# Phage therapy for Klebsiella pneumoniae: Understanding bacteria-phage interactions for therapeutic innovations

Julie Le Bris, Nathalie Chen, Adeline Supandy, Olaya Rendueles, Daria van Tyne

► To cite this version:

Julie Le Bris, Nathalie Chen, Adeline Supandy, Olaya Rendueles, Daria van Tyne. Phage therapy for Klebsiella pneumoniae: Understanding bacteria-phage interactions for therapeutic innovations. PLoS Pathogens, 2025, 21 (4), pp.e1012971. 10.1371/journal.ppat.1012971. hal-05027834

HAL Id: hal-05027834

https://hal.science/hal-05027834v1

Submitted on 13 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

REVIEW

# Phage therapy for *Klebsiella pneumoniae*: Understanding bacteria-phage interactions for therapeutic innovations

Julie Le Bris¹,²*, Nathalie Chen³*, Adeline Supandy³*, Olaya Rendueles⁴*, Daria Van Tyne⁵

1 Institut Pasteur, Université Paris Cité, CNRS UMR3525, Microbial Evolutionary Genomics, Paris, France,

2 Sorbonne Université, Collège Doctoral, Ecole Doctorale Complexité du Vivant, Paris, France, 3 Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America, 4 Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), CNRS UMR5100, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III - Paul Sabatier (UT3), Toulouse, France, 5 Center for Evolutionary Biology and Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America

* These authors contributed equally to this work.

* olaya.rendueles-garcia@univ-tlse3.fr (OR); vantyne@pitt.edu (DVT)

## Abstract

*Klebsiella pneumoniae* (KP) is a Gram-negative bacterium that commonly resides in the human gastrointestinal tract and can also act as an opportunistic pathogen and cause extra-intestinal infections. KP poses a global health threat because it causes both hospital- and community-acquired infections in immune-competent and immunocompromised hosts. These infections can be multidrug-resistant and/or hypervirulent, making KP infections difficult to treat and deadly. In the absence of effective treatments for recalcitrant KP infections, bacteriophage (phage) therapy is gaining attention as a promising alternative. In this review, we evaluate KP epidemiology and epitope diversity, discuss interactions between KP-targeting phages and their bacterial hosts from an eco-evolutionary perspective, and summarize recent efforts in phage therapy for treating KP infections. We also discuss novel approaches, including genetic engineering and machine learning, as initial steps toward developing KP-targeting phage therapy as a precision medicine approach for an emerging and dangerous pathogen.

## Introduction

*Klebsiella pneumoniae* (KP) are gut commensals that can also cause opportunistic infections. KP can be categorized into two distinct pathotypes, called classical and hypervirulent. Classical strains are associated with infections in hospitalized and immunocompromised patients, are frequently multidrug-resistant, and cause hospital-associated infections such as urinary tract infections, pneumonia, and surgical site infections (Fig 1A) [1]. In contrast, hypervirulent KP strains are usually community-acquired, infect healthy individuals, are often susceptible to antibiotics, and are able to cause highly invasive infections like liver and splenic abscesses, endophthalmitis, and meningitis (Fig 1A) [1]. While genomic studies have shown that the classical and hypervirulent KP pathotypes have followed independent evolutionary

<figure><img src="image_3.png" /></figure>

### OPEN ACCESS

**Citation:** Le Bris J, Chen N, Supandy A, Rendueles O, Van Tyne D (2025) Phage therapy for *Klebsiella pneumoniae*: Understanding bacteria-phage interactions for therapeutic innovations. PLoS Pathog 21(4): e1012971.
https://doi.org/10.1371/journal.ppat.1012971

**Editor:** Alison K Criss, University of Virginia, UNITED STATES OF AMERICA

**Published:** April 8, 2025

**Copyright:** © 2025 Le Bris et al. This is an open access article distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Funding:** This work was supported by grant T32AI138954 (to NC) and by the Department of Medicine at the University of Pittsburgh School of Medicine (to DVT). OR and JLB are supported by an ANR (Agence National de Recherche) grant [ANR 22 CE20 00181 BETinCAP] and by the Sorbonne Université, École Doctorale Complexité du Vivant, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
**Competing interests:** The authors have declared that no competing interests exist.

trajectories [2], recent studies report a worrisome convergence of multidrug-resistant and hypervirulent traits in some strains [3]. This poses a challenge, as the pace of new antimicrobial discovery and approval has not kept up with the increasing emergence and spread of high-risk KP clones [4].

To fight high-risk KP clones, therapeutic strategies have been developed to target different surface antigens, including capsular polysaccharide (CPS) and lipopolysaccharide (LPS) [5,6]. While vaccination and antibody therapies have been a primary focus in recent decades [7,8], bacteriophage (hereafter referred to as phage) therapy is gaining widespread attention as a new approach for treating KP infections [9]. Phages are bacteria-targeting viruses that can be used to treat infections. Naturally occurring phages and their bacterial hosts are constantly entangled in an evolutionary arms race, and prior work has suggested that the diversity of bacterial antigens like CPS and LPS is likely driven by phage predation [10,11]. Understanding the evolutionary dynamics between phages and bacterial antigens is critical in the deployment of phage therapy as a successful alternative therapeutic strategy for treating infections, like those caused by KP. Here, we review the diversity of surface antigens recognized by KP-targeting phages, analyze phage-host dynamics, explore the use of phage therapy to treat KP infections, and discuss how phages could be further harnessed as an alternative antimicrobial strategy.

## Klebsiella pneumoniae surface polysaccharides

KP is well known for producing a CPS, which is a major contributor to its virulence [12]. CPS is attached to the outer membrane with a lipid anchor, but may also be retained at the bacterial surface through interactions with other surface molecules such as LPS [13]. The KP CPS is composed of repeating oligosaccharide units that together form the K-antigen, which defines the K-type of a KP strain. In this review, the terms K-antigen and CPS will be used interchangeably. CPS can be composed of a variety of different carbohydrates including glucose, galactose, galactofuranose, fucose, mannose, and rhamnose [14]. These sugar moieties may be additionally modified by CPS-modifying enzymes such as acetyltransferases and pyruvyl transferases [14]. CPS composition is diverse and varies between different KP strains. Additionally, hypervirulent strains typically produce more CPS than classical strains and are often found to be hypermucoviscous [1]. The other dominant surface-associated polysaccharide, LPS, is composed of a lipid A molecule embedded in the bacterial outer membrane, a core oligosaccharide domain, and a variable O-antigen made of repeating sugar units that is used to define the O-antigen type. The O-antigen is composed of sugars such as galactose, galactofuranose, mannose, ribofuranose, and N-acetyl-D-glucosamine [15] and can be additionally modified (e.g., acetylated) to generate subvariants of O-antigens [16,17].

Typing and tracking of K-antigens and O-antigens can help identify which antigen types are more commonly associated with KP infections, and thus identify the K-types and O-types that should be prioritized for the development of new therapeutics. Historically, both CPS and LPS were typed using antisera reactive to specific and immunologically defined K-antigen or O-antigen types termed serotypes. K-antigen typing was initially performed with the Quellung capsular swelling reaction, in which typing serum is added to bacteria and then observed under a microscope for capsular swelling, which happens upon binding of type-specific antibodies to the K-antigen [18]. Several other methods were later developed to increase speed, accuracy, and efficiency, including indirect immunofluorescence, slide agglutination, double-diffusion gel precipitation (Ouchterlony test), countercurrent immunoelectrophoresis, and latex agglutination [19–21]. However, the requirement for antisera to perform these tests limited comprehensive identification, and many KP isolates were unable to be typed due to the limited number of antisera available. Additionally, cross-reactivity between different
<figure><img src="image_4.png" /><figcaption>**Fig 1.** *Klebsiella pneumoniae* infections and cell surface phage receptors. (A) Classical KP strains are typically associated with pneumonia, urinary tract infections (UTIs), and surgical site infections. Hypervirulent KP strains are associated with invasive infections such as meningitis, soft tissue infections, liver and splenic abscesses, and bacteremia. (B) The primary cell surface receptor for KP phage is the capsular polysaccharide (CPS). Other phage receptors include lipopolysaccharide (LPS), outer membrane porins (OMPs), and conjugative plasmid-encoded pili. (C) Phages bind to cell surface receptors using receptor-binding proteins on their tail fibers. These proteins can contain catalytic domains (e.g., depolymerase domains) that aid in targeting cell surface receptors. This figure was created using BioRender.</figcaption></figure>

https://doi.org/10.1371/journal.ppat.1012971.g001

serotypes made precise identification challenging. Like K-antigen typing, O-antigen typing was also traditionally performed using antisera in a tube or latex agglutination test [22], but this had the additional challenge of requiring acapsular mutants, as the K-antigen often masks the O-antigen. An enzyme-linked immunosorbent assay that did not require acapsular mutants was later developed, thereby facilitating the O-antigen typing process [23].
More recently, genetic methods based on PCR or whole genome sequencing (WGS) were
developed for both K-antigen and O-antigen typing. PCR-based methods include restriction
fragment length polymorphism analysis of the entire CPS locus to determine a “C-pattern,”
and typing based on the sequences of specific CPS biosynthesis genes such as wzi, wzc, and
wzy [24–26]. Similarly, O-antigen typing can be performed using PCR to identify specific
alleles in the wzm-wzt genes in the O-antigen locus, as well as alleles in the wbbY region
[27]. As WGS has become more accessible in recent years, genomic typing of K-antigen and
O-antigen loci is now preferred as it is more precise and comprehensive. Software tools like
Kaptive have been instrumental in the development of a standardized typing scheme for KP
isolates, even in the presence of genetic mutations and locus disruptions [28,29]. Kaptive is
regularly updated and currently enables the identification of 163 genetically defined K-antigen
types (also called K-loci) and 11 different O loci [28].

Despite the ability to assign K-antigen types from WGS data, additional work is still needed to link K-antigen locus genotypes to biochemical structures, as these cannot be predicted based solely on genomic sequence. Among the 163 different K-antigen types, only about half have a determined structure [14,30,31]. While K-antigen structures have historically been identified using gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy, a recent study used Fourier transform infrared spectroscopy to predict K-antigen structure based on similarity to known K-antigens [32]. As more structures are elucidated, it is tempting to speculate that one day new K-antigen types could be inferred from genome sequencing data alone, though biochemical validation would still be necessary to confirm polysaccharide composition.

CPS epidemiology and impact on virulence

The high diversity of KP CPS types appears to be a major determinant of the adaptive suc-
cess of KP, and the distribution of K-antigen types varies by geography [33,34]. Common
K-antigen types described in the literature include KL2, KL10, KL15, KL16, KL17, KL21,
KL22, KL24, KL25, KL28, KL30, KL54, KL62, and KL64. For this review, we surveyed 16,475
KP genomes deposited in NCBI (accessed October 16, 2023) that were collected from humans
and sampled from urine, blood, or the respiratory tract. Among these genomes, we found that
the ten most frequently observed K-loci were KL2, KL24, KL25, KL47, KL51, KL64, KL102,
KL106, KL107, and KL112. We also observed enrichment of different K-loci on different con-
tinents, confirming regional differences in prevalence (Fig 2A).

CPS is an important virulence factor for KP [12]. Experimental disruption of CPS in a
variety of KP strains has been shown to decrease virulence in mouse models of infection
compared to encapsulated parent strains [35,36]. CPS has also been shown to mediate evasion
of phagocytosis and complement-mediated lysis [37,38], and limits the inflammatory response
to KP infection [36]. Beyond simply the presence of CPS, the amount and composition of
the CPS also impact KP virulence. For example, CPS associated with hypervirulent strains
is often hypermucoviscous, and this characteristic has been demonstrated to correlate with
increased virulence [12,39]. Hypermucoviscosity is associated with specific K-antigen types,
including KL1, KL2, KL4, and KL5 [40]. Of these, KL1 and KL2 have been particularly well
characterized and shown to confer hypervirulence, defined as causing lethal infection in
mice at a low bacterial inoculum (10³ bacteria) and the ability to cause disease in otherwise
healthy humans. KL1 and KL2 antigen types have also been linked to more invasive disease
and increased resistance to phagocytosis, killing by neutrophils, and capture by liver-resident
macrophages [41–44]. The ability of CPS loci to be horizontally transferred between geneti-
cally distinct KP strains has caused some researchers to propose that KP virulence is associ-
ated with particular genetic lineages rather than specific K-antigen types [40,45]. To rigorously
<figure><img src="image_5.png" /><figcaption>**Fig 2.** K- and O- locus type diversity across different geographic regions. 16,475 KP genomes derived from human blood, urinary tract, or respiratory specimens were accessed from NCBI on October 16, 2023. Genomes were typed using Kaptive to determine K-locus types (A) and O-locus types (B) [28,29]. The most prevalent types are labeled; all other types are grouped as “Other.” K-loci and O-loci flagged as “unknown” by Kaptive are included in the “Other” category.</figcaption></figure>

https://doi.org/10.1371/journal.ppat.1012971.g002

study CPS-specific mechanisms without confounding from the genetic background, some recent studies have performed CPS swap experiments. Notably, Huang and colleagues found that “high-virulence” K-antigen types conferred the ability to evade capture by liver-resident macrophages more than “low-virulence” types [42]. However, other studies have shown that the transfer of a hypervirulent CPS to a less virulent strain does not fully recapitulate virulence [46-48]. Additionally, it is not uncommon to observe clinical KP strains with disruptions in CPS biosynthesis genes such as *wcaJ* and *wbaP* [49,50], further highlighting the complex relationship between K-antigen type and KP virulence.

## Lipopolysaccharide structure, epidemiology, and associated virulence

In contrast to the 163 K-antigen types currently known, only 11 primary O-antigen types have been described, including O1, O2a, O2ac, O2aeh, O3, O4, O5, O7, O8, O11, and O12 [16]. All except O11 have published structures [31], and four additional types (OL101, OL102, OL103, and OL104) have been genetically identified but not yet structurally characterized [33]. One final O-antigen type, O2afg, is associated with the ST258 lineage and is also considered to be
a distinct type [16,51]. Unlike K-antigens, whose structure is dictated entirely by the K-locus, O-antigen structures are determined by both the O-locus and additional genes outside the locus (*wbbY*, *gmlABD*, and *wbVW*) [28]. For example, the WbbY glycosyltransferase modifies the O1 antigen type and converts it to the O2 antigen type. Additionally, several O-antigens are structurally similar and differ by the presence of an additional subunit or by a modification such as acetylation, resulting in O-antigen subvariants [16,17,52]. Of the different O-antigen types, only a few are commonly found in clinical KP strains. Four O-antigen types, O1, O2ab, O3, and O5, accounted for 82% of strains tested in two German university hospitals and 92% of human-derived strains tested in Japan [22,53]. In line with these seroepidemiological studies, among the 16,475 KP genomes we accessed from NCBI, we found that the O1/O2v1 and O1/O2v2 loci were the most common O-loci, followed by O3b, OL101, O4, and O5. There was less regional variation in O-loci compared to K-loci, however, we did find the O4 locus to be more common in South America and the OL101 locus to be predominantly found in Asia (Fig 2B).

LPS, specifically lipid A, is strongly immunogenic and an activator of the pattern recognition receptor TLR4 [12,54]. Some KP strains are able to dampen this immunogenicity by masking LPS with specific CPS antigens [12,38]. LPS has also been implicated in the virulence of KP and contributes to bacterial resistance to complement-mediated killing by binding complement protein C3b far from the cell membrane and thus preventing the formation and insertion of the membrane attack complex [55]. The O1 serotype in particular is associated with more invasive and hypervirulent strains [28], and contributes to bacteremia in a murine model of pneumonia [56]. Finally, lipid A may also contribute to virulence by conferring protection against cationic antimicrobial peptides [12,57]. Overall, while LPS is more immunogenic than CPS, both CPS and LPS are highly abundant surface polysaccharides that contribute to KP virulence in different ways.

## Diversity of KP surface receptors from a phage therapy perspective

Antibiotics are currently the first line of treatment against KP infections. Due to the quick acquisition of antibiotic resistance by KP [58], antibiotics are no longer effective in clearing some infections and alternative approaches are required. Phage therapy is gaining attention as an alternative treatment for antibiotic-resistant bacterial infections. In contrast to broad-spectrum antimicrobials, phage therapy can specifically target pathogens and preserve beneficial bacteria in the microbiome [59,60], sparing patients from the microbial dysbiosis that can accompany antibiotic treatment.

KP-targeting phages have been isolated from a broad range of sources where KP bacteria are prevalent, including water, soil, and clinical samples [61]. The first KP phage was identified over a hundred years ago [62], and since then, more than 10,000 have been isolated [63]. KP phages belong mainly to the *Caudoviricetes* class of viruses [64], which are tailed viruses with double-stranded DNA (dsDNA) genomes ranging in size from 5 to 300+ kilobases [65]. *Caudoviricetes* phages are composed of (i) a head or capsid, which encases the dsDNA, (ii) a helical tail that injects DNA into the bacterial cytoplasm, (iii) a portal complex which links the head and tail, and (iv) tail fiber and tailspike proteins attached to the baseplate which interact with bacterial cell surface receptors to initiate infection. Until recently, phages were classified by their morphological characteristics, with tailed phages belonging to three families: myoviruses (long contractile tails), siphoviruses (long non-contractile tails), and podoviruses (short non-contractile tails) [66]. However, this classification scheme did not accurately reflect the evolutionary history of phages, and a new genome-based classification was recently proposed by the International Committee on the Taxonomy of Viruses [64]. Despite the significant
number of phages that remain to be classified, *Caudoviricetes* is now divided into 47 different families. According to this new classification, phages infecting KP are distributed across the phylogeny of dsDNA phages, with most belonging to the *Ackermannviridae*, *Autographiviridae*, *Demerecviridae*, *Drexlerviridae*, and *Straboviridae* families [67,68]. Recently, an open-source expandable phage and strain collection, known as KlebPhaCol, was initiated to collect, store, and distribute *Klebsiella* spp. bacterial strains and phages [69]. As researchers continue to study *Klebsiella*-targeting phages, their known diversity is likely to increase accordingly.

The initial steps in phage infection of bacterial cells involve the recognition of surface receptors and subsequent phage adsorption (i.e., binding) to the cell. These first steps are required for productive infection and are the primary determinants of the range of hosts that a particular phage can infect. Initiation of phage infection can happen in a single step by irreversible binding to a receptor [66], or in two steps, where initial reversible binding to a primary receptor is followed by irreversible binding to a secondary surface protein in proximity to the bacterial membrane [70]. Because the presence of a host cell receptor and irreversible binding are required for the release of phage genetic material into the cell, phages use highly variable receptor-binding proteins (RBPs) that recognize specific bacterial surface receptors to initiate infection (Fig 1C). The presence of suitable surface receptors, however, does not guarantee a successful infection because many bacteria encode genome defense systems that have evolved to protect against phage predation. In recent years, a myriad of additional anti-phage defense systems have been described [71,72]. On average, a KP genome encodes six anti-phage defense systems and they are often non-redundant [73]. These defense systems include restriction-modification [74], CRISPR-Cas [75], and abortive infection [76], among others. In the context of phage therapy, and for this review, only productive infection, whereby phages replicate and produce infectious viral progeny, will be considered. Other viral infection strategies like lysogeny or pseudolysogeny are not considered here but are reviewed elsewhere [77].

## Bacterial epitopes as receptors for *K. pneumoniae* phages

Because of its abundance and protrusion into the extracellular space, the first bacterial structure that interacts with KP phages is very likely the CPS. In some bacterial species, phage infection is hindered by CPS presence [78], which acts as a passive barrier that hides other cellular receptors. In contrast, most KP phages are dependent on CPS presence to adsorb efficiently onto host cells [67]. *In vitro* evolution experiments in which KP strains were exposed to infectious phages revealed that resistance occurred most often through mutations that resulted in a lack of CPS production [79,80]. Even when a secondary phage receptor was required and exposed on the cell surface, phage infection was still hindered in the absence of CPS [70], suggesting that it is crucial for successful infection by many KP-targeting phages (Fig 1B).

Because host tropism of KP phages appears to be mainly driven by CPS serotype [67,79] the infectivity of a given phage is likely limited to relatively few strains. The high specificity of phages for CPS has been used historically for serotype determination, as a complement to the traditional methods described above [81]. A recent study used 42 KP-targeting phages from various genera and tested their ability to infect 138 strains belonging to 59 different K-types [67]. The results showed that if a phage could infect one strain, there was a 92% chance it could also infect other strains with the same K-type. In agreement with this study, changing the K-type of a KP strain conferred resistance to phages that previously could infect the strain, and conferred susceptibility to phages to which the strain was formerly resistant [10] (Fig 3A). Other than CPS serotype, more subtle CPS variations also alter phage host range (Fig 3A). For example, insertion sequence (IS) disruption of the CPS locus or mutations in a putative acetyltransferase-encoding gene leading to reduced CPS acetylation both caused a change
A

Capsule dynamics under phage predation

<figure><img src="image_6.png" /></figure>

B

Phage tail modularity

<figure><img src="image_7.png" /><figcaption>**Fig 3.** **KP-targeting phage receptor dynamics.** (A) Because most phages are dependent on CPS presence to adsorb efficiently, pressure from phage preda- tion selects bacteria with reduced, altered, or no CPS production. Bacteria evolve to resist phages but become sensitized to other phages. (B) The arrange- ment of phage tail fiber and tailspike genes in a cassette-like organization enables rapid adaption to changes in bacterial epitopes. Phage specificity can rapidly evolve by mutating residues in the catalytic pocket (vertical evolution) or through horizontal gene transfer (HGT). HGT events between phages can result in the acquisition of enzymatic domains and the exchange of tailspike modules.</figcaption></figure>

https://doi.org/10.1371/journal.ppat.1012971.g003

in phage host range and/or reduced phage adsorption [82,83]. Thus, even if CPS is properly expressed, fine-tuning of monosaccharide linkage or chemical modification can alter phage affinity for the capsular receptor.

While CPS-targeting KP phages are prevalent, the CPS is not the only phage receptor described [67,84]. Transposon-directed insertion site sequencing (TraDIS, a powerful tool to
generate large loss-of-function mutant libraries) was used to identify alternative/secondary phage receptors essential for successful infection of KP and found that some phages required full-length LPS biosynthesis for infection [85] (Fig 1B). Similarly, mutations in the O-antigen biosynthesis genes *wecA* and *wecG* decreased phage adsorption and infection efficiency [86]. It remains unknown, however, whether LPS serves as a secondary phage receptor or if LPS is instead required for proper CPS assembly, anchoring, and/or positioning [13,85]. Because of the lower diversity of O-antigen types (Fig 2B), phages targeting LPS might be predicted to target a broader range of KP strains.

Beyond CPS and LPS, several surface-associated proteins have been identified as receptors for phage infection. These include the siderophore receptor FepA and the major porin OmpK36, commonly referred to as OmpC [70,87] (Fig 1B). Phage receptors can also be encoded on mobile genetic elements like plasmids, whereby some phages recognize specific components of the mating pair formation system to initiate adsorption [88,89] (Fig 1B). These plasmid-targeting phages can infect bacteria carrying IncF and IncP conjugative plasmids [88,89], many of which carry antibiotic-resistance genes [90,91]. A consequence of plasmid-dependent phage predation is that rather than targeting a particular bacterial strain, the phage exerts strong selective pressure against plasmid carriage and reduces dissemination throughout the population [92,93]. Given that antibiotic resistance and, more recently, virulence factors [94], are known to be encoded on conjugative plasmids, such counterselection constitutes a beneficial by-product of phage therapy by reducing virulence [95,96].

## Host recognition by receptor-binding proteins and other phage tail modules

The major determinants of phage host range are RBPs, which are commonly located at the distal part of the phage tail (Fig 1C). A typical RBP of a KP-targeting phage is composed of three main sections: (i) an N-terminal domain that anchors it to the phage baseplate or another structural element of the tail, (ii) a C-terminal domain that acts either as an autochaperone or as a noncatalytic carbohydrate-binding module [85], and (iii) a mid-section β-helical domain with enzymatic activity, such as a depolymerase domain that cleaves surface polysaccharides (Fig 1C). Identification and characterization of phage RBP depolymerases is relatively recent [97,98], and knowledge about their diversity and mechanisms of action is scarce. For instance, it was previously believed that the trimeric state of tailspikes was crucial for enzyme stability, however recent biochemical studies showed that monomeric versions of the catalytic domain were also stable and active [99].

Phage RBP-encoded depolymerases cleave glycosidic bonds of polysaccharides, including CPS, LPS, or biofilm matrix, and thus facilitate the early steps of phage infection (Fig 1B). Phage depolymerases fall into two major categories: glycoside hydrolases, including O-antigen endoglycosidases and CPS endosialidases; and lyases, including pectate and alginate lyases that specifically cleave LPS, extracellular polymeric substances, CPS, or biofilm matrix [99]. Substrate specificity is determined by the depolymerase enzymatic pocket, which recognizes precise polysaccharide residues. Thus, even subtle changes in receptor structure or composition can confer phage resistance [83]. Despite this specificity, predicting depolymerase activity from RBP gene sequences alone is challenging because single mutations in the catalytic site can strongly impact enzymatic activity [99]. Additionally, different RBPs that can degrade the same polysaccharide can exhibit low sequence similarity, suggesting the use of alternative cleavage sites or convergent evolution [100].
Within phage genomes, tail fiber, tailspike, and lyase genes tend to be clustered and arranged in a cassette-like organization (Fig 3B). This organization likely facilitates rapid RBP evolution to modify residues in the catalytic pocket via horizontal gene transfer and recombination [101], resulting in the acquisition of new enzymatic domains or the exchange of tail modules between phages. The modularity of RBPs is predicted to enhance phage adaptability through rapid modification to expand the functional repertoire (Fig 3B), i.e. host range, thereby increasing phage fitness. A recent model proposed that anchor and enzymatic domains of RBPs could function as interchangeable building blocks [102], facilitating extensive mosaicism (Fig 3B) [103]. Such domain shuffling in RBPs appears to occur despite taxonomic and ecological barriers. The potential for varied combinations of RBPs appears to only be limited by the constraints posed by the virion assembly process. Most phages carry one or two RBPs with different depolymerase domains, thereby restricting their host range to only one or a few KP serotypes [104]. However, some phages carry multiple depolymerases targeting different K-types [84], such as the broad host range phage ΦK64-1 which encodes up to eleven depolymerases [100]. The fitness advantage of such generalist phages is strongly influenced by the ecological conditions and the severity of trade-offs, which can fluctuate over extended periods of co-evolution [105,106]. Indeed, a study in *Escherichia coli* showed that generalist phages with broad host ranges exhibited higher fitness compared to specialist phages with narrower host ranges, despite their slower adaptation rate [105]. This remains to be addressed in KP. Taken together, RBPs play a critical role in phage infection, and understanding how they interact with bacterial surface receptors can yield important new insights for phage therapy.

**Bacteria-phage dynamics: Evolving to escape from one another**

Phages and bacteria are engaged in a co-evolutionary battle, with bacteria trying to resist phage infection and phages trying to infect their hosts more efficiently. While bacterial surface receptors and other defenses (i.e., anti-phage defense systems) have evolved to limit phage attacks, phages also diversify their targets through module shuffling and acquisition of new mechanisms to overcome bacterial defenses. There are two main models to explain phage-bacteria coevolution: “arms-race” dynamics and fluctuating selection dynamics [107] (Fig 4A, 4B). In the arms-race model, the continuous adaptation of both phage and bacteria leads to the accumulation of bacterial resistances and new phage infectivities (Fig 4A). Under this model, genotypes are replaced by successive selective sweeps that lead to phages with increased host ranges and bacteria with a large repertoire of phage resistance mechanisms. Evolved bacteria remain resistant to phages with ancestral traits, and evolved phages can still infect ancestral bacteria. Because of this, arms-race dynamics would very likely result in high fitness costs, ultimately leading to population extinction of either phage or bacteria.

On the other hand, the fluctuating selection model posits that phages evolve to overcome bacterial defenses at the cost of no longer being able to infect ancestral bacteria (Fig 4B). In this model, bacteria evolve to resist new phages, but in doing so, they may become newly sensitized to phages that they were previously resistant to. This is exemplified by K-type swaps, in which K-type exchanges allow a bacterial strain to resist infection by a given phage, but also result in sensitivity to other phages to which the strain was previously resistant [10]. The fluctuating selection model implies that phages maintain a narrow host range, with large selective sweeps being rare. Consequently, this model predicts a coexistence of numerous phage and bacterial genotypes whose dynamics are driven by negative frequency-dependent selection, wherein fitness changes over time as a function of allele frequency and rare genotypes have an advantage [108].
<figure><img src="image_8.png" /><figcaption>**Fig 4.** Models of co-evolution between bacterial and phage populations. (A) In the arms-race model, continuous adaptation by bacteria and phage leads to frequent selective sweeps and accumulation of new bacterial resistances and phage infectivities. (B) In the fluctuating selection model, phages maintain a narrow host range with infrequent selective sweeps. This enables the co-existence of multiple phage and bacterial genotypes, whose frequencies are driven by negative frequency-dependent selection and where rare genotypes have a fitness advantage.</figcaption></figure>

https://doi.org/10.1371/journal.ppat.1012971.g004

## Phage therapy for KP infections: Promise and challenges

Despite its use in Eastern Europe for nearly a century, phage therapy has emerged in Western medicine in the last decade as a potentially viable treatment approach for recurrent, recalcitrant, and multidrug-resistant bacterial infections. There are over a dozen reports of recent phage therapy treatment for KP infections in humans (Table 1). Successful reports include treatment of recurrent UTIs with KP-targeting phage cocktails [109,110] and clearance of KP biofilm in a prosthetic joint infection [111]. Additionally, over 30 studies have tested the treatment efficacy of phages using animal models of infection (Table 2), with most studies showing promising results. While phage therapy is often considered a last-resort salvage therapy for patients with no other viable treatment options, these therapeutic successes underscore the high potential of phages as next-generation antimicrobials.

At the same time, the development of phage therapy for widespread use faces several challenges. First, there are several disconnects between phage studies conducted in animals and humans. While numerous animal studies have demonstrated the therapeutic efficacy of phages, these have largely focused on acute systemic infections like pneumonia and bacteremia, however, applications in humans have thus far targeted chronic infections like UTIs and joint infections (Tables 1 and 2). Extrapolating outcomes from animal studies to human patients can be complicated as chronic infections introduce additional challenges such as biofilm formation, development of phage-resistant mutants, or phage neutralization by the host immune system. These issues are not typically encountered in acute infections. Additionally, animal studies often use hypervirulent KP strains, while most patient case reports describe the treatment of classical and multidrug-resistant strains. Another challenge is the variety of
<table><caption>Table 1. KP bacteriophage therapy studies in humans.</caption><thead><tr><th>Study</th><th>Infection type</th><th>KP ST<sup>1</sup></th><th>KP K-type<sup>1</sup></th><th>Co-administered antibiotics</th><th>Phage(s) used (Family: Name)<sup>2</sup></th><th>Phage dose (PFU/ dose)</th><th>Administration route(s)</th><th>Outcome(s)</th></tr></thead><tbody><tr><td>Kuipers and colleagues (2019) [153]</td><td>UTI</td><td>ND</td><td>ND</td><td>Meropenem</td><td>NR</td><td>NR</td><td>Oral; bladder irrigation; intravesical</td><td>Successful KP eradication</td></tr><tr><td>Bao and colleagues (2020) [109]</td><td>UTI</td><td>ST11</td><td>ND</td><td>Trimethoprim-sulfamethoxazole</td><td>NR: SZ-1, SZ-2, SZ-3, SZ-6, SZ-8, Kp165, Kp166, Kp167, Kp158, Kp169, Kp152, Kp154, Kp155, Kp164, Kp6377, HD001</td><td>2.5 x 10<sup>10</sup></td><td>Bladder irrigation</td><td>Successful KP eradication</td></tr><tr><td>Corbellino and colleagues (2020) [113]</td><td>Permanent ureteral stent</td><td>ST307</td><td>ND</td><td>None</td><td>Tevenvirinae: vB_KpnM_GF</td><td>1 x 10<sup>7</sup> (oral); 1 x 10<sup>6</sup> (intrarectal)</td><td>Oral; intrarectal</td><td>Successful KP eradication</td></tr><tr><td>Rubalskii and colleagues (2020) [154]</td><td>Lung infection</td><td>ND</td><td>ND</td><td>Ceftazidime, Linezolid, Avibactam, Colistin, Meropenem, Cotrimoxazole, Tobramycin</td><td>Autographivirinae: KPV811 Tevenvirinae: KPV15</td><td>2 x 10<sup>8</sup> (inhaled); 1.8 x 10<sup>9</sup> (nasogastric)</td><td>Inhaled; nasogastric</td><td>Temporary KP eradication</td></tr><tr><td>Cano and colleagues (2021) [111]</td><td>PJI</td><td>ND</td><td>ND</td><td>Minocycline</td><td>NR: KpJH46Φ2</td><td>6.3 x 10<sup>10</sup></td><td>IV</td><td>Resolution of symptoms</td></tr><tr><td>Qin and colleagues (2021) [155]</td><td>UTI</td><td>ST15</td><td>KL131</td><td>Piperacillin-Tazobactam</td><td>Podoviridae: JD902, JD907, JD908, JD910 Myoviridae: JD905</td><td>2.5 x 10<sup>10</sup> (bladder); 5 x 10<sup>9</sup> (kidney)</td><td>Bladder/kidney irrigation</td><td>Successful KP eradication</td></tr><tr><td>Rostkowska and colleagues (2021) [156]</td><td>UTI</td><td>ND</td><td>ND</td><td>Meropenem</td><td>NR</td><td>NR</td><td>Intrarectal</td><td>UTI cleared after nephrectomy</td></tr><tr><td>Zaldastanishvili and colleagues (2021) [157]</td><td>UTI</td><td>ND</td><td>ND</td><td>Metronidazole, Polymixin B, Neomycin</td><td>NR</td><td>NR</td><td>Oral; intravaginal</td><td>Temporary KP eradication</td></tr><tr><td>Doub and colleagues (2022) [158]</td><td>PJI</td><td>ND</td><td>ND</td><td>Ertapenem</td><td>NR: KP1, KP2</td><td>1 x 10<sup>9</sup>; 2 x 10<sup>8</sup></td><td>IV</td><td>Resolution of symptoms</td></tr><tr><td>Eskenazi and colleagues (2022) [115]</td><td>Fracture infection</td><td>ST893</td><td>K20</td><td>Meropenem, Colistin</td><td>Tevenvirinae: vB_KpnM_M1</td><td>1 x 10<sup>7</sup></td><td>Local instillation via catheter</td><td>Improved wound condition</td></tr><tr><td>Federici and colleagues (2022) [159]</td><td>IBD</td><td>ND</td><td>ND</td><td>None</td><td>Demerecviridae: 1.2-3 s Autographiviridae: MCoc5c</td><td>2.8 x 10<sup>10</sup></td><td>Oral</td><td>Phage administered as Phase 1 clinical trial, no off-target dysbiosis observed</td></tr><tr><td>Le and colleagues (2023) [110]</td><td>UTI</td><td>ST307; ST3647; ST1015</td><td>KL102, K30, K21, K17</td><td>Ciprofloxacin</td><td>Tevenvirinae: Metamorpho, Mineola, pKp20</td><td>5 x 10<sup>9</sup></td><td>IV</td><td>Successful eradication of bacterial burden, partial serum neutralization</td></tr><tr><td>Li and colleagues (2023) [112]</td><td>Lung infection</td><td>ST15</td><td>KL112</td><td>Amikacin, Ceftazidime-Avibactam</td><td>Podoviridae: Kp_GWPB35, Kp_GWPA139</td><td>>5 x 10<sup>9</sup></td><td>Inhaled</td><td>Decreased bacterial load, resolution of symptoms</td></tr></tbody></table>

<footnote n="1">¹Sequence types and K-locus types were provided in the published study, or genome sequences of reported KP strain(s) were analyzed with Kleborate.</footnote>

<footnote n="2">²Phage subfamily information is reported instead of family name if available. Phage classification was reported as described in the published study and may not follow current genome-based classification schemes.</footnote>

<footnote>UTI, urinary tract infection; PJI, prosthetic joint infection; IBD, inflammatory bowel disease; ND, not determined; NR, not reported; IV, intravenous.</footnote>

<footnote>https://doi.org/10.1371/journal.ppat.1012971.t001</footnote>
<table><caption>Table 2. KP bacteriophage therapy studies in mouse infection models.</caption><thead><tr><th>Study</th><th>Infection type</th><th>KP ST<sup>1</sup></th><th>KP K-type<sup>1</sup></th><th>Phage(s) used (Family: Name)<sup>2</sup></th><th>Phage dose (PFU/ dose)</th><th>Administration route(s)</th><th>Administration timing</th></tr></thead><tbody><tr><td>Hung and colleagues (2011) [160]</td><td>Liver abscess; Bacteremia</td><td>ND</td><td>K2</td><td>Podoviridae: NK5</td><td>2 × 10<sup>5</sup>–2 × 10<sup>8</sup></td><td>Intragastric; Intraperitoneal</td><td>30 mpi; 6 hpi; 24 hpi</td></tr><tr><td>Kumari and colleagues (2011) [161]</td><td>Burn wound</td><td>ND</td><td>ND</td><td>NR: Kpn5</td><td>5 × 10<sup>9</sup></td><td>Topical</td><td>Simultaneous</td></tr><tr><td>Gu and colleagues (2012) [162]</td><td>Bacteremia</td><td>ND</td><td>K2</td><td>Myoviridae: GH-K2<br>NR: GH-K1, GH-K3</td><td>3 × 10<sup>4</sup>–3 × 10<sup>7</sup></td><td>Intraperitoneal</td><td>30 mpi</td></tr><tr><td>Cao and colleagues (2015) [163]</td><td>Pneumonia</td><td>ND</td><td>ND</td><td>Siphoviridae:1513</td><td>2 × 10<sup>7</sup>–2 × 10<sup>9</sup></td><td>Intranasal</td><td>2 hpi</td></tr><tr><td>Chadha and colleagues (2016) [164]</td><td>Burn Wound</td><td>ST66</td><td>K2</td><td>Myoviridae: Kpn1, Kpn2, Kpn3, Kpn4, Kpn5</td><td>5 × 10<sup>6</sup></td><td>Topical</td><td>7 dpi</td></tr><tr><td>Chadha and colleagues (2017) [165]</td><td>Burn Wound</td><td>ND</td><td>ND</td><td>NR: KØ1, KØ2, KØ3, KØ4, KØ5</td><td>NR</td><td>Liposome loaded; Intraperitoneal</td><td>30 mpi; 24 hpi</td></tr><tr><td>Anand and colleagues (2020) [166]</td><td>Pneumonia</td><td>ST375</td><td>K2</td><td>NR: VTCCBPA43</td><td>2 × 10<sup>9</sup></td><td>Intranasal</td><td>2 hpi</td></tr><tr><td>Horváth and colleagues (2020) [167]</td><td>Peritonitis</td><td>ST15</td><td>K24</td><td>Siphoviridae: vB_KpnS_Kp13</td><td>1.75 × 10<sup>8</sup></td><td>Intraperitoneal</td><td>10 mpi; 1 hpi; 3 hpi</td></tr><tr><td>Soleimani and colleagues (2020) [168]</td><td>Pneumonia</td><td>ND</td><td>ND</td><td>Myoviridae: vB_KpnM-Teh.1</td><td>1 × 10<sup>8</sup>–1 × 10<sup>9</sup></td><td>Intraperitoneal</td><td>Simultaneous; 24 hpi</td></tr><tr><td>Dhungana and colleagues (2021) [169]</td><td>Peritonitis</td><td>ND</td><td>ND</td><td>Podoviridae: Kp_Pokalde_002</td><td>2.4 × 10<sup>7</sup></td><td>Intraperitoneal; Oral</td><td>Simultaneous</td></tr><tr><td>Fayez and colleagues (2021) [170]</td><td>Burn Wound</td><td>ND</td><td>ND</td><td>Siphoviridae: ZCKP8</td><td>NR</td><td>Suspension or gel</td><td>7 dpi</td></tr><tr><td>Hesse and colleagues (2021) [171]</td><td>Bacteremia</td><td>ST258</td><td>KL107</td><td>Podoviridae: Pharr<br>Siphoviridae: KpNIH-2</td><td>2.5 × 10<sup>7</sup> or 5 × 10<sup>7</sup></td><td>Intraperitoneal</td><td>1 hpi; 8 hpi; 24 hpi</td></tr><tr><td>Hao and colleagues (2021) [172]</td><td>Peritonitis</td><td>ND</td><td>K47</td><td>Autographivirinae: SRD2021</td><td>1 × 10<sup>8</sup></td><td>Intragastric</td><td>12 hpi</td></tr><tr><td>Luo and colleagues (2021) [173]</td><td>Pneumonia</td><td>ND</td><td>ND</td><td>Siphoviridae: vB_Kpn_B01</td><td>2 × 10<sup>8</sup></td><td>Intraperitoneal</td><td>4 hpi</td></tr><tr><td>Shi and colleagues (2021) [174]</td><td>Bacteremia</td><td>ST11</td><td>ND</td><td>NR: kpssk3</td><td>1 × 10<sup>6</sup>–1 × 10<sup>7</sup></td><td>Intraperitoneal</td><td>3 hpi</td></tr><tr><td>Wang and colleagues (2021) [175]</td><td>Pneumonia</td><td>ND</td><td>K47</td><td>Myoviridae: vB_KpnM_P-KP2</td><td>1 × 10<sup>7</sup>–1 × 10<sup>9</sup></td><td>Intranasal</td><td>1 hpi</td></tr><tr><td>Zhang and colleagues (2021) [176]</td><td>Pneumonia</td><td>ND</td><td>K20</td><td>Autographiviridae: vB_KpnP_Bp5</td><td>2 × 10<sup>8</sup></td><td>Intraperitoneal</td><td>Simultaneous; 2 hours before infection; 2 hpi</td></tr><tr><td>Asghar and colleagues (2022) [177]</td><td>Bacteremia</td><td>ND</td><td>ND</td><td>Myoviridae: A¥L<br>Siphoviridae: A¥M</td><td>NR</td><td>Intraperitoneal</td><td>Simultaneous</td></tr><tr><td>Bai and colleagues (2022) [178]</td><td>NR</td><td>ST2237</td><td>KL19</td><td>Myoviridae: vB_kpnM_17-11</td><td>5 × 10<sup>8</sup></td><td>Injection</td><td>Simultaneous</td></tr><tr><td>Federici and colleagues (2022) [159]</td><td>GI tract colonization</td><td>ST323</td><td>KL21</td><td>Autographiviridae: Mcoc5c<br>Demerecviridae: 1.2-3s, 8M-7<br>Myoviridae: PKP-55, KP-2-5-1</td><td>1 × 10<sup>5</sup> and 1 × 10<sup>9</sup></td><td>Oral</td><td>6 hpi; 9 hpi; 12 hpi</td></tr><tr><td>Gan and colleagues (2022) [179]</td><td>Pneumonia</td><td>ST11; ST383</td><td>ND</td><td>Podoviridae: pKp11<br>Siphoviridae: pKp383</td><td>1 × 10<sup>9</sup></td><td>NR</td><td>2 hpi</td></tr><tr><td>Pu and colleagues (2022) [180]</td><td>Pneumonia</td><td>ST23</td><td>K1</td><td>Siphoviridae: BUCT541</td><td>2 × 10<sup>4</sup>–2 × 10<sup>7</sup></td><td>Nasal drip</td><td>6 hpi</td></tr><tr><td>Singh and colleagues (2022) [181]</td><td>Bacteremia</td><td>ND</td><td>ND</td><td>Siphoviridae: KpBHU4, KpBHU14<br>Tectiviridae: KpBHU7</td><td>1 × 10<sup>2</sup>–1 × 10<sup>5</sup>, 1 × 10<sup>10</sup></td><td>Intraperitoneal</td><td>Simultaneous; 6 hours before infection; 6 hpi</td></tr></tbody></table>

(Continued)
<table><caption>**Table 2.** (Continued)</caption><thead><tr><th>Study</th><th>Infection type</th><th>KP ST<sup>1</sup></th><th>KP K-type<sup>1</sup></th><th>Phage(s) used (Family: Name)<sup>2</sup></th><th>Phage dose (PFU/ dose)</th><th>Administration route(s)</th><th>Administration timing</th></tr></thead><tbody><tr><td>Volozhantsev and colleagues (2022) [182]</td><td>Soft tissue</td><td>ST86; ST493</td><td>K2</td><td>NR: KpV74</td><td>1 × 10<sup>8</sup></td><td>Intraperitoneal</td><td>1 hour before infection; 3 hpi; 24 hpi</td></tr><tr><td>Fang and colleagues (2023) [183]</td><td>Pneumonia</td><td>ST259</td><td>K54</td><td>Autographiviridae: vB_KpnA_SCNJ1-Z Siphoviridae: vB_KpnS_SCNJ1-C Myoviridae: vB_KpnM_SCNJ1-Y</td><td>1 × 10<sup>8</sup></td><td>Nasal drip</td><td>2 hpi</td></tr><tr><td>Gan and colleagues (2023) [184]</td><td>Steatohepatitis</td><td>ST1536</td><td>ND</td><td>Podoviridae: phiW14</td><td>1 × 10<sup>4</sup>–1 × 10<sup>6</sup></td><td>Intragastric</td><td>Simultaneous</td></tr><tr><td>Ichikawa and colleagues (2023) [116]</td><td>Primary sclerosing cholangitis</td><td>ST37; ST145</td><td>K80; K3</td><td>Drexlerviridae: KP13-2 Stephanstirmvirinae: KP13-16 Straboviridae: KP13MC5-1 Autographiviridae: KP13MC5-2</td><td>Oral: 1 × 10<sup>9</sup>; IV: 1 × 10<sup>8</sup></td><td>Oral; IV</td><td>Simultaneous</td></tr><tr><td>Liang and colleagues (2023) [185]</td><td>Bacteremia</td><td>ND</td><td>K54</td><td>Straboviridae: BL02</td><td>1 × 10<sup>8</sup></td><td>Intraperitoneal</td><td>1 hpi</td></tr><tr><td>Rahimi and colleagues (2023) [186]</td><td>Pneumonia</td><td>ST273</td><td>K2</td><td>Drexlerviridae: PSKP16</td><td>2 × 10<sup>7</sup></td><td>Nasal drip</td><td>30 mpi; 24 hpi</td></tr><tr><td>Tang and colleagues (2023) [125]</td><td>NR</td><td>ST86; ST23; ST489</td><td>K20; K1; K6</td><td>Slopekvirinae: FK1979</td><td>NR</td><td>NR</td><td>Simultaneous</td></tr><tr><td>Feng and colleagues (2024) [187]</td><td>Wound</td><td>ST11</td><td>KL64</td><td>Ackermannviridae: PH1/P01 Autographiviridae: P24 Drexlerviridae: P39</td><td>6 × 10<sup>7</sup></td><td>NR</td><td>30 mpi</td></tr><tr><td>Kelishomi and colleagues (2024) [188]</td><td>Burn Wound</td><td>ST782</td><td>K2</td><td>Drexlerviridae: PSKP16</td><td>1.5 × 10<sup>8</sup></td><td>Topical</td><td>2 hpi</td></tr><tr><td>Li and colleagues (2024) [189]</td><td>NR</td><td>ST307; ND</td><td>K5</td><td>Drexlerviridae: P1011</td><td>1 × 10<sup>9</sup></td><td>Intraperitoneal</td><td>2 hpi</td></tr><tr><td>Tang and colleagues (2024) [190]</td><td>Bacteremia</td><td>ST86</td><td>K2</td><td>Slopekvirinae: FK1979 NR: phiR3</td><td>FK1979: 2 × 10<sup>5</sup>, 2 × 10<sup>5</sup>–2 × 10<sup>8</sup>, 2 × 10<sup>11</sup> phiR3: NR</td><td>Intraperitoneal</td><td>2 hpi</td></tr></tbody></table>

phages used across different studies, as well as the use of single phages (i.e., monophage therapy) versus phage cocktails containing mixtures of distinct phages. Most clinical reports used phage cocktails to treat KP infections, and some suggest that this approach reduces the emergence of phage-resistant bacteria [109,110,112]. Other clinical reports have suggested that monophage therapy is sufficient to resolve KP infection [111,113]. The use of antibiotics in combination with phages in some studies also complicates the interpretation of study results and makes it challenging to determine the independent contribution of phages to infection clearance. Lastly, a lack of standardized treatment protocols and outcome measures makes it difficult to compare studies. Overall, while the available literature suggests that phage therapy has good efficacy and a favorable safety profile, well-controlled clinical trials are needed to robustly measure the broader utility of this therapeutic modality. No currently enrolling

<footnote n="1">¹Sequence types and K-locus types were provided in the published study, or genome sequences of reported KP strain(s) were analyzed with Kleborate.</footnote>

<footnote n="2">²Phage subfamily information is reported instead of family name if available. Phage classification was reported as described in the published study and may not follow current genome-based classification schemes.</footnote>

<footnote>GI, gastrointestinal; NR, not reported; ND, not determined; hpi, hours post-infection; mpi, minutes post-infection; dpi, days post-infection; IV, intravenous.</footnote>

<footnote>https://doi.org/10.1371/journal.ppat.1012971.t002</footnote>
clinical trials are focused on KP infections specifically, however, the growing interest in this field may lead to the creation of such trials in the future.

An important step in developing phage therapy is determining the phages to be used. This determination is primarily based on phage host range. It is thought that the K-type specificity of most KP phages might be a double-edged sword; narrow host range enables the phages to target specific isolates while minimizing bacterial cross-resistance, however, the diversity of KP K-types likely reduces the overall species coverage of any individual CPS-targeting phage. To circumvent this limitation, there is increasing interest in CPS-independent phages. These phages recognize the O-antigen or surface-associated proteins, which tend to be more conserved across different KP strains, thus increasing phage host range [28,84,114]. *In vitro* evolution can also be leveraged to generate more efficient phages. One method involves preadapting phages by iteratively passaging them in the presence of a target KP strain to evolve a more active phage [115]. Another method uses *in vitro* evolution to generate KP strains that are resistant to an initial phage. These are then used as bait to isolate additional phages capable of targeting the phage-resistant KP strains. These additional phages can then be used in combination with the initial phage to create a cocktail that can target both the original KP strain and anticipated phage-resistant mutants [116]. Using phages that target different bacterial receptors and have different host ranges can also reduce the occurrence of phage resistance [117], which often evolves more rapidly *in vitro* compared with resistance to small molecule antibiotics [118,119]. Currently, alongside difficulties in the identification and production of suitable phages, obtaining regulatory approval for phage therapy can cause additional time delays between a compassionate use phage therapy request and the administration of phage to patients, a process that takes a median of 170 days [59]. These delays can be further lengthened in regions with limited resources, which lack access to phage therapy and often suffer from an increased burden of antimicrobial resistance.

The emergence of bacterial resistance to phage predation is often evoked as a concerning challenge to the potential success of phage therapy. While evolved phage resistance may limit therapeutic efficacy, it can also lead to beneficial trade-offs. For example, evolved phage resistance may result in increased bacterial antibiotic susceptibility, altered susceptibility to other phages, and changes in bacterial virulence [110,117,120]. Thus, even if phage therapy cannot directly clear KP infection, it can be used to steer the bacterial population toward a more treatable phenotype. Because KP-targeting phages typically rely on the CPS for adsorption, the emergence of phage resistance frequently involves the alteration or loss of the bacterial CPS (Fig 3A), which can have variable effects [87,102]. Acapsular KP variants have higher rates of conjugation and thus greater potential to acquire multidrug resistance [10]. CPS loss can also enhance tolerance to membrane-targeting antimicrobial peptides [121] allowing bacterial regrowth even under high-dose antibiotic treatment. Furthermore, a recent report found that *in vitro* phage exposure led to the formation of KP persister cells that had a 6-log increase in survival when exposed to lethal concentrations of antibiotics [122]. These persister cells also slow the pace of bacteria-phage coevolution and selection [123], promote anti-phage defenses [124], and evade antibiotic killing, thereby enabling regrowth post-treatment. On the other hand, acapsular KP variants have decreased rates of gastrointestinal tract colonization and diminished virulence compared with encapsulated strains [112,125]. Overall, understanding trade-offs driven by phage exposure and evolution of phage resistance can inform the development of phage therapies, alone or in combination with antibiotics, that effectively leverage these trade-offs for maximal therapeutic benefit.

## Looking ahead: The future of phage therapy for KP infections

With the growing popularity of phage therapy, there is also increased interest in phage-derived strategies, such as phage enzymes with antibacterial properties. These enzymes, namely lysins and depolymerases, have shown promise in combatting bacterial infections, with little to no
adverse effects (Fig 5A) [126,127]. Lysins are phage-encoded enzymes that digest the peptidoglycan of bacterial cell walls. Studies have shown that exogenous addition of lysins exhibits antibacterial activity both *in vitro* and *in vivo* [128]. Additionally, resistance to lysins is rare, likely due to their targeting of a highly conserved region of the cell wall [129,130]. Several lysins with activity against KP have been described [131,132]. Depolymerases, on the other hand, degrade carbohydrates and exhibit high substrate specificity for CPS, LPS, or other extracellular polysaccharides. These enzymes have therapeutic potential as standalone agents, as they can degrade KP biofilms and make the bacteria more sensitive to antimicrobials or the immune system, thus promoting infection clearance [133–135]. However, the large molecular mass of depolymerases may limit their tissue penetration, and as proteins, they are likely to stimulate an immune response and might prompt the generation of neutralizing antibodies that would likely reduce their effectiveness over time. Additionally, the effectiveness of depolymerases, as with phage therapy in general, can be limited by the emergence of resistance due to modifications or variations in bacterial surface-associated polysaccharides.

Phages can also be used as gene transfer agents that can deliver pre-determined “cargo” to bacterial cells (Fig 5B). Initial efforts have focused on the delivery of CRISPR-Cas systems that

<figure><img src="image_9.png" /><figcaption>**Fig 5.** Looking ahead—The future of phage therapy for KP infections. (A) Phage-derived enzymes like lysins and depolymerases can degrade peptidoglycans and carbohydrates, including those in the bacterial CPS and KP biofilm matrix. (B) Phages could be used as gene transfer agents to deliver predetermined “cargo” to bacterial cells and use CRISPR-Cas systems to kill directly or edit the bacterial genome. (C) Extracellular Contractile Ejection Systems (eCIS) are syringe-like macromolecular systems that deliver toxins into adjacent cells. eCIS could be reprogrammed to change their specificity and/or express alternative payload molecules to combat bacterial infections. (D) Phage genome editing can be performed through a process called “recombineering”. Recombineering enables modification, reduction, or broadening of phage host range. (E) Computational tools are being developed to predict interactions between phages and potential bacterial hosts. Machine learning and modeling allow rapid identification of candidate phages for a given bacterial infection, enabling the design of highly specific and optimized phage cocktails for use in clinical settings.</figcaption></figure>

https://doi.org/10.1371/journal.ppat.1012971.g005
can either kill bacteria outright or eliminate undesirable genes from the bacterial population [136]. More recently, a phage-derived particle was used to perform *in situ* base editing of *E. coli* and KP colonizing the mouse gut [137]. Additionally, extracellular contractile injection systems (eCISs) have been described as an additional phage-derived antimicrobial system (Fig. 5C). These are syringe-like macro-molecular systems that deliver toxins into adjacent cells and appear to have evolved from bacteriophage tails [138–140]. Like phages, these systems recognize specific receptors in the target cell and subsequently release a broad range of toxins that inhibit microbial growth. Recent studies have shown that eCISs can be “reprogrammed” and engineered to deliver a variety of different payloads in a strain-specific manner. Given the large number of eCISs currently described (>1,200) [139], these systems could constitute a novel and untapped source of phage-based antimicrobial strategies for further development. The potential of these phage-derived strategies to degrade KP biofilms and sensitize bacteria to other antimicrobials are particularly attractive features, thus their characterization and further development warrants additional study.

As modern medicine becomes increasingly personalized, the development of phage therapy has prompted the use of phage genome editing (Fig 5D). These techniques have largely focused on engineering genomes through recombination (i.e., recombineering), initially through the use of phage lambda as a model system to integrate linear DNA into the viral genome [141,142]. Additional methods such as BRED (bacteriophage recombineering of electroporated DNA) have been developed to facilitate genome manipulation and precise mutation of phage genes [143,144]. The integration of the CRISPR-Cas system has proven effective in both enhancing recombination efficiency [145] and selectively editing phage genomes [146]. Furthermore, plasmids encoding lambda-red recombinase have been employed as a strategy to further increase recombination efficiency [100]. These diverse recombineering approaches represent a substantial leap forward in the field of phage genome modification and pave the way toward finer specificity of phage therapy to modulate phage host range.

An alternative approach to phage genome editing is the use of computational approaches to rapidly predict and identify suitable phages based on bacterial and phage genome sequences (Fig 5E). Two recently developed computational tools to predict interactions between phages and potential bacterial hosts, iPHoP [147] and CHERRY [148], aim to accurately predict an individual phage's host at the genus and species level, respectively. In the context of phage therapy, however, prediction of activity at the strain level is likely required. While initial studies of *in silico* prediction of KP depolymerase specificity showed some uncertainty and generated many incorrect predictions [99], a more recent *in silico* RBP protein clustering-based method accurately forecasted a majority of productive infections in KP [67]. These predictions were limited to tropism driven by CPS type, however, and did not account for alternative KP receptors or phage resistance post-adsorption. Nonetheless, it appears that adsorption factors alone could be sufficient to predict many phage-bacteria interactions [149,150]. Expansion of these tools with larger collections of phages and KP strains would be beneficial to increase their accuracy and robustness.

The implementation of machine learning and modeling approaches in experimental labs, and potentially in the clinic, also opens new possibilities for the design of effective phage-based therapeutics. For example, recently developed algorithms designed to determine optimal phage cocktails to target specific *E. coli* strains based on predicted phage-bacteria interactions could be easily adapted to KP [150]. Additionally, a model-based approach using experimental data for four different multidrug-resistant KP isolates was recently used to select optimal combinatorial phage regimes [151]. When functionally tested, the predicted regimes were able to effectively reduce bacterial loads to a pre-specified target threshold. Access to automated computational pipelines could help design optimized strategies that take into
account a large number of variables, including but not restricted to: phage RBPs, inter-phage interactions [152], the presence and expression of phage receptors on targeted pathogens, pharmacokinetics, pharmacodynamics, and patient-specific factors. We expect that the development of automated methods to predict highly specific and optimized phage cocktails will pave the way toward large-scale, precise, and personalized phage therapy.

## Conclusions

The dramatic increase in multidrug-resistant KP strains worldwide, as well as their increasing convergence with hypervirulent traits, calls for new strategies to fight these worrisome infections. The phage therapy field is booming, and the resulting enthusiasm should be harnessed to propel the field forward to develop therapeutically effective protocols for clinical applications. Open-source initiatives, community engagement, and active crosstalk between researchers and clinicians are also crucial to bring phage therapy out of the laboratory and into the clinic. Standardized procedures and testing, rational therapeutic design, and leveraging the power of predictive computational tools will all facilitate this process. Additionally, the integration of evolutionary approaches and mathematical modeling with clinically relevant observations can help increase our understanding of what will make phage therapy an effective antimicrobial strategy. We are working toward a future where we can reliably predict the evolutionary trajectories of individual bacterial hosts upon exposure to phage predators, and can harness trade-offs of phage resistance to limit bacterial virulence and potentiate the effects of antibiotics and the immune system. We hope that rationally designed phage therapies will soon be possible and that they will improve the treatment and control of KP infections around the world.

## Acknowledgments

We gratefully acknowledge Emma Mills for assistance with accessing publicly available KP genomes.

## Author contributions

**Conceptualization:** Julie Le Bris, Nathalie Chen, Adeline Supandy, Olaya Rendueles, Daria Van Tyne.

**Data curation:** Adeline Supandy, Daria Van Tyne.

**Formal analysis:** Nathalie Chen.

**Funding acquisition:** Daria Van Tyne.

**Investigation:** Nathalie Chen.

**Methodology:** Olaya Rendueles.

**Project administration:** Daria Van Tyne.

**Supervision:** Daria Van Tyne.

**Visualization:** Julie Le Bris, Nathalie Chen, Olaya Rendueles, Daria Van Tyne.

**Writing – original draft:** Julie Le Bris, Nathalie Chen, Adeline Supandy, Olaya Rendueles.

**Writing – review & editing:** Julie Le Bris, Nathalie Chen, Adeline Supandy, Olaya Rendueles, Daria Van Tyne.

## References

1. Gonzalez-Ferrer S, Peñaloza HF, Budnick JA, Bain WG, Nordstrom HR, Lee JS, et al. Finding order in the chaos: outstanding questions in *Klebsiella pneumoniae* pathogenesis. *Infect Immun.* 2021;89(4):e00693-20. https://doi.org/10.1128/IAI.00693-20 PMID: 33558323
2. Wyres KL, Wick RR, Judd LM, Froumine R, Tokolyi A, Gorrie CL, et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of *Klebsiella pneumoniae*. PLoS Genet. 2019;15(4):e1008114. https://doi.org/10.1371/journal.pgen.1008114 PMID: 30986243

3. Arcari G, Carattoli A. Global spread and evolutionary convergence of multidrug-resistant and hypervirulent *Klebsiella pneumoniae* high-risk clones. Pathog Glob Health. 2023;117(4):328–41. https://doi.org/10.1080/20477724.2022.2121362 PMID: 36089853

4. WHO. Lack of innovation set to undermine antibiotic performance and health gains [Internet]. 2022 [cited 2024 Feb 21]. Available from: https://www.who.int/news/item/22-06-2022-22-06-2022-lack-of-innovation-set-to-undermine-antibiotic-performance-and-health-gains.

5. Opoku-Temeng C, Kobayashi SD, DeLeo FR. *Klebsiella pneumoniae* capsule polysaccharide as a target for therapeutics and vaccines. Comput Struct Biotechnol J. 2019;17(1):1360–6.

6. Wantuch P, Knoot C, Robinson L, Vinogradov E, Scott N, Harding C. Heptavalent O-antigen bioconjugate vaccine exhibiting differential functional antibody responses against diverse *Klebsiella pneumoniae* isolates. J Infect Dis. 2024;2024(2):jiae097. https://doi.org/jiae097

7. Douradinha B. Exploring the journey: a comprehensive review of vaccine development against *Klebsiella pneumoniae*. Microbiol Res. 2024;287(1):127837.

8. Feldman MF, Mayer Bridwell AE, Scott NE, Vinogradov E, McKee SR, Chavez SM, et al. A promising bioconjugate vaccine against hypervirulent *Klebsiella pneumoniae*. Proc Natl Acad Sci U S A. 2019;116(37):18655–63. https://doi.org/10.1073/pnas.1907833116 PMID: 31455739

9. Herridge WP, Shibu P, O'Shea J, Brook TC, Hoyles L. Bacteriophages of *Klebsiella* spp., their diversity and potential therapeutic uses. J Med Microbiol. 2020;69(2):176–94.

10. Haudiquet M, Le Bris J, Nucci A, Bonnin R, Domingo-Calap P, Rocha E. Capsules and their traits shape phage susceptibility and plasmid conjugation efficiency. Nat Commun. 2024;15(1):2032. https://doi.org/10.1038/s41467-024-2032-1

11. Mostowy RJ, Holt KE. Diversity-generating machines: genetics of bacterial sugar-coating. Trends Microbiol. 2018;26(12):1008–21. https://doi.org/10.1016/j.tim.2018.06.006 PMID: 30037568

12. Paczosa MK, Mecsas J. *Klebsiella pneumoniae*: going on the offense with a strong defense. Microbiol Mol Biol Rev. 2016;80(3):629–61. https://doi.org/10.1128/MMBR.00078-15 PMID: 27307579

13. Singh S, Wilksch JJ, Dunstan RA, Mularski A, Wang N, Hocking D, et al. LPS O antigen plays a key role in *Klebsiella pneumoniae* capsule retention. Microbiol Spectr. 2022;10(4):e0151721. https://doi.org/10.1128/spectrum.01517-21 PMID: 35913154

14. Pan Y, Lin T, Chen C, Chen Y, Hsieh P, Hsu C. Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of *Klebsiella* spp. Sci Reports. 2015;5(1):15573. https://doi.org/10.1038/srep15573

15. Patro L, Rathinavelan T. Targeting the sugary armor of *Klebsiella* species. Front Cell Infect Microbiol. 2019;9:367. https://doi.org/10.3389/fcimb.2019.00367

16. Clarke BR, Ovchinnikova OG, Kelly SD, Williamson ML, Butler JE, Liu B, et al. Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in *Klebsiella pneumoniae*. J Biol Chem. 2018;293(13):4666–79. https://doi.org/10.1074/jbc.RA117.000646 PMID: 29602878

17. Kelly RF, Severn WB, Richards JC, Perry MB, MacLean LL, Tomás JM, et al. Structural variation in the O-specific polysaccharides of *Klebsiella pneumoniae* serotype O1 and O8 lipopolysaccharide: evidence for clonal diversity in rfb genes. Mol Microbiol. 1993;10(3):615–25. https://doi.org/10.1111/j.1365-2958.1993.tb00933.x PMID: 7526122

18. Casewell MW. Experiences in the use of commercial antisera for the capsular typing of *Klebsiella* species. J Clin Pathol. 1972;25(8):734–7. https://doi.org/10.1136/jcp.25.8.734 PMID: 4561950

19. Onokodi JK, Wauters G. Capsular typing of Klebsiellae by coagglutination and latex agglutination. J Clin Microbiol. 1981;13(4):609–12. https://doi.org/10.1128/jcm.13.4.609-612.1981 PMID: 7014611

20. Palfreyman JM. *Klebsiella* serotyping by counter-current immunoelectrophoresis. J Hyg (Lond). 1978;81(2):219–25. https://doi.org/10.1017/s0022172400025043 PMID: 701786

21. Riser E, Noone P, Poulton T. A new serotyping method for *Klebsiella* species: development of the technique. J Clin Pathol. 1976;29(4):296–304.

22. Fujita S, Matsubara F. Latex agglutination text for O serogrouping of *Klebsiella* species. Microbiol Immunol. 1984;28(6):731–4. https://doi.org/10.1111/j.1348-0421.1984.tb00728.x PMID: 6384742

23. Albertí S, Hernández-Allés S, Gil J, Reina J, Martínez-Beltrán J, Camprubí S, et al. Development of an enzyme-linked immunosorbent assay method for typing and quantitation of *Klebsiella pneumoniae* lipopolysaccharide: application to serotype O1. J Clin Microbiol. 1993;31(5):1379–81. https://doi.org/10.1128/jcm.31.5.1379-1381.1993 PMID: 8501248
24. Brisse S, Issenhuth-Jeanjean S, Grimont PAD. Molecular serotyping of *Klebsiella* species isolates by restriction of the amplified capsular antigen gene cluster. J Clin Microbiol. 2004;42(8):3388-98. https://doi.org/10.1128/JCM.42.8.3388-3398.2004 PMID: 15297473

25. Brisse S, Passet V, Haugaard A, Babosan A, Kassis-Chikhani N, Struve C. wzi gene sequencing, a rapid method for determination of capsular type for *Klebsiella* strains. J Clin Microbiol. 2013;51(12):4073-8. https://doi.org/10.1128/JCM.02073-13

26. Pan Y-J, Lin T-L, Chen Y-H, Hsu C-R, Hsieh P-F, Wu M-C, et al. Capsular types of *Klebsiella pneumoniae* revisited by wzc sequencing. PLoS One. 2013;8(12):e80670. https://doi.org/10.1371/journal.pone.0080670 PMID: 24349011

27. Fang C-T, Shih Y-J, Cheong C-M, Yi W-C. Rapid and accurate determination of lipopolysaccharide O-antigen types in *Klebsiella pneumoniae* with a novel PCR-based O-genotyping method. J Clin Microbiol. 2016;54(3):666-75. https://doi.org/10.1128/JCM.02494-15 PMID: 26719438

28. Lam MMC, Wick RR, Judd LM, Holt KE, Wyres KL. Kaptive 2.0: updated capsule and lipopolysaccharide locus typing for the *Klebsiella pneumoniae* species complex. Microb Genom. 2022;8(3):000800. https://doi.org/10.1099/mgen.0.000800 PMID: 35311639

29. Wyres KL, Wick RR, Gorrie C, Jenney A, Follador R, Thomson NR, et al. Identification of *Klebsiella* capsule synthesis loci from whole genome data. Microb Genom. 2016;2(12):e000102. https://doi.org/10.1099/mgen.0.000102 PMID: 28348840

30. Bellich B, Ravenscroft N, Rizzo R, Lagatolla C, D'Andrea M, Rossolini G. Structure of the capsular polysaccharide of the KPC-2-producing *Klebsiella pneumoniae* strain KK207-2 and assignment of the glycosyltransferases functions. Int J Biol Macromol. 2019;130:536-44.

31. Patro L, Sudhakar K, Rathinavelan T. K-PAM: a unified platform to distinguish *Klebsiella* species K- and O-antigen types, model antigen structures and identify hypervirulent strains. Scientific Reports. 2020;10(1):16732.

32. Rodrigues C, Sousa C, Lopes JA, Novais Â, Peixe L. A front line on *Klebsiella pneumoniae* Capsular polysaccharide knowledge: Fourier transform infrared spectroscopy as an accurate and fast typing tool. mSystems. 2020;5(2):e00386-19. https://doi.org/10.1128/mSystems.00386-19 PMID: 32209717

33. Follador R, Heinz E, Wyres KL, Ellington MJ, Kowarik M, Holt KE, et al. The diversity of *Klebsiella pneumoniae* surface polysaccharides. Microb Genom. 2016;2(8):e000073. https://doi.org/10.1099/mgen.0.000073 PMID: 28348868

34. Gorrie CL, Mirčeta M, Wick RR, Judd LM, Lam MMC, Gomi R, et al. Genomic dissection of *Klebsiella pneumoniae* infections in hospital patients reveals insights into an opportunistic pathogen. Nat Commun. 2022;13(1):3017. https://doi.org/10.1038/s41467-022-30717-6 PMID: 35641522

35. Lawlor MS, Hsu J, Rick PD, Miller VL. Identification of *Klebsiella pneumoniae* virulence determinants using an intranasal infection model. Mol Microbiol. 2005;58(4):1054-73. https://doi.org/10.1111/j.1365-2958.2005.04918.x PMID: 16262790

36. Yoshida K, Matsumoto T, Tateda K, Uchida K, Tsujimoto S, Yamaguchi K. Role of bacterial capsule in local and systemic inflammatory responses of mice during pulmonary infection with *Klebsiella pneumoniae*. J Med Microbiol. 2000;49(11):1003-10.

37. Clements A, Gaboriaud F, Duval JFL, Farn JL, Jenney AW, Lithgow T, et al. The major surface-associated saccharides of *Klebsiella pneumoniae* contribute to host cell association. PLoS One. 2008;3(11):e3817. https://doi.org/10.1371/journal.pone.0003817 PMID: 19043570

38. Domenico P, Salo RJ, Cross AS, Cunha BA. Polysaccharide capsule-mediated resistance to opsonophagocytosis in *Klebsiella pneumoniae*. Infect Immun. 1994;62(10):4495-9. https://doi.org/10.1128/iai.62.10.4495-4499.1994 PMID: 7927714

39. Yu WL, Ko WC, Cheng KC, Lee HC, Ke DS, Lee CC, et al. Association between rmpA and magA genes and clinical syndromes caused by *Klebsiella pneumoniae* in Taiwan. Clin Infect Dis. 2006 May 15;42(10):1351-8.

40. Brisse S, Fevre C, Passet V, Issenhuth-Jeanjean S, Tournebize R, Diancourt L, et al. Virulent clones of *Klebsiella pneumoniae*: identification and evolutionary scenario based on genomic and phenotypic characterization. PLoS One. 2009;4(3):e4982. https://doi.org/10.1371/journal.pone.0004982 PMID: 19319196

41. Fang CT, Lai SY, Yi WC, Hsueh PR, Liu KL, Chang SC. *Klebsiella pneumoniae* genotype K1: an emerging pathogen that causes septic ocular or central nervous system complications from pyogenic liver abscess. Clinical Infectious Diseases. 2007;45(3):284-93.

42. Huang X, Li X, An H, Wang J, Ding M, Wang L, et al. Capsule type defines the capability of *Klebsiella pneumoniae* in evading Kupffer cell capture in the liver. PLoS Pathog. 2022;18(8):e1010693. https://doi.org/10.1371/journal.ppat.1010693 PMID: 35914009
43. Lin JC, Chang FY, Fung CP, Xu JZ, Cheng HP, Wang JJ, et al. High prevalence of phagocytic-resistant capsular serotypes of *Klebsiella pneumoniae* in liver abscess. Microbes Infect. 2004 Nov 1;6(13):1191-8.

44. Wang L, Shen D, Wu H, Ma Y. Resistance of hypervirulent *Klebsiella pneumoniae* to both intracellular and extracellular killing of neutrophils. PLoS One. 2017;12(3):e0173638. https://doi.org/10.1371/journal.pone.0173638 PMID: 28282434

45. Haudiquet M, Buffet A, Rendueles O, Rocha EPC. Interplay between the cell envelope and mobile genetic elements shapes gene flow in populations of the nosocomial pathogen *Klebsiella pneumoniae*. PLoS Biol. 2021;19(7):e3001276. https://doi.org/10.1371/journal.pbio.3001276 PMID: 34228700

46. Kabha K, Nissimov L, Athamna A, Keisari Y, Parolis H, Parolis LA, et al. Relationships among capsular structure, phagocytosis, and mouse virulence in *Klebsiella pneumoniae*. Infect Immun. 1995;63(3):847-52. https://doi.org/10.1128/iai.63.3.847-852.1995 PMID: 7868255

47. Lin C, Chen F, Huang L, Chang J, Chen J, Tsai Y. Effect in virulence of switching conserved homologous capsular polysaccharide genes from *Klebsiella pneumoniae* serotype K1 into K20. Virulence. 2016;8(5):487-93.

48. Ofek I, Kabha K, Athamna A, Frankel G, Wozniak D, Hasty D. Genetic exchange of determinants for capsular polysaccharide biosynthesis between *Klebsiella pneumoniae* strains expressing serotypes K2 and K21a. Infection and Immunity. 1993;61(10):4208-16.

49. Bain W, Ahn B, Peñaloza H, McElheny C, Tolman N, Van Der Geest R. In vivo evolution of a *Klebsiella pneumoniae* capsule defect with *wcaJ* mutation promotes complement-mediated opsonophagocytosis during recurrent infection. J Infect Dis. 2024;2024(1):jiae003. https://doi.org/jiae003

50. Ernst CM, Braxton JR, Rodriguez-Osorio CA, Zagieboyo AP, Li L, Pironti A. Adaptive evolution of virulence and persistence in carbapenem-resistant *Klebsiella pneumoniae*. Nature Medicine. 2020;26(5):705-11.

51. Szijártó V, Guachalla L, Hartl K, Varga C, Banerjee P, Stojkovic K. Both clades of the epidemic KPC-producing *Klebsiella pneumoniae* clone ST258 share a modified galactan O-antigen type. Int J Med Microbiol. 2016;306(2):89-98.

52. Wick RR, Heinz E, Holt KE, Wyres KL. Kaptive web: user-friendly capsule and lipopolysaccharide serotype prediction for *Klebsiella Genomes*. J Clin Microbiol. 2018;56(6):e00197-18. https://doi.org/10.1128/JCM.00197-18 PMID: 29618504

53. Trautmann M, Ruhnke M, Rukavina T, Held TK, Cross AS, Marre R, et al. O-antigen seroepidemiology of *Klebsiella* clinical isolates and implications for immunoprophylaxis of *Klebsiella* infections. Clin Diagn Lab Immunol. 1997;4(5):550-5. https://doi.org/10.1128/cdli.4.5.550-555.1997 PMID: 9302204

54. Standiford L, Standiford T, Newstead M, Zeng X, Ballinger M, Kovach M. TLR4-dependent GM-CSF protects against lung injury in Gram-negative bacterial pneumonia. Am J Physiol - Lung Cell Mol Physiol. 2012;302(5):L447-54.

55. Merino S, Camprubí S, Albertí S, Benedí VJ, Tomás JM. Mechanisms of *Klebsiella pneumoniae* resistance to complement-mediated killing. Infect Immun. 1992;60(6):2529-35. https://doi.org/10.1128/iai.60.6.2529-2535.1992 PMID: 1587619

56. Shankar-Sinha S, Valencia GA, Janes BK, Rosenberg JK, Whitfield C, Bender RA, et al. The *Klebsiella pneumoniae* O antigen contributes to bacteremia and lethality during murine pneumonia. Infect Immun. 2004;72(3):1423-30. https://doi.org/10.1128/IAI.72.3.1423-1430.2004 PMID: 14977947

57. Llobet E, Martínez-Moliner V, Moranta D, Dahlström KM, Regueiro V, Tomás A, et al. Deciphering tissue-induced *Klebsiella pneumoniae* lipid A structure. Proc Natl Acad Sci U S A. 2015;112(46):E6369-78. https://doi.org/10.1073/pnas.1508820112 PMID: 26578797

58. De Oliveira D, Forde B, Kidd T, Harris P, Schembri M, Beatson S. Antimicrobial resistance in ESKAPE pathogens. Clinical Microbiology Reviews. 2020;33(3):e00181-19. https://doi.org/10.1128/CMR.00181-19

59. Aslam S, Lampley E, Wooten D, Karris M, Benson C, Strathdee S, et al. Lessons learned from the first 10 consecutive cases of intravenous bacteriophage therapy to treat multidrug-resistant bacterial infections at a single center in the United States. Open Forum Infect Dis. 2020;7(9):ofaa389. https://doi.org/10.1093/ofid/ofaa389 PMID: 33005701

60. Lamy-Besnier Q, Chaffringeon L, Lourenço M, Payne R, Trinh J, Schwartz J. Prophylactic administration of a bacteriophage cocktail is safe and effective in reducing *Salmonella enterica serovar Typhimurium* burden in vivo. Microbiol Spectr. 2021;9(1):e00497-21.

61. Thorpe HA, Booton R, Kallonen T, Gibbon MJ, Couto N, Passet V. A large-scale genomic snapshot of *Klebsiella spp.* isolates in Northern Italy reveals limited transmission between clinical and non-clinical settings. Nat Microbiol. 2022;7(12):2054-67. https://doi.org/10.1038/s41564-022-01180-0
62. Caublot P. Le bacteriophage du pneumobacille Friedlander. Compt Rend Soc Biol. n.d.;90622-3.

63. Wang R, Yang S, Liu Z, Zhang Y, Wang X, Xu Z. PhageScope: a well-annotated bacteriophage database with automatic analyses and visualizations. Nucleic Acids Res. 2024;52(D1):D756-61.

64. Turner D, Shkoporov AN, Lood C, Millard AD, Dutilh BE, Alfenas-Zerbini P, et al. Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee. Arch Virol. 2023;168(2):74. https://doi.org/10.1007/s00705-022-05694-2 PMID: 36683075

65. Gulyaeva A, Garmaeva S, Kurilshikov A, Vich Vila A, Riksen NP, Netea MG, et al. Diversity and ecol-
ogy of caudoviricetes phages with genome terminal repeats in fecal metagenomes from four Dutch
cohorts. Viruses. 2022;14(10):2305. https://doi.org/10.3390/v14102305 PMID: 36298860

66. Nobrega FL, Vlot M, de Jonge PA, Dreesens LL, Beaumont HJE, Lavigne R, et al. Targeting mech-
anisms of tailed bacteriophages. Nat Rev Microbiol. 2018;16(12):760-73. https://doi.org/10.1038/
s41579-018-0070-8 PMID: 30104690

67. Beamud B, García-González N, Gómez-Ortega M, González-Candelas F, Domingo-Calap P, Sanjuan R. Genetic determinants of host tropism in *Klebsiella phages*. Cell Rep. 2023;42(2):112048. https://doi.org/10.1016/j.celrep.2023.112048 PMID: 36753420

68. Martins WMBS, Cino J, Lenzi MH, Sands K, Portal E, Hassan B, et al. Diversity of lytic bacterio-phages against XDR *Klebsiella pneumoniae* sequence type 16 recovered from sewage samples in different parts of the world. Sci Total Environ. 2022;839:156074. https://doi.org/10.1016/j.scitotenv.2022.156074 PMID: 35623509

69. University of Southampton MIL. KlebPhaCol: an open collection of phages targeting Klebsiella spp. for your research [Internet]. figshare; 2023 [cited 2024 Feb 28]. p. 6533076 Bytes. Available from: https://figshare.com/articles/poster/KlebPhaCol_an_open_collection_of_phages_targeting_i_Klebsiella_i_spp_for_your_research/23823657/1

70. Dunstan RA, Bamert RS, Tan KS, Imbulgoda U, Barlow CK, Taiaroa G, et al. Epitopes in the capsular polysaccharide and the porin OmpK36 receptors are required for bacteriophage infection of *Klebsiella pneumoniae*. Cell Rep. 2023;42(6):112551. https://doi.org/10.1016/j.celrep.2023.112551 PMID: 37224021

71. Tesson F, Planel R, Egorov A, Georjon H, Vaysset H, Brancotte B, et al. A Comprehensive Resource for Exploring Antiphage Defense: DefenseFinder Webservice, Wiki and Databases [Internet]. Genomics; 2024 Jan [cited 2024 Feb 28]. Available from: http://biorxiv.org/lookup/doi/10.1101/2024.01.25.577194

72. Georjon H, Bernheim A. The highly diverse antiphage defence systems of bacteria. Nat Rev Microbiol. 2023;21(10):686-700. https://doi.org/10.1038/s41579-023-00934-x PMID: 37460672

73. Tesson F, Hervé A, Mordret E, Touchon M, d'Humières C, Cury J, et al. Systematic and quantitative view of the antiviral arsenal of prokaryotes. Nat Commun. 2022;13(1):2561. https://doi.org/10.1038/s41467-022-30269-9 PMID: 35538097

74. Tock MR, Dryden DTF. The biology of restriction and anti-restriction. Curr Opin Microbiol. 2005;8(4):466-72. https://doi.org/10.1016/j.mib.2005.06.003 PMID: 15979932

75. Hille F, Richter H, Wong SP, Bratovič M, Ressel S, Charpentier E. The biology of CRISPR-Cas: backward and forward. Cell. 2018;172(6):1239-59. https://doi.org/10.1016/j.cell.2017.11.032 PMID: 29522745

76. Lopatina A, Tal N, Sorek R. Abortive infection: bacterial suicide as an antiviral immune strategy. Annu Rev Virol. 2020;7(1):371-84.

77. Mäntynen S, Laanto E, Oksanen HM, Poranen MM, Díaz-Muñoz SL. Black box of phage-bacterium interactions: exploring alternative phage infection strategies. Open Biol. 2021;11(9):210188. https://doi.org/10.1098/rsob.210188 PMID: 34520699

78. Scholl D, Adhya S, Merril C. *Escherichia coli* K1's capsule is a barrier to bacteriophage T7. Appl Environ Microbiol. 2005;71(8):4872-4. https://doi.org/10.1128/AEM.71.8.4872-4874.2005 PMID: 16085886

79. de Sousa JAM, Buffet A, Haudiquet M, Rocha EPC, Rendueles O. Modular prophage interactions driven by capsule serotype select for capsule loss under phage predation. ISME J. 2020;14(12):2980-96. https://doi.org/10.1038/s41396-020-0726-z PMID: 32732904

80. Tan D, Zhang Y, Qin J, Le S, Gu J, Chen L-K, et al. A frameshift mutation in wcaJ associated with phage resistance in *Klebsiella pneumoniae*. Microorganisms. 2020;8(3):378. https://doi.org/10.3390/microorganisms8030378 PMID: 32156053

81. ADAMS MH, PARK BH. An enzyme produced by a phage-host cell system. II. The properties of the polysaccharide depolymerase. Virology. 1956;2(6):719-36. https://doi.org/10.1016/0042-6822(56)90054-x PMID: 13392519
82. Rendueles O, De Sousa J, Rocha E. Competition between lysogenic and sensitive bacteria is determined by the fitness costs of the different emerging phage-resistance strategies. eLife. 2023;12:e83479. https://doi.org/10.7554/eLife.e83479

83. Venturini C, Ben Zakour NL, Bowring B, Morales S, Cole R, Kovach Z, et al. Fine capsule variation affects bacteriophage susceptibility in *Klebsiella pneumoniae* ST258. FASEB J Off Publ Fed Am Soc Exp Biol. 2020 Aug;34(8):10801-17.

84. Lourenço M, Osbelt L, Passet V, Gravey F, Megrian D, Strowig T, et al. Phages against non-capsulated *Klebsiella pneumoniae*: broader host range, slower resistance. Microbiol Spectr. 2023;11(4):e0481222. https://doi.org/10.1128/spectrum.04812-22 PMID: 37338376

85. Dunstan R, Bamert R, Belousoff M, Short F, Barlow C, Pickard D. Mechanistic insights into the capsule-targeting depolymerase from a *Klebsiella pneumoniae* bacteriophage. Microbiol Spectr. 2021;9(1):e0102321.

86. Hao G, Yuan C, Shu R, Jia Y, Zhao S, Xie S, et al. O-antigen serves as a two-faced host factor for bacteriophage NJS1 infecting nonmucoid *Klebsiella pneumoniae*. Microb Pathog. 2021;155:104897. https://doi.org/10.1016/j.micpath.2021.104897 PMID: 33878399

87. Hesse S, Rajaure M, Wall E, Johnson J, Bliskovsky V, Gottesman S. Phage resistance in multidrug-resistant *Klebsiella pneumoniae* ST258 evolves via diverse mutations that culminate in impaired adsorption. mBio. 2020;11(1):e02530-19.

88. Parra B, Cockx B, Lutz V, Brøndsted L, Smets B, Dechesne A. Isolation and characterization of novel plasmid-dependent phages infecting bacteria carrying diverse conjugative plasmids. Microbiol Spectr. 2024;12(1):e02537-23.

89. Quinones-Olvera N, Owen SV, McCully LM, Marin MG, Rand EA, Fan AC, et al. Diverse and abundant phages exploit conjugative plasmids. Nat Commun. 2024;15(1):3197. https://doi.org/10.1038/s41467-024-47416-z PMID: 38609370

90. Hospenthal MK, Costa TRD, Waksman G. A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nat Rev Microbiol. 2017;15(6):365-79. https://doi.org/10.1038/nrmicro.2017.40 PMID: 28496159

91. León-Sampedro R, Dela Fuente J, Díaz-Agero C, Crellen T, Musicha P, Rodríguez-Beltrán J. Pervasive transmission of a carbapenem resistance plasmid in the gut microbiota of hospitalized patients. Nat Microbiol. 2021;6(5):606-16. https://doi.org/10.1038/s41564-021-00873-5

92. Colom J, Batista D, Baig A, Tang Y, Liu S, Yuan F. Sex pilus specific bacteriophage to drive bacterial population towards antibiotic sensitivity. Sci Reports. 2019;9(1):12616. https://doi.org/10.1038/s41598-019-49000-5

93. Jalasvuori M, Friman V, Nieminen A, Bamford J, Buckling A. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids. Biol Lett. 2011;7(6):902-5. https://doi.org/10.1098/rsbl.2011.0860

94. Fu L, Tang L, Wang S, Liu Q, Liu Y, Zhang Z, et al. Co-location of the blaKPC-2, blaCTX-M-65, rmtB and virulence relevant factors in an IncFII plasmid from a hypermucoviscous *Klebsiella pneumoniae* isolate. Microb Pathog. 2018;124:301-4. https://doi.org/10.1016/j.micpath.2018.08.001

95. Ojala V, Laitalainen J, Jalasvuori M. Fight evolution with evolution: plasmid-dependent phages with a wide host range prevent the spread of antibiotic resistance. Evol Appl. 2013;6(6):925-32. https://doi.org/10.1111/eva.12063

96. Penttinen R, Given C, Jalasvuori M. Indirect selection against antibiotic resistance via specialized plasmid-dependent bacteriophages. Microorganisms. 2021;9(2):280. https://doi.org/10.3390/microorganisms9020280 PMID: 33572937

97. Hsu C-R, Lin T-L, Pan Y-J, Hsieh P-F, Wang J-T. Isolation of a bacteriophage specific for a new capsular type of *Klebsiella pneumoniae* and characterization of its polysaccharide depolymerase. PLoS One. 2013;8(8):e70092. https://doi.org/10.1371/journal.pone.0070092 PMID: 23936379

98. Lin T-L, Hsieh P-F, Huang Y-T, Lee W-C, Tsai Y-T, Su P-A, et al. Isolation of a bacteriophage and its depolymerase specific for K1 capsule of *Klebsiella pneumoniae*: implication in typing and treatment. J Infect Dis. 2014;210(11):1734-44. https://doi.org/10.1093/infdis/jiu332 PMID: 25001459

99. Maciejewska B, Squeglia F, Latka A, Privitera M, Olejniczak S, Switala P, et al. *Klebsiella phage* KP34gp57 capsular depolymerase structure and function: from a serendipitous finding to the design of active mini-enzymes against K. pneumoniae. mBio. 2023;14(5):e0132923. https://doi.org/10.1128/mbio.01329-23 PMID: 37707438

100. Pan Y-J, Lin T-L, Chen C-C, Tsai Y-T, Cheng Y-H, Chen Y-Y, et al. *Klebsiella Phage* ΦK64-1 encodes multiple depolymerases for multiple host capsular types. J Virol. 2017;91(6):e02457-16. https://doi.org/10.1128/JVI.02457-16 PMID: 28077636
101. Leiman P, Molineux I. Evolution of a new enzyme activity from the same motif fold. Mol Microbiol. 2008;69(2):287-90.

102. Latka A, Lemire S, Grimon D, Dams D, Maciejewska B, Lu T. Engineering the modular receptor-binding proteins of *Klebsiella phages* switches their capsule serotype specificity. mBio. 2021;12(3):e00455-21. https://doi.org/10.1128/mbio.00455-21

103. Smug BJ, Szczepaniak K, Rocha EPC, Dunin-Horkawicz S, Mostowy RJ. Ongoing shuffling of protein fragments diversifies core viral functions linked to interactions with bacterial hosts. Nat Commun. 2023;14(1):7460. https://doi.org/10.1038/s41467-023-43236-9 PMID: 38016962

104. Latka A, Leiman P, Drulis-Kawa Z, Briers Y. Modeling the architecture of depolymerase-containing receptor binding proteins in *Klebsiella phages*. Front Microbiol. 2019;10:2649. https://doi.org/10.3389/fmicb.2019.02649

105. Sant DG, Woods LC, Barr JJ, McDonald MJ. Host diversity slows bacteriophage adaptation by selecting generalists over specialists. Nat Ecol Evol. 2021;5(3):350-9.

106. Bisesi AT, Möbius W, Nadell CD, Hansen EG, Bowden SD, Harcombe WR. Bacteriophage specificity is impacted by interactions between bacteria. mSystems. 2024;9(3):e01177-23.

107. Koskella B, Brockhurst MA. Bacteria-phage coevolution as a driver of ecological and evolutionary processes in microbial communities. FEMS Microbiol Rev. 2014;38(5):916-31. https://doi.org/10.1111/1574-6976.12072 PMID: 24617569

108. Gómez P, Buckling A. Bacteria-phage antagonistic coevolution in soil. Science. 2011;332(6025):106-9.

109. Bao J, Wu N, Zeng Y, Chen L, Li L, Yang L. Non-active antibiotic and bacteriophage synergism to successfully treat recurrent urinary tract infection caused by extensively drug-resistant *Klebsiella pneumoniae*. Emerg Microb Infect. 2020;9(1):771-4.

110. Le T, Nang S, Zhao J, Yu H, Li J, Gill J. Therapeutic potential of intravenous phage as stand-alone therapy for recurrent drug-resistant urinary tract infections. Antimicrob Agents Chemother. 2023;67(4):e00037-23. https://doi.org/10.1128/AAC.00037-23

111. Cano E, Caflisch K, Bollyky P, Van Belleghem J, Patel R, Fackler J. Phage therapy for limb-threatening prosthetic knee *Klebsiella pneumoniae* infection: case report and in vitro characterization of anti-biofilm activity. Clin Infect Dis. 2021;73(1):e144-151.

112. Li J, Yan B, He B, Li L, Zhou X, Wu N, et al. Development of phage resistance in multidrug-resistant *Klebsiella pneumoniae* is associated with reduced virulence: a case report of a personalised phage therapy. Clin Microbiol Infect. 2023;29(12):1601.e1-1601.e7. https://doi.org/10.1016/j.cmi.2023.08.022 PMID: 37652124

113. Corbellino M, Kieffer N, Kutateladze M, Balarjishvili N, Leshkasheli L, Askilashvili L, et al. Eradication of a multidrug-resistant, carbapenemase-producing *Klebsiella pneumoniae* isolate following oral and intra-rectal therapy with a custom made, lytic bacteriophage preparation. Clin Infect Dis. 2020;70(9):1998-2001.

114. Concha-Eloko R, Barberán-Martínez P, Sanjuán R, Domingo-Calap P. Broad-range capsule-dependent lytic Sugarlandvirus against *Klebsiella* sp. Microbiol Spectr. 2023;11(6):e0429822. https://doi.org/10.1128/spectrum.04298-22 PMID: 37882584

115. Eskenazi A, Lood C, Wubbolts J, Hites M, Balarjishvili N, Leshkasheli L, et al. Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant *Klebsiella pneumoniae*. Nat Commun. 2022;13(1):302. https://doi.org/10.1038/s41467-021-27656-z PMID: 35042848

116. Ichikawa M, Nakamoto N, Kredo-Russo S, Weinstock E, Weiner I, Khabra E. Bacteriophage therapy against pathological *Klebsiella pneumoniae* ameliorates the course of primary sclerosing cholangitis. Nat Commun. 2023;14(1):3261.

117. Majkowska-Skrobek G, Markwitz P, Sosnowska E, Lood C, Lavigne R, Drulis-Kawa Z. The evolutionary trade-offs in phage-resistant *Klebsiella pneumoniae* entail cross-phage sensitization and loss of multidrug resistance. Environ Microbiol. 2021;23(12):7723-40. https://doi.org/10.1111/1462-2920.15476 PMID: 33754440

118. Burmeister A, Fortier A, Roush C, Lessing A, Bender R, Barahman R. Pleiotropy complicates a trade-off between phage resistance and antibiotic resistance. Proc Natl Acad Sci U S A. 2020;117(21):11207-16.

119. Wright R, Friman V, Smith M, Brockhurst M. Resistance evolution against phage combinations depends on the timing and order of exposure. mBio. 2019;10(5):e01652-19.

120. Uddin MJ, Kim B, Dawan J, Ding T, Kim J-C, Ahn J. Assessment of antibiotic resistance in bacteriophage-insensitive *Klebsiella pneumoniae*. Microb Pathog. 2019;135:103625. https://doi.org/10.1016/j.micpath.2019.103625 PMID: 31325570
121. D'Angelo F, Rocha E, Rendueles O. The capsule increases susceptibility to last-resort poly-myxins, but not to other antibiotics, in *Klebsiella pneumoniae*. Antimicrob Agents Chemother. 2023;67(4):e00127-23.

122. Fernández-García L, Kirigo J, Huelgas-Méndez D, Benedik MJ, Tomás M, García-Contreras R, et al. Phages produce persisters. Microb Biotechnol. 2024;17(8):e14543. https://doi.org/10.1111/1751-7915.14543 PMID: 39096350

123. Schwartz DA, Shoemaker WR, Măgălie A, Weitz JS, Lennon JT. Bacteria-phage coevolution with a seed bank. ISME J. 2023;17(8):1315-25. https://doi.org/10.1038/s41396-023-01449-2 PMID: 37286738

124. Meeske A, Nakandakari-Higa S, Marraffini L. Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage. Nature. 2019;570(7760):241-5. https://doi.org/10.1038/s41586-019-1364-3

125. Tang M, Huang Z, Zhang X, Kong J, Zhou B, Han Y, et al. Phage resistance formation and fitness costs of hypervirulent *Klebsiella pneumoniae* mediated by K2 capsule-specific phage and the corresponding mechanisms. Front Microbiol. 2023;14:1156292.

126. Jun SY, Jang IJ, Yoon S, Jang K, Yu K-S, Cho JY, et al. Pharmacokinetics and tolerance of the phage endolysin-based candidate drug SAL200 after a single intravenous administration among healthy volunteers. Antimicrob Agents Chemother. 2017;61(6):e02629-16. https://doi.org/10.1128/AAC.02629-16 PMID: 28348152

127. Navarro F, Muniesa M. Phages in the human body. Front Microbiol. 2017;8:566. https://doi.org/10.3389/fmicb.2017.00566

128. Oliveira H, Melo LDR, Santos SB, Nóbrega FL, Ferreira EC, Cerca N, et al. Molecular aspects and comparative genomics of bacteriophage endolysins. J Virol. 2013;87(8):4558-70. https://doi.org/10.1128/JVI.03277-12 PMID: 23408602

129. Fischetti V. Bacteriophage endolysins: a novel anti-infective to control Gram-positive pathogens. Int J Med Microbiol. 2010;300(6):357-62.

130. Fischetti VA. Development of phage lysins as novel therapeutics: a historical perspective. Viruses. 2018;10(6):310. https://doi.org/10.3390/v10060310 PMID: 29875339

131. Antonova NP, Vasina DV, Lendel AM, Usachev EV, Makarov VV, Gintsburg AL, et al. Broad bactericidal activity of the myoviridae bacteriophage lysins LysAm24, LysECD7, and LysSi3 against Gram-negative ESKAPE pathogens. Viruses. 2019;11(3):284. https://doi.org/10.3390/v11030284 PMID: 30901901

132. Euler CW, Raz A, Hernandez A, Serrano A, Xu S, Andersson M, et al. PlyKp104, a novel phage lysin for the treatment of *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and other Gram-negative ESKAPE pathogens. Antimicrob Agents Chemother. 2023;67(5):e0151922. https://doi.org/10.1128/aac.01519-22 PMID: 37098944

133. Majkowska-Skrobek G, Łątka A, Berisio R, Maciejewska B, Squeglia F, Romano M, et al. Capsule-targeting depolymerase, derived from *Klebsiella* KP36 phage, as a tool for the development of anti-virulent strategy. Viruses. 2016;8(12):324. https://doi.org/10.3390/v8120324 PMID: 27916936

134. Sun X, Pu B, Qin J, Xiang J. Effect of a depolymerase encoded by Phage168 on a carbapenem-resistant *Klebsiella pneumoniae* and its biofilm. Pathogens. 2023;12(12):1396. https://doi.org/10.3390/pathogens12121396 PMID: 38133282

135. Zhao R, Jiang S, Ren S, Yang L, Han W, Guo Z, et al. A novel phage putative depolymerase, Depo16, has specific activity against K1 capsular-type *Klebsiella pneumoniae*. Appl Environ Microbiol. 2024;90(4):e0119723. https://doi.org/10.1128/aem.01197-23 PMID: 38551353

136. Fage C, Lemire N, Moineau S. Delivery of CRISPR-Cas systems using phage-based vectors. Curr Opin Biotechnol. 2021;68174-80. https://doi.org/10.1016/j.copbio.2020.11.012 PMID: 33360715

137. Brödel AK, Charpenay LH, Galtier M, Fuche FJ, Terrasse R, Poquet C, et al. In situ targeted base editing of bacteria in the mouse gut. Nature. 2024 Jul 10;

138. Backman T, Latorre SM, Symeonidi E, Muszyński A, Bleak E, Eads L, et al. A phage tail-like bacteriocin suppresses competitors in metapopulations of pathogenic bacteria. Science. 2024;384(6701):eado0713. https://doi.org/10.1126/science ado0713 PMID: 38870284

139. Geller AM, Pollin I, Zlotkin D, Danov A, Nachmias N, Andreopoulos WB, et al. The extracellular contractile injection system is enriched in environmental microbes and associates with numerous toxins. Nat Commun. 2021;12(1):3743. https://doi.org/10.1038/s41467-021-23777-7 PMID: 34145238

140. Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, et al. Programmable protein delivery with a bacterial contractile injection system. Nature. 2023;616(7956):357-64. https://doi.org/10.1038/s41586-023-05870-7 PMID: 36991127
141. Marinelli LJ, Hatfull GF, Piuri M. Recombineering: A powerful tool for modification of bacteriophage genomes. Bacteriophage. 2012;2(1):5–14. https://doi.org/10.4161/bact.18778 PMID: 22666652

142. Oppenheim AB, Rattray AJ, Bubunenko M, Thomason LC, Court DL. *In vivo* recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides. Virology. 2004;319(2):185–9. https://doi.org/10.1016/j.virol.2003.11.007 PMID: 14980479

143. Fehér T, Karcagi I, Blattner FR, Pósfai G. Bacteriophage recombineering in the lytic state using the lambda red recombinases. Microb Biotechnol. 2012;5(4):466–76. https://doi.org/10.1111/j.1751-7915.2011.00292.x PMID: 21910851

144. Marinelli LJ, Piuri M, Swigonová Z, Balachandran A, Oldfield LM, van Kessel JC, et al. BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One. 2008;3(12):e3957. https://doi.org/10.1371/journal.pone.0003957 PMID: 19088849

145. Martel B, Moineau S. CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages. Nucleic Acids Res. 2014;42(14):9504–13. https://doi.org/10.1093/nar/gku628 PMID: 25063295

146. Kiro R, Shitrit D, Qimron U. Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system. RNA Biol. 2014;11(1):42–4. https://doi.org/10.4161/rna.27766 PMID: 24457913

147. Roux S, Camargo AP, Coutinho FH, Dabdoub SM, Dutilh BE, Nayfach S, et al. iPHoP: An integrated machine learning framework to maximize host prediction for metagenome-derived viruses of archaea and bacteria. PLoS Biol. 2023;21(4):e3002083. https://doi.org/10.1371/journal.pbio.3002083 PMID: 37083735

148. Shang J, Sun Y. CHERRY: a Computational metHod for accuratE pRediction of virus-pRokarYotic interactions using a graph encoder-decoder model. Brief Bioinform. 2022;23(5):bbac182. https://doi.org/10.1093/bib/bbac182 PMID: 35595715

149. Boeckaerts D, Stock M, Ferriol-González C, Oteo-Iglesias J, Sanjuán R, Domingo-Calap P, et al. Prediction of Klebsiella phage-host specificity at the strain level. Nat Commun. 2024;15(1):4355. https://doi.org/10.1038/s41467-024-48675-6 PMID: 38778023

150. Gaborieau B, Vaysset H, Tesson F, Charachon I, Dib N, Bernier J, et al. Prediction of strain level phage-host interactions across the Escherichia genus using only genomic information. Nat Microbiol. 2024;9(11):2847–61. https://doi.org/10.1038/s41564-024-01832-5 PMID: 39482383

151. Smith NM, Nguyen TD, Lodise TP, Chen L, Kaur JN, Klem JF, et al. Machine Learning-Led Optimization of Combination Therapy: Confronting the Public Health Threat of Extensively Drug Resistant Gram-Negative Bacteria. Clin Pharmacol Ther. 2024;115(4):896–905. https://doi.org/10.1002/cpt.3134 PMID: 38062797

152. Leeks A, Bono L, Ampolini E, Souza L, Höfler T, Mattson C, et al. Open questions in the social lives of viruses. Journal of Evolutionary Biology. 2023;36(11):1551–67.

153. Kuipers S, Ruth MM, Mientjes M, de Sévaux RGL, van Ingen J. A Dutch Case Report of Successful Treatment of Chronic Relapsing Urinary Tract Infection with Bacteriophages in a Renal Transplant Patient. Antimicrob Agents Chemother. 2019;64(1):e01281-19. https://doi.org/10.1128/AAC.01281-19 PMID: 31611357

154. Rubalskii E, Ruemke S, Salmoukas C, Boyle EC, Warnecke G, Tudorache I, et al. Bacteriophage Therapy for Critical Infections Related to Cardiothoracic Surgery. Antibiotics (Basel). 2020;9(5):232. https://doi.org/10.3390/antibiotics9050232 PMID: 32380707

155. Qin J, Wu N, Bao J, Shi X, Ou H, Ye S. Heterogeneous Klebsiella pneumoniae co-infections complicate personalized bacteriophage therapy. Front Cell Infect Microbiol. 2021;10(1):608402. https://doi.org/10.3389/fcimb.2021.608402

156. Rostkowska OM, Międzybrodzki R, Miszewska-Szyszkowska D, Górski A, Durlik M. Treatment of recurrent urinary tract infections in a 60-year-old kidney transplant recipient. The use of phage therapy. Transpl Infect Dis. 2021;23(1):e13391. https://doi.org/10.1111/tid.13391 PMID: 32599666

157. Zaldastanishvili E, Leshkasheli L, Dadiani M, Nadareishvili L, Askilashvili L, Kvatadze N, et al. Phage therapy experience at the Eliava Phage Therapy Center: three cases of bacterial persistence. Viruses. 2021;13(10):1901. https://doi.org/10.3390/v13101901 PMID: 34696331

158. Doub JB, Shishido A, Srikumaran U, Haskoor J, Tran-Nguyen P, Lee M, et al. Salphage: salvage bacteriophage therapy for a recalcitrant Klebsiella pneumoniae prosthetic shoulder infection—a case report. Acta Orthop. 2022;93(5):756–9.

159. Federici S, Kredo-Russo S, Valdés-Mas R, Kviatcovsky D, Weinstock E, Matiuhin Y, et al. Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation. Cell. 2022;185(16):2879–2898.e24.

160. Hung CH, Kuo CF, Wang CH, Wu CM, Tsao N. Experimental phage therapy in treating Klebsiella pneumoniae-mediated liver abscesses and bacteremia in mice. Antimicrob Agents Chemother. 2011;55(4):1358–65.
161. Kumari S, Harjai K, Chhibber S. Bacteriophage versus antimicrobial agents for the treatment of murine burn wound infection caused by Klebsiella pneumoniae B5055. J Med Microbiol. 2011;60(2):205-10.

162. Gu J, Liu X, Li Y, Han W, Lei L, Yang Y, et al. A method for generation phage cocktail with great therapeutic potential. PLoS One. 2012;7(3):e31698. https://doi.org/10.1371/journal.pone.0031698 PMID: 22396736

163. Cao F, Wang X, Wang L, Li Z, Che J, Wang L, et al. Evaluation of the efficacy of a bacteriophage in the treatment of pneumonia induced by multidrug resistance Klebsiella pneumoniae in mice. Biomed Res Int. 2015;2015:752930. https://doi.org/10.1155/2015/752930 PMID: 25879036

164. Chadha P, Katare OP, Chhibber S. *In vivo* efficacy of single phage versus phage cocktail in resolving burn wound infection in BALB/c mice. Microb Pathog. 2016 Oct;99:68-77.

165. Chadha P, Katare O, Chhibber S. Liposome loaded phage cocktail: Enhanced therapeutic potential in resolving Klebsiella pneumoniae mediated burn wound infections. Burns. 2017;43(7):1532-43.

166. Anand T, Virmani N, Kumar S, Mohanty A, Pavulraj S, Bera Bc. Phage therapy for treatment of virulent Klebsiella pneumoniae infection in a mouse model. J Glob Antimicrob Resist. 2020;21(1):34-41.

167. Horváth M, Kovács T, Koderivalappil S, Ábrahám H, Rákhely G, Schneider G. Identification of a newly isolated lytic bacteriophage against K24 capsular type, carbapenem resistant Klebsiella pneumoniae isolates. Sci Reports. 2020;10(1):5891. https://doi.org/10.1038/s41598-020-62832-3

168. Soleimani Sasani M, Eftekhar F. Potential of a bacteriophage isolated from wastewater in treatment of lobar pneumonia infection induced by Klebsiella pneumoniae in mice. Curr Microbiol. 2020;77(10):2650-5.

169. Dhungana G, Nepal R, Regmi M, Malla R. Pharmacokinetics and pharmacodynamics of a novel virulent Klebsiella phage Kp_Pokalde_002 in a mouse model. Front Cell Infect Microbiol. 2021;11:684704. https://doi.org/10.3389/fcimb.2021.684704

170. Fayez MS, Hakim TA, Agwa MM, Abdelmoteleb M, Aly RG, Montaser NN, et al. Topically applied bacteriophage to control multi-drug resistant Klebsiella pneumoniae infected wound in a rat model. Antibiotics (Basel). 2021;10(9):1048. https://doi.org/10.3390/antibiotics10091048 PMID: 34572629

171. Hesse S, Malachowa N, Porter AR, Freedman B, Kobayashi SD, Gardner DJ, et al. Bacteriophage treatment rescues mice infected with multidrug-resistant Klebsiella pneumoniae ST258. mBio. 2021;12(1):e00034-21. https://doi.org/10.1128/mBio.00034-21 PMID: 33622728

172. Hao G, Shu R, Ding L, Chen X, Miao Y, Wu J, et al. Bacteriophage SRD2021 recognizing capsular polysaccharide shows therapeutic potential in serotype K47 Klebsiella pneumoniae infections. Antibiotics (Basel). 2021;10(8):894. https://doi.org/10.3390/antibiotics10080894 PMID: 34438943

173. Luo Z, Geng S, Lu B, Han G, Wang Y, Luo Y. Isolation, genomic analysis, and preliminary application of a bovine Klebsiella pneumoniae bacteriophage vB_Kpn_B01. Front Vet Sci. 2021;8:622049.

174. Shi Y, Peng Y, Zhang Y, Chen Y, Zhang C, Luo X, et al. Safety and efficacy of a phage, kpssk3, in an in vivo model of carbapenem-resistant hypermucoviscous Klebsiella pneumoniae bacteremia. Front Microbiol. 2021;12:613356. https://doi.org/10.3389/fmicb.2021.613356

175. Wang Z, Cai R, Wang G, Guo Z, Liu X, Guan Y, et al. Combination therapy of phage vB_KpnM_P-KP2 and gentamicin combats acute pneumonia caused by K47 serotype Klebsiella pneumoniae. Front Microbiol. 2021;12:674068. https://doi.org/10.3389/fmicb.2021.674068

176. Zhang C, Yuan J, Guo C, Ge C, Wang X, Wei D. Identification and complete genome of lytic "Kp34likevirus" phage vB_KpnP_Bp5 and therapeutic potency in the treatment of lethal Klebsiella pneumoniae infections in mice. Virus Res. 2021;297:198348.

177. Asghar S, Ahmed A, Khan S, Lail A, Shakeel M. Genomic characterization of lytic bacteriophages A¥L and A¥M infecting ESBL K. pneumoniae and its therapeutic potential on biofilm dispersal and in-vivo bacterial clearance. Microbiol Res. 2022 Sep 1;262:127104.

178. Bai J, Zhang F, Liang S, Chen Q, Wang W, Wang Y, et al. Isolation and characterization of vB_kpnM_17-11, a novel phage efficient against carbapenem-resistant Klebsiella pneumoniae. Front Cell Infect Microbiol. 2022;12:897531.

179. Gan L, Fu H, Tian Z, Cui J, Yan C, Xue G, et al. Bacteriophage effectively rescues pneumonia caused by prevalent multidrug-resistant Klebsiella pneumoniae in the early stage. Microbiol Spectr. 2022;10(5):e02358-22. https://doi.org/10.1128/microbiolspec.e02358-22

180. Pu M, Li Y, Han P, Lin W, Geng R, Qu F, et al. Genomic characterization of a new phage BUCT541 against Klebsiella pneumoniae K1-ST23 and efficacy assessment in mouse and Galleria mellonella larvae. Front Microbiol. 2022 Sep 16;13:950737.

181. Singh A, Singh A, Rathor N, Chaudhry R, Singh S, Nath G. Evaluation of bacteriophage cocktail on septicemia caused by colistin-resistant Klebsiella pneumoniae in mice model. Front Pharmacol. 2022;13:778676. https://doi.org/10.3389/fphar.2022.778676
182. Volozhantsev NV, Borzilov AI, Shpirt AM, Krasilnikova VM, Verevkin VV, Denisenko EA. Comparison of the therapeutic potential of bacteriophage KpV74 and phage-derived depolymerase (β-glucosidase) against Klebsiella pneumoniae capsular type K2. Virus Res. 2022;322(1):198951. https://doi.org/10.1016/j.virusres.2022.198951

183. Fang C, Dai X, Xiang L, Qiu Y, Yin M, Fu Y, et al. Isolation and characterization of three novel lytic phages against K54 serotype carbapenem-resistant hypervirulent Klebsiella pneumoniae. Front Cell Infect Microbiol. 2023;13:1265011.

184. Gan L, Feng Y, Du B, Fu H, Tian Z, Xue G. Bacteriophage targeting microbiota alleviates non-alcoholic fatty liver disease induced by high alcohol-producing Klebsiella pneumoniae. Nat Commun. 2023;14:3215.

185. Liang Z, Shi Y, Peng Y, Xu C, Zhang C, Chen Y. BL02, a phage against carbapenem- and polymyxin-B resistant Klebsiella pneumoniae, isolated from sewage: a preclinical study. Virus Research. 2023;331:199126.

186. Rahimi S, Bakht M, Javadi A, Foroughi F, Marashi SMA, Nikkhahi F. Characterization of novel bacteriophage PSKP16 and its therapeutic potential against β-lactamase and biofilm producer strain of K2-Hypervirulent Klebsiella pneumoniae pneumonia infection in mice model. BMC Microbiol. 2023 Aug 23;23:233.

187. Feng Y, Fang Q, Luo H, Li J, Yin X, Zong Z. Safety and efficacy of a phage cocktail on murine wound infections caused by carbapenem-resistant Klebsiella pneumoniae. Int J Antimicrob Agents. 2024;63(2):107088.

188. Kelishomi F, Nikkhahi F, Amereh S, Ghayyaz F, Marashi S, Javadi A. Evaluation of the therapeutic effect of a novel bacteriophage in the healing process of infected wounds with Klebsiella pneumoniae in mice. J Glob Antimicrob Resist. 2024;36(1):371-8.

189. Li P, Guo G, Zheng X, Xu S, Zhou Y, Qin X, et al. Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection. Veterinary Research. 2024;55:59.

190. Tang M, Yao Z, Liu Y, Ma Z, Zhao D, Mao Z. Host immunity involvement in the outcome of phage therapy against hypervirulent Klebsiella pneumoniae infections. Antimicrob Agents Chemother. 2024;68(6):e01429-23. https://doi.org/10.1128/AAC.01429-23