsible for: (i) dissolved and particulate organic matter circulation via host cell lysis, (ii) regulation of numbers and biodiversity of populations, (iii) horizontal gene transfer (HGT) via transduction, or indirectly via transformation of bacterial DNA released during cell lysis, and, finally, (iv) lysogenic conversion by temperate phages. Therefore, phages greatly affect microbial diversification as an integral part of each ecological niche, including the human body. The tremendous dynamics of phage–host interactions result in the continuous flow of genetic material, which drives the co-evolution of both entities.Viruses “Phage–Bacteria Interplay in Health and Disease” focuses primarily on the regulation and functioning of human/mammal microbial ecosystems as the consequence of specific and non-specific virus–bacteria interactions, bacterial defence against phages which can drive the outcome of the disease/infection, phages as human immunomodulators, and the description of innovative experimental techniques characterizing the phage traces in mammals.Advances in recent years in molecular biology, multi-omics, and bioinformatic approaches have contributed to the deeper insight into and functional analyses of a wide range of phage–bacteria interactions at the individual and population levels, and their consequences on health and disease. The Special Issue of A total of 13 manuscripts, including 10 original and 3 review papers, have been published in this Special Issue. The collection contains recent papers that could be roughly classified into three themes comprising the following: (1) survival strategies of bacteria in response to phage infection as well as the impact of phages on bacterial virulence and pathogenicity ; (2) influence of lytic and temperate phages on the human microbiome as well as human virome composition ; (3) phages as potential antibacterial therapeutics . All papers reflect the impact of phage–bacteria as well as phage–bacteria–host interactions on human health, disease, and economic activities. They also highlight the great importance and relevance of the topic addressed.Streptococcus pyogenes is a Gram-positive β-hemolytic pathogen that strictly infects humans and can cause superficial skin infections, pharyngitis, toxin-mediated diseases ), and invasive diseases of subcutaneous tissues [S. pyogenes to protect against Phage A1. Phage A1, as a temperate phage, was able to infect S. pyogenes strains, resulting in complete resistance against subsequent phage infections, most likely by providing superinfection immunity. Furthermore, the lysogenization did not influence the humoral host immune response or bacterial virulence and did not induce ampicillin tolerance to this common antistreptococcal antibiotic. The authors demonstrated that the type II-A CRISPR-Cas system of S. pyogenes acquires new spacers upon phage infection, which are increasingly detectable in the absence of a capsule. Finally, the authors showed that the number of infecting phages is limited through binding to released streptococcal outer membrane vesicles. The authors proposed a multistage interaction model between S. pyogenes and Phage A1 [ tissues . Bacteri tissues . Beerens tissues  describePseudomonas aeruginosa antiviral strategy based on outer membrane vesicles (OMVs) against the LPS-specific phages KT28 and LUZ7. To investigate the passive and active role played by OMVs towards these phages, the OMVs derived from the phage-sensitive wild-type PAO1 strain, and an LPS-deficient mutant (∆wbpl PAO1) resistant to both phages, were used. It turned out that naturally formed OMVs efficiently protected P. aeruginosa from phage infection. Next, it was verified whether OMVs derived from the wild-type PAO1 strain were able to sensitize the LPS-deficient mutant (Δwbpl PAO1) to the tested phages. The growth kinetic curves and one-step growth assay revealed no sensitization event [Outer membrane vesicles (OMVs) of Gram-negative bacteria are important virulence factors as decoys against a variety of antibacterials but are also an element in host–bacteria interplay, and intra- or inter-species bacterial communication ,4. In thon event .Clostridioides difficile is one of the most common causes of antibiotic-related nosocomial infections with symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis and/or toxic megacolon [C. difficile phages, with a particular interest in phage CDHS-1 propagating on the hypervirulent 027 ribotype C. difficile R20291. The study focused on the transcriptomic takeover of phage CDHS-1 during infection, and analyses revealed that the majority of the bacterial genes connected with metabolism and toxin production were downregulated at the early phase of CDHS-1 replication, whereas genes related to DNA synthesis and ATP production were among those upregulated at this stage. The holin, endolysin, and structural genes were upregulated towards the mid-log and late phases of the phage replication. The retrieved phage-resistant clones and lysogens showed relatively low virulence in the larval model of Galleria mellonella compared to the wild-type strain. The data suggest that phage infection both reduces bacterial colonization and negatively impacts bacterial pathogenicity, supporting the therapeutic potential of the phage for human and animal use [egacolon . Therefoimal use .Enterococcus faecalis strains. The report indicated that vB_EfaS-271 can significantly decrease the number of viable E. faecalis cells in biofilms formed on catheters and in liquid cultures and revealed no considerable toxicity to mammalian cells, influencing neither their viability nor morphology. The efficiency of phage resistance development was especially significant under conditions of high MOI values; nevertheless, phage vB_EfaS-271 was considered promising for phage therapy [Topka-Bielecka et al.  describeStreptococcus agalactiae (GBS) clinical isolates to verify the prevalence of prophages versus the bacterial virulence potential. Based on the whole genome sequencing and PCR methods, the authors identified eight groups of prophages, amongst which the highest prevalence was observed for a prophage from group A (71%) and a satellite prophage from group B (62%). They observed that the prophage distribution did not differ between clinical and screening strains, but it was unevenly distributed in MLST (multilocus sequence typing) sequence types. This study implies that prophages could be beneficial for the host bacterium [Lichvariková et al.  performeacterium .Siphoviridae, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. The results pointed out that some prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. Shaping the biodiversity of bacterial populations, these viruses contribute to local fluctuations in the vaginal microbiome [Phages have an important role in shaping bacterial communities. Phages also impact human health by infecting bacteria forming human microbiomes in the gut, respiratory tract, skin, or vagina. Happel et al.  investigcrobiome .Lactobacillus (L.) spp. abundance and increased colonization of facultative anaerobes, such as Gardnerella spp. [L. crispatus and L. iners) as well as with known pathobionts (Gardnerella spp. and Atopobium vaginae) [Bacterial vaginosis (BV) is characterized by a reduction in lla spp. . The paplla spp.  related vaginae) .®) on the ex vivo human gut microbiome composition and function. Using a novel in vitro assay called RapidAIM as well as 16S rRNA gene sequencing and metaproteomic approaches, the authors documented that the ex vivo human gut microbiota composition and function were unaffected by BAFASAL® treatment, which proves the GRAS provision  [Mayne et al.  investigas safe) .At present, traditional antimicrobials are becoming ineffective against multidrug-resistant bacterial pathogens. Therefore, phages are increasingly beginning to be recognized as an alternative or supportive therapeutic agent .Staphylococcus aureus phage in the treatment of atopic dermatitis (AD), the most common inflammatory skin disease, in an atopic mouse model. As previously documented, phage SaGU1 can infect a broad range of S. aureus in AD patients, whereas it does not kill strains of the symbiotic bacterium S. epidermidis. In this work, the authors showed that administration of SaGU1 to the back skin of mice reduced both S. aureus counts and the disease exacerbation caused by these bacteria. Furthermore, the application of S. epidermidis in combination with SaGU1 inhibited the emergence of phage-resistant S. aureus, indicating that synergistic use of probiotics and phages can be promising and effective in the treatment of S. aureus-associated AD [Shimamori et al.  investigiated AD .E. coli lytic phages as a new tool for the investigation of phage interactions with cells and tissues. The deposition of an RFP-displaying phage in multiple murine organs ex vivo after various routes  of phage administration was verified. The most effective intravenous administration led to effective distribution kinetics with phage presence in the lymph nodes, lungs, and liver after 20 min, whereas in the muscles and spleen/lymph nodes, this occurred 30 min and 1 h after administration, respectively [Since interactions between phages and mammals strongly affect the possible applications of phages, tools to study how phages circulate in the body and can be deposited in tissues are highly desirable. Understanding this need, Kaźmierczak et al.  proposedectively .Iszatt et al.  providedS. aureus, Klebsiella pneumoniae, P. aeruginosa, and E. faecalis infection models. It also illustrated appropriate phage selection criteria, as well as recommendations for successful therapy. The main conclusions from the analysis of the selected literature were that phage applications (single or in cocktails) are effective and safe, can work synergistically in combination with certain antibiotics, and may induce the emergence of phage resistance [An interesting review of Al Ishaq et al.  addressesistance .Salmonella spp., E. coli, Campylobacter spp., Vibro spp., and P. aeruginosa, and other pathogens, such as Staphylococcus spp., Streptococcus spp., Klebsiella spp., Acinetobacter spp., and Mycobacterium sp. The varied therapeutic and immunomodulatory activities of phages on humoral and cellular immune response mechanisms were also discussed [In parallel with attempts to use phages against nosocomial human pathogens, there has been much work conducted on the use of these viruses as alternative methods to control zoonotic and foodborne pathogens. This problem was addressed in a review paper written by Alomari et al. . The autiscussed .


Background: Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus. Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 1011 PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages. Conclusions: In sum, we propose a quality-by-design approach for the amplification of broad-spectrum anti-S. aureus phages, facilitating the subsequent steps of the manufacturing process; namely, purification and quality control. Staphylococcus aureus is a major human pathogen responsible for a wide range of diseases [S. aureus is able to form biofilms that prevent the access of antibiotics and immune cells to bacteria, which are thus associated with therapeutic failures [S. aureus lytic Caudovirales bacteriophages, targeting a wide range of hosts, have been previously isolated [S. aureus phages belong to various families and genera, including Herelleviridae  and Podoviridae, with Silviavirus phages having the broadest activity spectrum [diseases . It is tdiseases ,3. In adfailures . In thisfailures ,6. Anti-isolated ,9,10,11.spectrum .To ensure the future success of phage therapy, phage production should comply with pharmaceutical standards, including a robust and safe process of phage amplification. Such standards still need to be established and validated by national and international drug agencies so that they can be adapted to the specific biological statuses of phages . HoweverS. aureus Silviavirus phages with broad activity against a large panel of S. aureus clinical strains. We also describe (i) the selection of optimal bacterial strains, aiming at limiting the production of bacterial contaminants upon phage amplification, and (ii) the optimization of experimental parameters for their optimal production, both in terms of amplification yields and safety.In the present study, we report the isolation and characterization of three novel anti-All bacterial strains included in the present study, both for host range assessment and for phage production, were obtained from the collection of the French National Reference Centre for Staphylococci . Clonal complexes of S. aureus strains were assigned using DNA microarray , following the manufacturer’s instructions. Whole-genome sequencing was also performed for the screening of candidate strains for phage production. Illumina libraries were prepared using the Nextera XT or DNA Prep kits  and sequenced on a MiSeq or NextSeq 500 instrument (Illumina) using a 300 or 150 bp paired-end protocol, respectively. Detection of virulence- and resistance-associated markers was performed from assembled genomes using Abricate (v0.7) and an in-house nucleotide-based database, built mainly from the Resfinder database (version 2020-06-02) and covering the known S. aureus toxins, including enterotoxins, exfoliatins, toxic shock syndrome toxin TSST-1, and leukocidins. Accession numbers for genomes of selected strains are provided in Bacterial genomic DNA extraction consisted of an initial incubation with 40 µg of lysostaphin , 200 µg of lysozyme (Sigma-Aldrich) and 200 µg of RNaseA  for 1 h at 37 °C, followed by incubation with 12 mAU of proteinase K  for 20 min at 60 °C, then an extraction using the MaxwellThree phages  were isolated from three different wastewater samples in Lyon, France. Briefly, 5 mL of the filtered water samples was incubated with 500 µL of Tryptic Soy Broth (TSB) 10X  and 10 µL of overnight bacterial culture . Then, the culture supernatant was filtered using a 0.22 µm syringe filter and diluted in double-layer agars, pouring a mix of 100 µL of this supernatant, 250 µL of bacterial culture, and 5 mL of TSB-soft agar (TSB containing 0.75% agar) over a TSA plate . An individual plaque-forming unit (PFU) was further purified with five rounds of serial passages and eventually propagated in liquid medium. The obtained phage lysates were filtered using a 0.22 µm syringe filter and stored at +4 °C.g  over 3 h at +4 °C in a three-layer CsCl (Sigma-Aldrich) gradient with densities of 1.6, 1.5, and 1.3. After centrifugation, phages were collected between the 1.5 and 1.6 layers and dialyzed  twice in 3 L of DPBS buffer (Sigma-Aldrich) at 4 °C for 6 h, with one buffer change. Lastly, phage suspensions were filtered using a 0.22 µm syringe filter and stored at +4 °C.A volume of 9 mL of each crude phage lysate was purified by ultracentrifugation at 120,000× g for 5 h and pellets were resuspended in 50 µL of NaCl 0.9%. Enzymatic treatment was then applied with 100 mU of benzonase® nuclease (Sigma-Aldrich) at 37 °C overnight to degrade extracellular bacterial DNA, followed by benzonase heat-inactivation at 95 °C for 30 min, a treatment with 4 µg of proteinase K (Sigma-Aldrich), and proteinase K heat-inactivation. Phage DNA was extracted using the DNA Extractor® WB kit  and sequenced on an Illumina NextSeq 500 instrument using a 150 bp paired-end protocol. Reads were trimmed  and the read mappings against the genome of the bacterial strain used for the amplification of phages  were removed. The remaining reads were assembled  and scaffolds smaller than 100 bp were removed. Taxonomic assignation was performed using kraken 1.1.1 with the minikraken database. Genomes were first annotated using PATRIC (v3.6.12) with parameters for bacteriophage annotation. The protein sequences of genes annotated as “hypothetical protein” or “phage protein” were further analysed: a blastp was performed against the PHROGs database to improve annotation, considering annotated proteins with identity and query cover percentages higher than 90%. Finally, Abricate (v0.8.13) was used for resistance and virulence gene detection using all the databases available. The lytic nature of phages was assessed using the Phage AI repository (https://app.phage.ai/phages/ (accessed on 17 March 2022)). Phage genomes were deposited in GenBank under the accession numbers ON814134, ON814135, and ON814136 for V1SA19, V1SA20, and V1SA22, respectively.A volume of 6 mL of phages was centrifuged at 14,000× S. aureus and S. argenteus strains genetically characterized using WGS and representative of clinical isolates collected in France between 2017 and 2020 , pH = 7, and then centrifuged again at 21,000× g for 1 h and finally re-suspended in 100 µL of the same buffer. Phage suspensions were adsorbed on 200 Mesh Nickel grids coated with formvar-C for 10 min at RT. Then, grids were coloured with Uranyless  for 1 min and observed on a transmission electron microscope  equipped with a Orius® 1000 camera  and Digital Micrograph software.A volume of 1 mL of the phage suspensions obtained after ultracentrifugation was centrifuged at 21,000× S. aureus or S. argenteus genomes from the French National Reference Centre for Staphylococci. First, strains harbouring genes encoding major virulence/resistance factors, such as enterotoxins, leukocidins, superantigens, and methicillin-resistance, were excluded. The remaining genomes were examined using prophage-prediction tools—namely, PHASTER (https://phaster.ca/statistics (accessed on 10 January 2021)) and ProPHET (v0.5.1)—allowing the exclusion of genomes harbouring intact prophages.Candidate strains for phage production were screened among a collection of more than 2000 −3 phage per bacteria in 10 mL of TSB. After 24 h of incubation, phage titres were measured.Using this in silico approach, only 10 candidate strains were selected for further experimental assessment. EOP ratios were determined for the three selected phages against these 10 strains as described above. Phage amplification yields were then assessed in small scale production conditions with a selection of the five strains presenting the highest EOP values for all phages and representative of each sequence type. For these experiments, bacteria in exponential phase and phages were mixed at two concentrations (multiplicity of infection (MOI)) of 1 and 10TM , and Superior BrothTM (Athenaes). To this end, bacteria were grown in 1 L of medium in 2.5 L Fernbach culture flasks with sided baffles  for 2 h until an exponential phase was reached. Then, phages were added at MOIs of 10−2 or 10−4. Phage titrations were performed at different time points during the incubation in order to follow the kinetics of phage amplification.Finally, the bacterial strain allowing the highest yield for each phage was then selected for production optimization in larger containers to test the impact of (i) the MOI or (ii) the medium used, comparing conventional media and animal protein-free media designed for pharmaceutical production of proteins, including TSB, LB Broth Lennox , Turbo Both−4 for graphical representation purposes. For phage production experiments, Student’s t-test was used to compare the mean phage titres. A p-value < 0.05 was considered significant. Statistical analyses were performed and figures were generated using GraphPad Prism, version 8.0.0 .Phage activity variations between clonal complexes were assessed by comparing EOP distributions using the Kruskal–Wallis test. Null EOP values were arbitrarily set to 10Herelleviridae family and the Silviavirus genus. Genome lengths were 138,507, 136, 919, and 133,701 bp for V1SA19, V1SA20, and V1SA22, respectively. The numbers of ORFs were 245, 234, and 225 for V1SA19, V1SA20, and V1SA22, respectively, including 74.6% to 76.4% of genes for which a putative function could not be attributed . Of note, V1SA20 was the only phage active against CC80 strains. Phages were poorly active against CC7, CC59, CC239, and CC398 strains.The host range of the three phages was determined using a collection of 150 strains chosen to be representative of (i) the major methicillin-susceptible (MSSA) or resistant (MRSA) clones circulating worldwide and (ii) the genetic and clinical diversity of fections . Phage Vfections A. Phage 10 PFU/mL  compared to conventional TSB or LB media were assessed. The Superior BrothTM yielded the maximal phage titres in shorter times compared to other media (10 to 1 × 1011 PFU/mL reached in 3 to 4 h). Of note, we observed significant mean phage titre drops, with all types of media, of 4 × 1010 and 7 × 1010 PFU/mL between 8 and 24 h of incubation for V1SA20 and V1SA22, respectively .The bacterial strains P2SA225 and P2SA237 were then selected for optimization of the amplification of phages V1SA19 or V1SA20 and V1SA22, respectively, in larger containers. Phage production yields were first assessed in conventional TSB medium at two MOI values: maximal phage titres were reached more quickly at MOI 10MOI 10−4 A,B. In aPhage therapy a promising alternative strategy to address antimicrobial resistance and improve prognoses in the treatment of staphylococcal infections. Although no marketing authorization has been issued for therapeutic phages as therapeutic products by the US Food and Drug Administration or the European Medicines Agency yet, several clinical trials are currently on-going and their current compassionate use, codified by the Declaration of Helsinki, is increasing very rapidly in diverse clinical settings ,24,25. TS. aureus phages belonging to the Silviavirus genus showing complementary activities: 82.0% of the bacterial collection were susceptible to one of the three phages, while 76.7% were susceptible to the most active phage V1SA20. Anti-S. aureus phages have previously been reported to have broad-spectrum activity within this bacterial species. However, among the numerous studies describing the isolation of such phages, very few of them have tested large, diverse, and clinically relevant genetically-characterized collections of clinical strains, ensuring the diversity of the tested strains, to more precisely describe the phage host range [S. aureus phages depended on the clonal complex (CC), which is in agreement with previous studies. This specificity has been linked to the presence of type I restriction-modification systems, which are also associated with the CC [In the present study, we characterized three novel, strictly lytic anti-st range ,27,28. IAlthough the status of phage products is not definitively established by the health authorities, the scientific community expects it to be adapted to the biological specificities of phages compared to conventional medicines. A quality-by-design approach has been recommended for the development of phage production in line with Good Manufacturing Practice (GMP) guidelines . It shouSilviavirus phages based on the simultaneous absence in their genomes of (i) a maximum of genes encoding virulence/resistance factors  and (ii) prophages. Very few other studies have previously addressed this topic [Staphylococcus xylosus strain, a low pathogenic staphylococcal species, for amplification of anti-S. aureus Kayvirus phages in order to limit the production of virulence factors and improve the safety of phage therapy [First, this implies the need to establish the possible contaminants of phage preparations that might affect the safety of the final product for phage therapy and to assess ways to control them . Indeed,is topic ,32. El H therapy . However therapy ,33. Thes therapy ,35. None therapy ,36.11 PFU/mL, and/or were reached more rapidly using these media compared to conventional TSB. Of note, shortening the time necessary for amplification could be of interest to limit the release of metabolites, notably bacterial DNA, which cannot be avoided despite the selection of optimal bacterial strains. These kinds of bacterial metabolites are known to be able to induce inflammation and their quantification is part of the quality controls that should be performed to assess the quality of phage therapy products [Consequently, propagation hosts must also allow the acquisition of high titres of phages. Interestingly, our study showed that high EOP ratios did not provide guarantees for a high level of phage amplification and, thus, that bacterial strains with high EOP ratios are not systematically the best candidates for phage production. Bacterial and phage inocula (MOI), the nutrient composition of culture media, stirring speed, oxygenation, and temperature strongly influence phage amplification yields . We notaproducts ,38.Staphylococcus aureus phages with large spectra of activity. We showed that it is possible to optimize phage production protocols to enhance amplification yields after the in silico selection of candidate strains, aiming at facilitating the subsequent steps of the manufacturing process—namely, purification and quality controls—in order to ensure patient safety.We reported the initial steps of the pharmaceutical development of three novel anti-


K. pneumoniae emphasizes the urgent need of new therapeutic strategies for the control of this pathogen. There is growing interest in the use of bacteriophages (or phages) to treat K. pneumoniae infections, and newly isolated phages are needed. Here, we report the isolation and physical/biological/molecular characterization of a novel lytic phage and its efficacy in the control of MDR K. pneumoniae. The phage vB_KpnS_Uniso31, referred to hereafter as phage Kpn31, was isolated from hospital wastewater using K. pneumoniae CCCD-K001 as the host. Phage Kpn31 presents a siphovirus-like morphotype and was classified as Demerecviridae; Sugarlandvirus based on its complete genome sequence. The 113,444 bp Kpn31 genome does not encode known toxins or antimicrobial resistance genes, nor does it encode depolymerases related sequences. Phage Kpn31 showed an eclipse time of 15 min and a burst size of 9.12 PFU/host cell, allowing us to conclude it replicates well in K. pneumoniae CCCD-K001 with a latency period of 30 min. Phage Kpn31 was shown to be effective against at least six MDR K. pneumoniae clinical isolates in in vitro antibacterial activity assays. Based on its features, phage Kpn31 has potential for controlling infections caused by MDR K. pneumoniae.The worldwide increase in serious infections caused by multidrug-resistant (MDR)  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., which have been integrated within the acronym “ESKAPE”, are the leading cause of hospital-acquired infections throughout the world . After three rounds of purification on a K. pneumoniae CCCD-K001 lawn, the isolated phage produced clear and translucent plaques with diameters of ≈1.0 mm (13 PFU/mL) of phage Kpn31 were obtained for further assays, aiming at its characterization.One phage clear lysis plaque was selected among the clear and turbid plaques of the same size obtained from the enrichment of a wastewater sample (sewage) collected at Hospital UNIMED Miguel Soeiro, Sorocaba, São Paulo, Brazil, using  ≈1.0 mm . The isoSiphoviridae-like tail morphotype [The TEM photomicrograph  clearly rphotype  belonginK. pneumoniae is 57.5% [The genome of phage Kpn31 was sequenced and assembled, resulting in a contig of 113,444 bp. The contig had 141 bp direct terminal repeats, indicating that the assembled phage genome is complete. The GC content of the Kpn 31 genome is 45.3%, whereas that of the is 57.5% . The oveThe genome of phage Kpn31 encodes 14 tRNAs and 188 protein coding genes  . A compaDemerecviridae or Sugarlandvirus. Therefore, phage vB_KpnS_Uniso31 (phage Kpn31) can be assigned to the Sugarlandvirus genus. The taxonomic classification of Kpn31 agrees with the TEM analysis . Thus, within 10 min after phage-host mixture, ~90% of the phage particles were adsorbed to the bacterial cells , in the first 8 h of incubation, the inactivation factor was higher for MOI 1000 . At a MOI of 1000, after 6, 8, 10, and 12 h of incubation, the reduction in K. pneumoniae counts  was significantly larger  than that produced with MOI 1 . K. pneumoniae regrowth was observed after 6 h of incubation. However, at the end of the experiment, the rate of bacterial regrowth with a MOI of 1 and 1000  was significantly lower  than the one obtained with the bacterial control (p > 0.05)  a.p < 0.05) for MOI 1 and MOI 1000, respectively  increased 5.1 log CFU/mL a, whereaectively b.The effect of the MOI value was more pronounced during the first 8–10 h of bacterial inactivation a, with M13 virions/mL) were studied via UV–VIS spectral scans and X-ray diffraction (XRD) analysis. Kpn31 = 2.2877 × 10−12 (PFU’s/mL)−1 cm−1, allowing for calculating the phage particle concentration based on the resulting calibration curve . An EOP of 100% means that every phage particle adsorbing to a susceptible bacterial cell can inject its DNA and produce a lysis plaque under ideal conditions [K. pneumoniae phages (vB_KpnS_FZ10 and vB_KpnP_FZ12) were active against a high percentage of K. pneumoniae strains  [K. pneumoniae phages showed that phages of the Siphoviridae and Podoviridae families lysed about 7–15% of strains, and only one phage of the Podoviridae family was effective against 22% of the strains tested [Myoviridae family were active against 4–22% of K. pneumoniae strains [K. pneumoniae strains, which confirms its potential for prophylaxis and for the treatment of bacterial infections caused by this bacterium. In the future, new phages need to be isolated and tested together with phage Kpn31 in order to produce a cocktail with a broader spectrum of activity against K. pneumoniae and other human pathogenic bacteria.One (if not the major) advantage of antimicrobial phage treatment lies in the high specificity of phage particles, even though they should be able to promote the lysis of the majority of strains of a given bacterial species ,64,65,66s tested . However low EOP , indicatnditions . Zurabovctively) . Howeverctively) . In anots tested . Phages  strains . Phage K−8–10−9 mL/min. Approximately 90% of phage Kpn31 particles adsorbed to K. pneumoniae CCCD—K001 cells after 10 min, and 100% of the phage particles adsorbed after 30 min, which confirms its potential for the treatment and prevention of bacterial infections. The adsorption rate of phage Kpn31 particles (1.700 × 10−9 PFU−1 CFU−1 mL−1 h−1) closely agrees with the results published by Zurabov et al. (2021) [K. pneumoniae phages .Before applying phages to control bacterial pathogens, the dynamics of phage–host replication should be well characterized in vitro. The first step in the phage infection process is the adsorption of the phage virion onto a susceptible bacterial cell ,70,71,72. (2021)  for K. pK. pneumoniae CCCD-K001 with a short latency period (30 min). The latent period of phage Kpn31 correlates well with published data on K. pneumoniae phages [K. pneumoniae, but its effect only started after 4 h of incubation. K. pneumoniae was effectively inactivated by phage Kpn31, reaching a maximum inactivation of 5.6 log CFU/mL and 7.5 log CFU/mL after 6 h of incubation at MOI 1 and 1000, respectively. After this time, although some host cells were not inactivated by phage Kpn31, after phage treatment, the cell growth was much slower. Between 8 h and 24 h of treatment with phage Kpn31 at MOI 1 and 1000, the concentration of host cells was significatively lower than that observed for the control.The growth parameters of Kpn31 phage particles showed a burst size of 9.12 PFU/host cell, allowing to conclude that the phage replicates well in e phages ,69,77. Te phages . The bure phages , but low5 to 106 PFU/mL, 8 to 1012 PFU/mL, 5 to 1010 and from 105 to 109+ CFU/mL, respectively, during the first 4 h of treatment , the iK. pneumoniae clinical isolates. However, the spot test may produce false positives due to bacterial cell lysis, without them being infected by the phage [The phage Kpn31 host range was initially evaluated by spot test to assess if it was able to produce clear plaques on strains of different bacterial genera and of MDR he phage , either he phage ,87,88. Ihe phage .K. pneumoniae strains paves the way for new studies, especially in vivo studies, aiming at the development of strategies to control infections caused by K. pneumoniae. The results of this study highlight the importance of isolating, characterizing, and testing the effectiveness of new phages to inactivate bacteria in clinically relevant settings.The high bacterial inactivation efficiency of phage Kpn31 combined with the safety of the phage and its efficiency against


Klebsiella pneumoniae.The development of alternative control measures, such as phage therapy or adjunctive therapy, is urgently needed to manage the dissemination of carbapenemase-producing K. pneumoniae clinical isolate in vitro in Kenya.This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of carbapenemase-producing Klebsiella phages. The efficacy of individual bacteriophages and phage cocktails as well as their combination with antibiotics were determined for their inhibitory activity on carbapenemase-producing K. pneumoniae (KP20).The study was conducted from February 2021 to October 2021 at the Institute of Primate Research, Nairobi, Kenya. Phage cocktails were formulated based on the morphology and biological properties of precipitated Myoviridae, Siphoviridae and Podoviridae. Regarding the evaluation of the phage cocktails, the absorbances at 600 nm of the bacterial culture treated with the two-phage cocktail (2φ MA) ranged from 0.173 to 0.246 at 16 h and 20 h whereas it peaked from 2.116 to 2.190 for the positive control. Moreover, the results of the adjunctive therapy showed that the optical density at 600 nm of the bacterial culture treated with 2φ MA was 0.186 at 24 h post-incubation time while it was 0.099 with the bacterial culture treated with imipenem in combination with 2φ MA.The precipitated bacteriophages were members of K. pneumoniae growth in vitro.This study demonstrated that the two-phage cocktail in combination with imipenem was able to synergistically delay the increase in carbapenemase-producing  Klebsiella pneumoniae because of the decrease in the effectiveness of antibiotic drugs.2 Bacteriophage therapy has, therefore, been proposed as an alternative strategy in controlling multidrug-resistant (MDR) bacterial infections, including MDR K. pneumoniae clinical isolates.4 Some of the beneficial effects of bacteriophages over antibiotics include their abundance, host specificity and exponential replication.5 However, the narrowness of phage host range and the ever emergence of novel pathogen variants will at minimum represent some limitations for phage therapy.7 This challenge can be managed by formulating phage cocktails that contain different phages infecting one species or by combining phages with antibiotics, which may result in a broad spectrum of activity.8 The characterisation of therapeutic phages in terms of their biology and bactericidal activity is mandatory before any preclinical trials because the application of non-characterised lytic bacteriophages may cause undesirable virulence as an adaptive response to bacteriophage infection.9 In other words, the development of effective strategies, such as phage growth parameters, host range spectrum, the presence of lytic proteins, formulation of phage cocktail and phage synergistic interaction with antibiotic to enhance phage efficacy, is a key prerequisite for optimal treatment of MDR infections caused by K. pneumoniae. Several studies have reported the advantages and drawbacks of phage cocktails in treating MDR bacteria.11 Unfortunately, bacteriophages employed as cocktail or adjunctive therapy have not been well investigated in the African continent to effectively control MDR bacterial infections.12 This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of CP K. pneumonia clinical isolate in vitro in Kenya.The development of alternative control measures is urgently needed to manage the dissemination of carbapenemase-producing (CP) This study was not human or animal research. However, clearance was issued by the Nairobi City Water & Sewerage Company Limited (NCWSC/HR/TRG.14/Vol.8/14/MMM/ak) for collection of waste water samples.K. pneumoniae (KP20) was obtained from the stock of the recently published data in Kenya.13 Bacteriophages were isolated from waste water samples of Dandora Estate Sewage Treatment Works (Ruai) and Rongai effluent, Nairobi, Kenya. They were purified from waste water samples through spot assay and double-layer plaque method as described, previously.15The study was conducted from February 2021 to October 2021 in the Phage Biology Research Laboratory at the Institute of Primate Research, Nairobi, Kenya. The clinical isolate of CP The minimum inhibitory concentrations of a panel of antibiotics including carbapenems for KP20 were determined using VITEK 2 Systems version 9.2 and AST-XN05 (bar code 1481424403205844) card , according to the manufacturer’s instructions. The detailed procedure is described in online Supplementary Method 1.8 PFU/mL or 109 PFU/mL) was propagated in large volume, concentrated and cleaned up in the presence of sodium chloride and polyethylene glycol as described elsewhere.16Phage lysate  uranyl acetate on 3 mm carbon-coated copper grids of the precipitated Klebsiella phages was conducted at the University of Leicester and the bacteriophages were visualised with a transmission electron microscope .17 Bacteriophages were then classified according to their morphology.18The morphological characterisation of the precipitated 19 Bacterial strains tested in our study included reference strains and MDR bacterial strains reported by Michodigni et al.13 and described in online Supplementary Methods 2.A spot assay was conducted to determine the host range of precipitated bacteriophages and phage cocktails as described by Kutter.20 Exponential-growth-phase culture of CP K. pneumoniae (≈ 2.98 × 108 CFU/mL) and phage precipitate (≈ 109–1010 PFU/mL) were mixed with bacterial suspension to obtain a multiplicity of infection (MOI) of 0.1.14 The suspension was incubated at 37 °C in a shaking incubator for 10 min at 120 rotations per minute following with centrifugation. The pellet was then suspended in 7 mL of fresh tryptic soy broth and incubated at 37 °C in a shaking incubator at 125 rotations per minute. At 3-min intervals, from 0 min to 36 min, 500 µL of the suspension was taken and phage titres were estimated.21A one-step growth experiment for determination of latent period, and burst size of precipitated bacteriophages, was performed as described elsewhere, with some modifications.Klebsiella phages, designated as CPRSA, CPRSB and ESBLA, were used for the formulation of two-phage cocktail (2φ MA) and three-phage cocktail (3φ MB). The two-phage cocktail comprised CPRSA and CPRSB while the three-phage cocktail consisted of CPRSA, CPRSB and ESBLA. Bacteriophage cocktails consisting of two and three tested phage lysates were formulated by combining in equal ratios of 1:1 and 1:1:1. The evaluation of the phage cocktails’ effectiveness was carried out based on the method described by Merabishvili with some modifications.22 Two-phage (2φ MA) and three-phage (3φ MB) cocktails were composed and their ability to inhibit the growth of KP20 at its exponential-growth-phase was evaluated. Subsequently, the efficacy of two individual bacteriophages, designated as Klebsiella phage CPRSA and CPRSB at MOI 1.0, 0.1 and 0.001, was also investigated. Only broth media (tryptic soy broth) and individual bacteriophages represented the negative controls. Bacterial culture with a final resulting concentration of ≈ 2.98 × 106 CFU/mL served as positive controls. The OD600 of the host bacterium was measured every 4 h for 24 h, and finally at 48 h, to assess the inhibitory activity of bacterial growth by both individual phages and phage cocktails. The different absorbances obtained at 600 nm were compared to the ones of the positive control sample. The different concentrations of individual bacteriophages and phage cocktails are summarised in online Supplementary Three 600 ≈ 0.6). The two antibiotics meropenem and tigecycline  were tested in combination with 2φ MA and 3φ MB. The phage cocktails 2φ MA and 3φ MB with optimal concentrations (MOI 0.1 and 0.001) were used in this experiment in combination with the single and double of the minimum inhibitory concentrations of imipenem and tigecycline. Sterile broth media (tryptic soy broth), bacterial culture treated with antibiotics, and bacteriophage cocktails represented the negative controls. Bacterial culture with a final resulting concentration of ≈ 2.98 × 108 CFU/mL served as a positive control sample. The growth of the host bacterium was monitored at 3-h intervals, between 0 h and 24 h, and at 48 h of incubation by measuring OD600. The optical densities obtained at 600 nm were compared to the ones of positive and negative control samples. The different concentrations of phage cocktails and antibiotics are indicated in online Supplementary The inhibitory activity of the phage cocktail alone, and in combination with imipenem or tigecycline was assessed on KP20 at its optimal exponential growth . A p < 0.05 was considered as significantly different.A two-tailed 13 The ESBL and carbapenemase genes produced by KP20 are indicated in The minimum inhibitory concentrations of imipenem and tigecycline to KP20 were 4 µg/mL and 1 µg/mL . We prevA transmission electron microscope showed that the bacteriophages included in this cocktail were all tailed phages . Among t11 ÷ 2.98 × 108), 1 (2.294 × 108 ÷ 2.98 × 108) and 10 (3.123 × 109 ÷ 2.98 × 108) for Klebsiella phage CPRSA, CPRSB, ESBLA. Regarding their latent period, Klebsiella phages CPRSA and CPRSB had the same latent period (9 min) and the one of ESBLA was 18 min as shown in online Supplementary The burst sizes of the precipitated phages in the cocktail were 610  and the three-phage cocktail (3φ MB). The two-phage cocktail comprised CPRSA and CPRSB while the three-phage cocktail consisted of CPRSA, CPRSB and ESBLA. The two-phage cocktail was the combination of two bacteriophages belonging to the family Myoviridae while the three-phage cocktail was the combination of 2φ MA and one belonging to the family Podoviridae. The results of the host range analysis showed that both the individual phages and phage cocktails (2φ MA and 3φ MB) were active on bacterial strains belonging to Klebsiella species. The three carbapenem-resistant K. pneumoniae were susceptible to only one individual phage, the Klebsiella phage CPRSA. The two-phage preparations were also active on the three CP K. pneumoniae strains, in addition to K2, which was described as an extended-spectrum beta-lactamase producer in our previous study.≈ 0.3 was determined at three different multiplicities of infection . It was observed in our study that there was an increase in CP K. pneumoniae growth to the individual phage CPRSB at its three different doses and to the phage CPRSA at MOIs 0.1 and 0.001 after 8 h and 16 h of incubation . At the most effective phage dose (MOI: 0.001), the absorbances at 600 nm of the bacterial culture treated with the individual phage CPRSA, and the two-phage cocktail (2φ MA), increased from 0.019 to 0.515, and 0.173 to 0.246 at 16 h and 20 h, while it rose from 2.116 to 2.190 for the positive control during the same interval of time (p = 0.12) at MOI 0.1 after 16 h of treatment time (p = 0.01) was observed between the two treatments at MOI 0.001.The ability of the mixture of two phages (MA) and three phages (3φ MB) to inhibit the growth of KP20 culture at an absorbance of cubation . The two to 24 h . This ba of time . For theent time . Signifi600 decreased from 0.294 (21 h) to 0.189 (24 h) for bacterial culture treated with tigecycline and the two-phage preparation at the lowest phage concentration, MOI 0001 (TG1 + 2φ MA3) while it increased slightly from 0.207 (21 h) to 0.331 (24 h) in the case of bacterial culture treated with tigecycline and the three-phage preparation (TG1 + 3φ MB3). The absorbances of the bacterial culture treated with tigecycline was 0.249 (21 h) and 0.314 (24 h) while it ranged from 1.212 (21 h) to 1.245 (24 h) for the bacterial culture treated with imipenem. The positive control sample exhibited an optical density of 1.166 (21 h) and 1.270 (24 h). The level of significance between the bacterial culture treated with 2φ MA and the bacterial culture treated with IMP2 + 2φ MA3 after 21 h of incubation are shown in p = 0.02). No statistical difference was observed between the results of the bacterial culture treated with a combination of phage and antibiotic and those of bacterial culture treated with tigecycline alone. A significant difference (p = 0.04) was observed between the bacterial culture treated with 2φ MA at MOI 0.001 and the adjunctive therapy .The in vitro studies showed that the same phage cocktails (2φ MA and 3φ MB) maintained their bactericidal activity after 24 h of incubation at the lowest phage concentration (MOI 0.001) . At diffMyoviridae with contractile tails consisting of a sheath and a central tube (25% of tailed phages), Siphoviridae with long and noncontractile tails (61%), and Podoviridae having short tails (14%).23Klebsiella tailed phages identified in our study belonged to the family of Myoviridae and Podoviridae.24 Previous studies reported similar observations regarding the morphological characters of Klebsiella phages.25This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of the KP20 clinical isolate. On one hand, the phenotypic results revealed that the precipitated phages obtained in this study had lytic activity with high titers and they were related to tailed phages. The latter are generally divided into three families including Klebsiella phage had a relatively short latency period of 18 min and its burst size was ~220 phage particles per infected bacteria.20 Indeed, a high burst size is key to achieve a productive adsorption and replication of bacteriophages and to reach the benefits that phages could have in comparison with antibiotics while a short latency period is recommended from a phage therapy perspective.26On the other hand, this study showed that one of the precipitated phages (CPRSA) had the shortest latency period (9 min) and high burst size (610). Our findings were supported by Horváth et al. in 2020. The authors reported that their isolated Klebsiella phages reported similar results.29 The major consequence of phage host specificity is that it demands an appropriate diagnosis of the bacteria involved in the infection before initiation of phage treatment.30 Nevertheless, this narrowness of phage host range to the strains of the same bacterial species could limit its lytic activity on microbiota.30 The phage host range is indeed affected by a number of factors including the absence of required accessory proteins for phage adsorption, restriction-modification and clustered regularly interspaced short palindromic repeats (CRISPR) systems.32 Despite their narrow host range, bacteriophages with lytic activity may still be useful in phage therapy and the use of bacteriophages as cocktails for adjunctive treatment can represent a highly attractive strategy.33Both the individual bacteriophages and phage cocktails were not susceptible to the reference bacterial strains tested in this study and, hence, displayed high specificity for their host bacteria, which were KP20 clinical isolates. Previous studies on host ranges of 22 Therefore, our study demonstrated that the mixture of two phages (2φ MA) was able to significantly delay the resurgence of bacterial cells in culture, as compared to the application of individual phage or the mixture of three phages (3φ MB) (p = 0.02). Our result was in line with the previous data published on phage cocktail efficacy.11 This synergistic activity of two-phage cocktail over the mixture of three phages in our study suggested that the individual phages might have employed different receptors to adsorb to the bacterial cells and, hence, effectively inhibited the bacterial growth. Some authors have reported that one phage in their two-phage cocktail used an outer membrane protein (OmpC) as a receptor, and another one employed a lipopolysaccharide component as its receptor to effectively control bacterial resistance.35 Moreover, the mixture of many phages may be less effective in the absence of identification of specific bacteriophage receptors because the same phages might share the same receptors and interfere with one another.37Besides the choice of bacteriophages with different bacterial cell wall receptor recognition sites in formulating a phage cocktail, the most important criterion for successful phage cocktail preparation also includes the compatibility of bacteriophages in mixtures.p = 0.04). At MOI 0.001, the two-phage cocktail combined with imipenem had significantly lysed the bacterial cell compared to the two-phage cocktail. This statistical difference might be related to the beneficial effect of bacteriophage treatment in adjunctive therapy. Our finding contradicted the study of Pacios et al., who indicated the inability of phage-imipenem combination to kill imipenem-resistant isolate harbouring OXA-245 b-lactamase.38 This divergence might be related to the use of a single lytic phage in adjunctive treatment instead of phage cocktail. Furthermore, an antibacterial effect was also observed between the phage cocktails in combination with the most effective antibiotic (tigecycline) without significant difference (p = 0.99). A number of studies have reported the positive effect of bacteriophages in combination with antibiotics.39 This synergistic activity between phage cocktail in combination with imipenem might have been due to the sensitivity of the bacterial strains to the select antibiotic after bacteriophage action. Indeed, it was reported in a previous study that phage-resistant bacterial strains are more susceptible to antibiotics and the rate of their growth is slower in comparison with wild bacterial strains.40 Our study also demonstrated the efficacy of phage cocktail in the absence of phage receptor analysis in formulating phage cocktails. Interestingly, this study pointed out the repurposing of imipenem using phage cocktail therapy, and further encourages the repurposing of the efficacy of Food and Drug Administration-approved antibiotics for acceptance of phage therapy worldwide, especially in Africa.The adjunctive treatment (IMP2 + 2φ MA3) significantly inhibited the growth of KP20 in vitro compared to the two-phage treatment alone  against carbapenem-resistant K. pneumoniae clinical isolate in vitro.This current study revealed the presence of lytic tailed