Essential phage component induces resistance of bacterial community.

Despite extensive knowledge on phage resistance at bacterium level, the resistance of bacterial communities is still not well-understood. Given its ubiquity, it is essential to understand resistance at the community level. We performed quantitative investigations on the dynamics of phage infection in Klebsiella pneumoniae biofilms. We found that the biofilms quickly developed resistance and resumed growth. Instead of mutations, the resistance was caused by unassembled phage tail fibers released by the phage-lysed bacteria. The tail fibers degraded the bacterial capsule essential for infection and induced spreading of capsule loss in the biofilm, and tuning tail fiber and capsule levels altered the resistance. Latent infections sustained in the biofilm despite resistance, allowing stable phage-bacteria coexistence. Last, we showed that the resistance exposed vulnerabilities in the biofilm. Our findings indicate that phage lysate plays important roles in shaping phage-biofilm interactions and open more dimensions for the rational design of strategies to counter bacteria with phage.

Evaluation of phage-based decontamination in respiratory intensive care unit environments using ddPCR and 16S rRNA targeted sequencing techniques.

Background: Klebsiella pneumoniae is a major cause of hospital-acquired infections (HAIs), primarily spread through environmental contamination in hospitals. The effectiveness of current chemical disinfectants is waning due to emerging resistance, which poses environmental hazards and fosters new resistance in pathogens. Developing environmentally friendly and effective disinfectants against multidrug-resistant organisms is increasingly important. Methods: This study developed a bacteriophage cocktail targeting two common carbapenem-resistant Klebsiella pneumoniae (CRKP) strains, ST11 KL47 and ST11 KL64. The cocktail was used as an adjunctive disinfectant in a hospital's respiratory intensive care unit (RICU) via ultrasonic nebulization. Digital PCR was used to quantify CRKP levels post-intervention. The microbial community composition was analyzed via 16S rRNA sequencing to assess the intervention's impact on overall diversity. Results: The phage cocktail significantly reduced CRKP levels within the first 24 hours post-treatment. While a slight increase in pathogen levels was observed after 24 hours, they remained significantly lower than those treated with conventional disinfectants. 16S rRNA sequencing showed a decrease in the target pathogens' relative abundance, while overall species diversity remained stable, confirming that phages selectively target CRKP without disrupting ecological balance. Discussion: The findings highlight the efficacy and safety of phage-based biocleaners as a sustainable alternative to conventional disinfectants. Phages selectively reduce multidrug-resistant pathogens while preserving microbial diversity, making them a promising tool for infection control.

Isolation and characterization of two novel bacteriophages against carbapenem-resistant Klebsiella pneumoniae.

The increase of antibiotic-resistant bacteria has become a global health emergency and the need to explore alternative therapeutic options arises. Phage therapy uses bacteriophages to target specific bacterial strains. Phages are highly specific and can target resistant bacteria. Currently, research in this regard is focused on ensuring reliability and safety to bring this tool into clinical practice. The first step is to conduct comprehensive preclinical research. In this work, we present two novel bacteriophages vB_Kpn_F13 and vB_Kpn_F14 isolated against clinical carbapenem-resistant Klebsiella pneumoniae strains obtained from hospital sewage. Multiple studies in vitro were conducted, such as sequencing, electron microscopy, stability, host range infectivity, planktonic effect and biofilm inhibition in order to discover their ability to be used against carbapenem-resistant K. pneumoniae pathogens causing difficult-to-treat infections.

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 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.

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.

Antibacterial effect of phage cocktails and phage-antibiotic synergy against pathogenic Klebsiella pneumoniae.

The global rise of antibiotic resistance has renewed interest in phage therapy, as an alternative to antibiotics to eliminate multidrug-resistant (MDR) bacterial pathogens. However, optimizing the broad-spectrum efficacy of phage therapy remains a challenge. In this study, we addressed this issue by employing strategies to improve antimicrobial efficacy of phage therapy against MDR Klebsiella pneumoniae strains, which are notorious for their resistance to conventional antibiotics. This includes the selection of broad host range phages, optimization of phage formulation, and combinations with last-resort antibiotics. Our findings unveil that having a broad host range was a dominant trait of isolated phages, and increasing phage numbers in combination with antibiotics significantly enhanced the suppression of bacterial growth. The decreased incidence of bacterial infection was explained by a reduction in pathogen density and emergence of bacterial resistance. Furthermore, phage-antibiotic synergy (PAS) demonstrated considerable broad-spectrum antibacterial potential against different clades of clinical MDR K. pneumoniae pathogens. The improved treatment outcomes of optimized PAS were also evident in a murine model, where mice receiving optimized PAS therapy demonstrated a reduced bacterial burden in mouse tissues. Taken together, these findings offer an important development in optimizing PAS therapy and its efficacy in the elimination of MDR K. pneumoniae pathogens. IMPORTANCE: The worldwide spread of antimicrobial resistance (AMR) has posed a great challenge to global public health. Phage therapy has become a promising alternative against difficult-to-treat pathogens. One important goal of this study was to optimize the therapeutic efficiency of phage-antibiotic combinations, known as phage-antibiotic synergy (PAS). Through comprehensive analysis of the phenotypic and genotypic characteristics of a large number of CRKp-specific phages, we developed a systematic model for phage cocktail combinations. Crucially, our finding demonstrated that PAS treatments not only enhance the bactericidal effects of colistin and tigecycline against multidrug-resistant (MDR) K. pneumoniae strains in in vitro and in vivo context but also provide a robust response when antibiotics fail. Overall, the optimized PAS therapy demonstrates considerable potential in combating diverse K. pneumoniae pathogens, highlighting its relevance as a strategy to mitigate antibiotic resistance threats effectively.

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.

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.

De novo gene synthesis by an antiviral reverse transcriptase.

Defense-associated reverse transcriptase (DRT) systems perform DNA synthesis to protect bacteria against viral infection, but the identities and functions of their DNA products remain largely unknown. We show that DRT2 systems encode an unprecedented immune pathway that involves de novo gene synthesis through rolling circle reverse transcription of a noncoding RNA (ncRNA). Programmed template jumping on the ncRNA generates a concatemeric cDNA, which becomes double-stranded upon viral infection. This DNA product constitutes a protein-coding, nearly endless open reading frame (neo) gene whose expression leads to potent cell growth arrest, restricting the viral infection. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.

Targeted phage hunting to specific Klebsiella pneumoniae clinical isolates is an efficient antibiotic resistance and infection control strategy.

Klebsiella pneumoniae is one of the most threatening multi-drug-resistant pathogens today, with phage therapy being a promising alternative for personalized treatments. However, the intrinsic capsule diversity in Klebsiella spp. poses a substantial barrier to the phage host range, complicating the development of broad-spectrum phage-based treatments. Here, we have isolated and genomically characterized phages capable of infecting each of the acquired 77 reference serotypes of Klebsiella spp., including capsular types widespread among high-risk K. pneumoniae clones causing nosocomial infections. We demonstrated the possibility of isolating phages for all capsular types in the collection, revealing high capsular specificity among taxonomically related phages, in contrast to a few phages that exhibited broad-spectrum infection capabilities. To decipher the determinants of the specificity of these phages, we focused on their receptor-binding proteins, with particular attention to depolymerases. We also explored the possibility of designing a broad-spectrum phage cocktail based on phages isolated in reference capsular-type strains and determining the ability to lyse relevant clinical isolates. A combination of 12 phages capable of infecting 55% of the reference Klebsiella spp. serotypes was tested on a panel of carbapenem-resistant K. pneumoniae clinical isolates. Thirty-one percent of isolates were susceptible to the phage cocktail. However, our results suggest that in a highly variable encapsulated bacterial host, phage hunting must be directed to the specific Klebsiella isolates. This work is a step forward in the understanding of the complexity of phage-host interactions and highlights the importance of implementing precise and phage-specific strategies to treat K. pneumoniae infections worldwide.IMPORTANCEThe emergence of resistant bacteria is a serious global health problem. In the absence of effective treatments, phages are a personalized and effective therapeutic alternative. However, little is still known about phage-host interactions, which are key to implementing effective strategies. Here, we focus on the study of Klebsiella pneumoniae, a highly pathogenic encapsulated bacterium. The complexity and variability of the capsule, where in most cases phage receptors are found, make it difficult for phage-based treatments. Here, we isolated a large collection of Klebsiella phages against all the reference strains and in a cohort of clinical isolates. Our results suggest that clinical isolates represent a challenge, especially high-risk clones. Thus, we propose targeted phage hunting as an effective strategy to implement phage-derived therapies. Our results are a step forward for new phage-based strategies to control K. pneumoniae infections, highlighting the importance of understanding phage-host interactions to design personalized treatments against Klebsiella spp.

Phage-triggered reverse transcription assembles a toxic repetitive gene from a noncoding RNA.

Reverse transcription has frequently been co-opted for cellular functions and in prokaryotes is associated with protection against viral infection, but the underlying mechanisms of defense are generally unknown. Here, we show that in the DRT2 defense system, the reverse transcriptase binds a neighboring pseudoknotted noncoding RNA. Upon bacteriophage infection, a template region of this RNA is reverse transcribed into an array of tandem repeats that reconstitute a promoter and open reading frame, allowing expression of a toxic repetitive protein and an abortive infection response. Biochemical reconstitution of this activity and cryo-electron microscopy provide a molecular basis for repeat synthesis. Gene synthesis from a noncoding RNA is a previously unknown mode of genetic regulation in prokaryotes.

[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.

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.

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.

Newly isolated Drexlerviridae phage LAPAZ is physically robust and fosters eradication of Klebsiella pneumoniae in combination with meropenem.

Due to the spread of multidrug resistance there is a renewed interest in using bacteriophages (briefly: phages) for controlling bacterial pathogens. The objective of this study was the characterization of a newly isolated phage (i.e. phage LAPAZ, vB_KpnD-LAPAZ), its antimicrobial activity against multidrug resistant Klebsiella pneumoniae and potential synergistic interactions with antibiotics. LAPAZ belongs to the family Drexlerviridae (genus: Webervirus) and lysed 30 % of tested strains, whereby four distinct capsular types can be infected. The genome consists of 51,689 bp and encodes 84 ORFs. The latent period is 30 min with an average burst size of 27 PFU/cell. Long-term storage experiments show that LAPAZ is significantly more stable in wastewater compared to laboratory media. A phage titre of 90 % persists up to 30 min at 50 ˚C and entire phage loss was seen only at temperatures > 66 ˚C. Besides stability against UV-C, antibacterial activity in liquid culture medium was consistent at pH values ranging from 4 to 10. Unlike exposure to phage or antibiotic alone, synergistic interactions and a complete bacterial eradication was achieved when combining LAPAZ with meropenem. In addition, synergism with the co-presence of ciprofloxacin was observed and phage resistance emergence could be delayed. Without co-addition of the antibiotic, phage resistant mutants readily emerged and showed a mixed pattern of drug sensitivity alterations. Around 88 % became less sensitive towards ceftazidime, meropenem and gentamicin. Conversely, around 44 % showed decreased resistance levels against ciprofloxacin. Whole genome analysis of a phage-resistant mutant with a 16-fold increased sensitivity towards ciprofloxacin revealed one de novo frameshift mutation leading to a gene fusion affecting two transport proteins belonging to the major facilitator-superfamily (MFS). Apparently, this mutation compromises ciprofloxacin efflux efficiency and further studies are warranted to understand how the non-mutated protein might be involved in phage-host adsorption.

Data-Driven Engineering of Phages with Tunable Capsule Tropism for Klebsiella pneumoniae.

Klebsiella pneumoniae, a major clinical pathogen known for causing severe infections, is attracting heightened attention due to its escalating antibiotic resistance. Phages are emerging as a promising alternative to antibiotics; however, their specificity to particular hosts often restricts their use. In this study, a collection of 114 phages is obtained and subjected to analysis against 238 clinical K. pneumoniae strains, revealing a spectrum of lytic behaviors. A correlation between putative tail protein clusters and lysis patterns leads to the discovery of six receptor-binding protein (RBP) clusters that determine host capsule tropism. Significantly, RBPs with cross-capsular lysis capabilities are identified. The newly-identified RBPs provide a toolbox for customizing phages to target diverse capsular types. Building on the toolbox, the engineered phages with altered RBPs successfully shifted and broadened their host capsule tropism, setting the stage for tunable phage that offer a precise and flexible solution to combat K. pneumoniae infections.

Bacteriophage-resistant carbapenem-resistant Klebsiella pneumoniae shows reduced antibiotic resistance and virulence.

Phage therapy has shown great promise in the treatment of bacterial infections. However, the effectiveness of phage therapy is compromised by the inevitable emergence of phage-resistant strains. In this study, a phage-resistant carbapenem-resistant Klebsiella pneumoniae strain SWKP1711R, derived from parental carbapenem-resistant K. pneumoniae strain SWKP1711 was identified. The mechanism of bacteriophage resistance in SWKP1711R was investigated and the molecular determinants causing altered growth characteristics, antibiotic resistance, and virulence of SWKP1711R were tested. Compared to SWKP1711, SWKP1711R showed slower growth, smaller colonies, filamentous cells visible under the microscope, reduced production of capsular polysaccharide (CPS) and lipopolysaccharide, and reduced resistance to various antibiotics accompanied by reduced virulence. Adsorption experiments showed that phage vB_kpnM_17-11 lost the ability to adsorb onto SWKP1711R, and the adsorption receptor was identified to be bacterial surface polysaccharides. Genetic variation analysis revealed that, compared to the parental strain, SWKP1711R had only one thymine deletion at position 78 of the open reading frame of the lpcA gene, resulting in a frameshift mutation that caused alteration of the bacterial surface polysaccharide and inhibition of phage adsorption, ultimately leading to phage resistance. Transcriptome analysis and quantitative reverse transcriptase PCR revealed that genes encoding lipopolysaccharide synthesis, ompK35, blaTEM-1, and type II and Hha-TomB toxin-antitoxin systems, were all downregulated in SWKP1711R. Taken together, the evidence presented here indicates that the phenotypic alterations and phage resistance displayed by the mutant may be related to the frameshift mutation of lpcA and altered gene expression. While evolution of phage resistance remains an issue, our study suggests that the reduced antibiotic resistance and virulence of phage-resistant strain derivatives might be beneficial in alleviating the burden caused by multidrug-resistant bacteria.