In situ targeted base editing of bacteria in the mouse gut.

Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health1. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells2, we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a ß-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.

Bacteriophage cocktail shows no toxicity and improves the survival of Galleria mellonella infected with Klebsiella spp.

Klebsiella spp. are causative agents of healthcare-associated infections in patients who are immunocompromised and use medical devices. The antibiotic resistance crisis has led to an increase in infections caused by these bacteria, which can develop into potentially life-threatening illnesses if not treated swiftly and effectively. Thus, new treatment options for Klebsiella are urgently required. Phage therapy can offer an alternative to ineffective antibiotic treatments for antibiotic-resistant bacteria infections. The aim of the present study was to produce a safe and effective phage cocktail treatment against Klebsiella pneumoniae and Klebsiella oxytoca, both in liquid in vitro culture and an in vivo Galleria mellonella infection model. The phage cocktail was significantly more effective at killing K. pneumoniae and K. oxytoca strains compared with monophage treatments. Preliminary phage cocktail safety was demonstrated through application in the in vivo G. mellonella model: where the phage cocktail induced no toxic side effects in G. mellonella. In addition, the phage cocktail significantly improved the survival of G. mellonella when administered as a prophylactic treatment, compared with controls. In conclusion, our phage cocktail was demonstrated to be safe and effective against Klebsiella spp. in the G. mellonella infection model. This provides a strong case for future treatment for Klebsiella infections, either as an alternative or adjunct to antibiotics.IMPORTANCEKlebsiella infections are a concern in individuals who are immunocompromised and are becoming increasingly difficult to treat with antibiotics due to their drug-resistant properties. Bacteriophage is one potential alternative therapy that could be used to tackle these infections. The present study describes the design of a non-toxic phage cocktail that improved the survival of Galleria mellonella infected with Klebsiella. This phage cocktail demonstrates potential for the safe and effective treatment of Klebsiella infections, as an adjunct or alternative to antibiotics.

Extending the Host Range of Bacteriophage Particles for DNA Transduction.

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant Gram-negative pathogens.

Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.

Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization.

BACKGROUND: This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections. METHODS: KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10. RESULTS: KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family. CONCLUSION: The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.

Bacteriophage vB_kpnS-Kpn15: Unveiling its potential triumph against extended-spectrum beta-lactamase-producing Klebsiella pneumoniae - Unraveling efficacy through innovative animal alternate models.

Aim -To isolate bacteriophages targeting extended-spectrum beta-lactamase-producing K. pneumoniae and evaluate their effectiveness across diverse models, incorporating innovative alternatives in animal testing. METHODS AND RESULTS: vB_kpnS-Kpn15 was isolated from sewage sample from Thane district. It produced a clear plaques on K. pneumoniae ATCC 700603. It has a flexible, non-contractile long tail and an icosahedral head and the Siphoviridae family of viruses in the order Caudovirales matched all of its structural criteria. Sequencing of vB_kpnS-Kpn15 revealed a 48,404 bp genome. The vB_KpnS-Kpn15 genome was found to contain 50 hypothetical proteins, of which 16 were found to possess different functions. The vB_KpnS-Kpn15 was also found to possess enzymes for its DNA synthesis. It was found to be lytic for the planktonic cells of K. pneumoniae and bactericidal for up to 48 h and potentially affected established K. pneumoniae biofilms. It demonstrated a broad host range and caused lytic zones on about 46 % of K. pneumoniae multi-drug resistant strains. In an in vitro wound and burn infection model, phage vB_kpnS-Kpn15 in combination with other phages resulted in successful cell proliferation and wound healing. Based on vB_kpnS-Kpn15's lytic properties, it can be incorporated in a bacteriophage cocktail to combat ESBL strains. CONCLUSIONS: The phages isolated during this research are better candidates for phage therapy, and therefore provide new and exciting options for the successful control of antibiotic-resistant bacterial infections in the future. The utilization of animal alternative models in this study elucidates cellular proliferation and migration, underscoring its significance in screening novel drugs with potential applications in the treatment of wound and burn infections. SIGNIFICANCE AND IMPACT OF THE RESEARCH: The findings of this research have implications for the creation of innovative, promising strategies to treat ESBL K. pneumoniae infections.

The potential use of bacteriophages as antibacterial agents against Klebsiella pneumoniae.

One of the most common bacteria that cause nosocomial infections is Klebsiella pneumonia (K. pneumoniae), especially in patients who are very sick and admitted to the intensive care unit (ICU). The frequency of multi-drug-resistant Klebsiella pneumoniae (MDRKP) has dramatically increased worldwide in recent decades, posing an urgent threat to public health. The Western world's bacteriophage (phage) studies have been revitalized due to the increasing reports of antimicrobial resistance and the restricted development and discovery of new antibiotics. These factors have also spurred innovation in other scientific domains. The primary agent in phage treatment is an obligately lytic organism (called bacteriophage) that kills the corresponding bacterial host while sparing human cells and lessening the broader effects of antibiotic usage on commensal bacteria. Phage treatment is developing quickly, leading to many clinical studies and instances of life-saving medicinal use. In addition, phage treatment has a few immunological adverse effects and consequences in addition to its usefulness. Since K. pneumoniae antibiotic resistance has made treating multidrug-resistant (MDR) infections challenging, phage therapy (PT) has emerged as a novel therapeutic strategy. The effectiveness of phages has also been investigated in K. pneumoniae biofilms and animal infection models. Compared with antibiotics, PT exhibits numerous advantages, including a particular lysis spectrum, co-evolution with bacteria to avoid the emergence of phage resistance, and a higher abundance and diversity of phage resources than found in antibiotics. Moreover, phages are eliminated in the absence of a host bacterium, which makes them the only therapeutic agent that self-regulates at the sites of infection. Therefore, it is essential to pay attention to the role of PT in treating these infections. This study summarizes the state of knowledge on Klebsiella spp. phages and provides an outlook on the development of phage-based treatments that target K. pneumoniae in clinical trials.

Characterization of a novel phage against multidrug-resistant Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) poses a significant challenge in global healthcare, underscoring the urgency for innovative therapeutic approaches. Phage therapy emerges as a promising strategy amidst rising antibiotic resistance, emphasizing the crucial need to identify and characterize effective phage resources for clinical use. In this study, we introduce a novel lytic phage, RCIP0100, distinguished by its classification into the Chaoyangvirus genus and Fjlabviridae family based on International Committee on Taxonomy of Viruses (ICTV) criteria due to low genetic similarity to known phage families. Our findings demonstrate that RCIP0100 exhibits broad lytic activity against 15 out of 27 tested MDR-KP strains, including diverse profiles such as carbapenem-resistant K. pneumoniae (CR-KP). This positions phage RCIP0100 as a promising candidate for phage therapy. Strains resistant to RCIP0100 also showed increased susceptibility to various antibiotics, implying the potential for synergistic use of RCIP0100 and antibiotics as a strategic countermeasure against MDR-KP.

Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection.

Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection.

Antimicrobial resistance: use of phage therapy in the management of resistant infections.

The emergence and increase in antimicrobial resistance (AMR) is now widely recognized as a major public health challenge. Traditional antimicrobial drugs are becoming increasingly ineffective, while the development of new antibiotics is waning. As a result, alternative treatments for infections are garnering increased interest. Among these alternatives, bacteriophages, also known as phages, are gaining renewed attention and are reported to offer a promising solution to alleviate the burden of bacterial infections. This review discusses the current successes of phage therapy (PT) against multidrug-resistant organisms (MDROs), such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter spp. The review also compares the efficacy of PT with that of chemical antibiotics, reporting on its benefits and limitations, while highlighting its impact on the human gut microbiome and immune system. Despite its potential, phage therapy is reported to face challenges such as the narrow antibacterial range, the complexity of developing phage cocktails, and the need for precise dosing and duration protocols. Nevertheless, continued research, improved regulatory frameworks, and increased public awareness are essential to realize its full potential and integration into standard medical practice, paving the way for innovative treatments that can effectively manage infections in an era of rising antimicrobial resistance.

Proteomic analysis of carbapenem-resistant Klebsiella pneumoniae outer membrane vesicles under the action of phages combined with tigecycline.

BACKGROUND: Klebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear. METHODS: To isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated blaNDM-1 and blaKPC-2 genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis. RESULTS: K. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group. CONCLUSIONS: This study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.

Biological and genomic characteristics of three novel bacteriophages and a phage-plasmid of Klebsiella pneumoniae.

Bacteriophages have emerged as promising candidates for the treatment of difficult-to-treat bacterial infections. The aim of this study is to isolate and characterize phages infecting carbapenem-resistant and extended-spectrum beta-lactamase producer Klebsiella pneumoniae isolates. Water samples were taken for the isolation of bacteriophages. One-step growth curve, the optimal multiplicity of infection (MOI), thermal and pH stabilities, transmission electron microscopy and whole-genome sequencing of phages were studied. Four phages were isolated and named Klebsiella phage Kpn02, Kpn17, Kpn74, and Kpn13. The optimal MOI and latent periods of phage Kpn02, Kpn17, Kpn74, and Kpn13 were 10, 1, 0.001, and 100 PFU/CFU and 20, 10, 20, and 30 min, respectively. Burst sizes ranged from 811 to 2363. No known antibiotic resistance and virulence genes were identified. No tRNAs were detected except Klebsiella phage Kpn02 which encodes 24 tRNAs. Interestingly, Klebsiella phage Kpn74 was predicted to be a lysogenic phage whose prophage is a linear plasmid molecule with covalently closed ends. Of the Klebsiella-infecting phages presented in current study, virulent phages suggest that they may represent candidate therapeutic agents against MDR K. pneumoniae, based on short latent period, high burst sizes and no known antibiotic resistance and virulence genes in their genomes.

In Vitro and In Vivo Assessments of Newly Isolated N4-like Bacteriophage against ST45 K62 Capsular-Type Carbapenem-Resistant Klebsiella pneumoniae: vB_kpnP_KPYAP-1.

The rise of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a significant global challenge in clinical and healthcare settings, severely limiting treatment options. This study aimed to utilize a bacteriophage as an alternative therapy against carbapenem-resistant K. pneumoniae. A novel lytic N4-like Klebsiella phage, vB_kpnP_KPYAP-1 (KPYAP-1), was isolated from sewage. It demonstrated efficacy against the K62 serotype polysaccharide capsule of blaOXA-48-producing K. pneumoniae. KPYAP-1 forms small, clear plaques, has a latent period of 20 min, and reaches a growth plateau at 35 min, with a burst size of 473 plaque-forming units (PFUs) per infected cell. Phylogenetic analysis places KPYAP-1 in the Schitoviridae family, Enquatrovirinae subfamily, and Kaypoctavirus genus. KPYAP-1 employs an N4-like direct terminal repeat mechanism for genome packaging and encodes a large virion-encapsulated RNA polymerase. It lacks integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and toxins, ensuring its safety for therapeutic use. Comparative genome analysis revealed that the KPYAP-1 genome is most similar to the KP8 genome, yet differs in tail fiber protein, indicating variations in host recognition. In a zebrafish infection model, KPYAP-1 significantly improved the survival rate of infected fish by 92% at a multiplicity of infection (MOI) of 10, demonstrating its potential for in vivo treatment. These results highlight KPYAP-1 as a promising candidate for developing phage-based therapies targeting carbapenemase-producing K. pneumoniae.

[The effect of wza gene deletion in Klebsiella pneumoniae on capsule formation and bacteriophage sensitivity].

Objective: To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods: The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δwza were detected by measuring the optical density OD600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δwza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δwza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δwza mutant strain. Results: The PCR results revealed that the Δwza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δwza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δwza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δwza mutant strain (45.963±2.795) µg/ml compared with wild-type strain W14 (138.800±5.201) µg/ml. There was a statistical difference between the two groups (t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion: Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections. OBJECTIVE: In this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically. METHODS: One hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at -80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst's latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk-v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student's t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05. RESULTS: Phage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3-11) and temperature (45-65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome. Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1). CONCLUSION: The broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

Isolation and characterization of lytic phage TUN1 specific for Klebsiella pneumoniae K64 clinical isolates from Tunisia.

BACKGROUND: Multidrug-resistant Klebsiella pneumoniae spp. (kp) are emerging agents of severe infections of the respiratory, urinary tract and wounds that can progress to fatal septicemia. The use of bacteriophages is currently being considered as an effective alternative or adjuvant to antibiotic therapy. RESULTS: In this study, we report capsule (K)-typing of 163 carbapenem-resistant Kp (CRKP) isolated 2014-2018 at the Military Hospital of Instruction of Tunis (MHT), Tunisia, by partial amplification and sequencing of the Kp wzi gene. The most prevalent K-type overall was K64 with 50.3% followed by K17 and K27 (22.7 and 11.0%, respectively). K64 Kp strains were most common and associated with increased case/fatality rates, especially at the intensive care unit (ICU). Using a K64 Kp strain we isolated and characterized a lytic Kp phage, vB_KpP_TUN1 (phage TUN1), from wastewater samples of the ICU at the MHT. TUN1 belongs to the Autographiviridae family and specifically digests K64 Kp capsules most probably via a depolymerase encoded by gp47. Furthermore, we successfully assembled phage TUN1 in a non-replicative host (E. coli) raising the possibility of in vitro assembly in the absence of live bacterial hosts. We propose that phage TUN1 is a promising candidate to be used as an adjuvant or an alternative to antibiotic therapy in CRKP infections, facilitating regulatory approval of phage therapy. CONCLUSIONS: K64, K17 and K27 are the most common wzi capsule types in this geographical location in Northern Africa. The lytic phage TUN1 efficiently lyses K64 Kp strains associated with increased case/fatality rates at body temperature. Together with its ability to be rescued in a non-replicative host these features enhance the utility of this phage as an antibacterial agent.

Fine capsule variation affects bacteriophage susceptibility in Klebsiella pneumoniae ST258.

Multidrug resistant (MDR) carbapenemase-producing (CP) Klebsiella pneumoniae, belonging to clonal group CG258, is capable of causing severe disease in humans and is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as therapeutic alternatives to antibiotics. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the genomes of 18 Kumoniae target strains and tested them against novel lytic bacteriophages (n = 65). The genomes of K pneumoniae carrying blaNDM and blaKPC were sequenced and CG258 isolates selected for bacteriophage susceptibility testing. The local K pneumoniae CG258 population was dominated by sequence type ST258 clade 1 (86%) with variations in capsular locus (cps) and prophage content. CG258-specific bacteriophages primarily targeted the capsule, but successful infection is also likely blocked in some by immunity conferred by existing prophages. Five tailed bacteriophages against K pneumoniae ST258 clade 1 were selected for further characterization. Our findings show that effective control of K pneumoniae CG258 with bacteriophage will require mixes of diverse lytic viruses targeting relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic bacteriophage candidates against this serious pathogen.

Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation.

BACKGROUND: Nowadays, hundreds of thousands of deaths per year are caused by antibiotic resistant nosocomial infections and the prognosis for future years is much worse, as evidenced by modern research. Bacteria of the Klebsiella genus are one of the main pathogens that cause nosocomial infections. Among the many antimicrobials offered to replace or supplement traditional antibiotics, bacteriophages are promising candidates. METHODS: This article presents microbiological, physicochemical and genomic characterization of 4 virulent bacteriophages belonging to Siphoviridae, Myoviridae and Podoviridae families. Phages were studied by electron microscopy; their host range, lytic activity, adsorption rate, burst size, latent period, frequency of phage-resistant forms generation, lysis dynamics and sensitivity of phage particles to temperature and pH were identified; genomes of all 4 bacteriophages were studied by restriction digestion and complete genome sequence. RESULTS: Studied phages showed wide host range and high stability at different temperature and pH values. In contrast with single phages, a cocktail of bacteriophages lysed all studied bacterial strains, moreover, no cases of the emergence of phage-resistant bacterial colonies were detected. Genomic data proved that isolated viruses do not carry antibiotic resistance, virulence or lysogenic genes. Three out of four bacteriophages encode polysaccharide depolymerases, which are involved in the degradation of biofilms and capsules. CONCLUSIONS: The bacteriophages studied in this work are promising for further in vivo studies and might be used in phage therapy as part of a complex therapeutic and prophylactic phage preparation. The conducted studies showed that the complex preparation is more effective than individual phages. The use of the complex phage cocktail allows to extend the lytic spectrum, and significantly reduces the possibility of phage-resistant forms generation.

Identification of a phage-derived depolymerase specific for KL64 capsule of Klebsiella pneumoniae and its anti-biofilm effect.

The increasing prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to global health. Phages and phage-derived enzymes gained increasing attention for controling CRKP infections. In this study, a lytic phage P510 infecting KL64 type K. pneumoniae was isolated and characterized. Whole genome analysis and electron microscopy analysis showed that phage P510 belonged to genus Przondovirus, family Autographiviridae, the order Caudovirales. The tail fiber protein of the phage was predicted to encode capsule depolymerase. Further analysis demonstrated that recombinant depolymerase P510dep had polysaccharide-degrading activity against KL64-types capsule of K. pneumoniae, and its lysis spectrum matched to host range of phage P510. We also demonstrated that the recombinant depolymerase was able to significantly inhibit biofilm formation. The discovery of the phage-derived depolymerase lays the foundation for controlling the spread of CRKPs.

Bacteriophage therapy for inhibition of multi drug-resistant uropathogenic bacteria: a narrative review.

Multi-Drug Resistant (MDR) uropathogenic bacteria have increased in number in recent years and the development of new treatment options for the corresponding infections has become a major challenge in the field of medicine. In this respect, recent studies have proposed bacteriophage (phage) therapy as a potential alternative against MDR Urinary Tract Infections (UTI) because the resistance mechanism of phages differs from that of antibiotics and few side effects have been reported for them. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are the most common uropathogenic bacteria against which phage therapy has been used. Phages, in addition to lysing bacterial pathogens, can prevent the formation of biofilms. Besides, by inducing or producing polysaccharide depolymerase, phages can easily penetrate into deeper layers of the biofilm and degrade it. Notably, phage therapy has shown good results in inhibiting multiple-species biofilm and this may be an efficient weapon against catheter-associated UTI. However, the narrow range of hosts limits the use of phage therapy. Therefore, the use of phage cocktail and combination therapy can form a highly attractive strategy. However, despite the positive use of these treatments, various studies have reported phage-resistant strains, indicating that phage-host interactions are more complicated and need further research. Furthermore, these investigations are limited and further clinical trials are required to make this treatment widely available for human use. This review highlights phage therapy in the context of treating UTIs and the specific considerations for this application.