Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence.

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome.

Characterization of phage evolution and phage resistance in drug-resistant Stenotrophomonas maltophilia.

Phage therapy has become a viable antimicrobial treatment as an alternative to antibiotic treatment, with an increase in antibiotic resistance. Phage resistance is a major limitation in the therapeutic application of phages, and the lack of understanding of the dynamic changes between bacteria and phages constrains our response strategies to phage resistance. In this study, we investigated the changing trends of mutual resistance between Stenotrophomonas maltophilia (S. maltophilia) and its lytic phage, BUCT603. Our results revealed that S. maltophilia resisted phage infection through mutations in the cell membrane proteins, while the evolved phage re-infected the resistant strain primarily through mutations in structure-related proteins. Compared with the wild-type strain (SMA118), the evolved phage-resistant strain (R118-2) showed reduced virulence, weakened biofilm formation ability, and reduced resistance to aminoglycosides. In addition, the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant S. maltophilia in vitro and significantly improve the survival rate of S. maltophilia-infected mice. Altogether, these results suggest that in vitro characterization of bacteria-phage co-evolutionary relationships is a useful research tool to optimize phages for the treatment of drug-resistant bacterial infections.IMPORTANCEPhage therapy is a promising approach to treat infections caused by drug-resistant Stenotrophomonas maltophilia (S. maltophilia). However, the rapid development of phage resistance has hindered the therapeutic application of phages. In vitro evolutionary studies of bacteria-phage co-cultures can elucidate the mechanism of resistance development between phage and its host. In this study, we investigated the resistance trends between S. maltophilia and its phage and found that inhibition of phage adsorption is the primary strategy by which bacteria resist phage infection in vitro, while phages can re-infect bacterial cells by identifying other adsorption receptors. Although the final bacterial mutants were no longer infected by phages, they incurred a fitness cost that resulted in a significant reduction in virulence. In addition, the combination treatment with phage and aminoglycoside antibiotics could prevent the development of phage resistance in S. maltophilia in vitro. These findings contribute to increasing the understanding of the co-evolutionary relationships between phages and S. maltophilia.

fENko-Kae01 is a flagellum-specific jumbo phage infecting Klebsiella aerogenes.

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

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability.

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

Screening of genes involved in phage-resistance of Escherichia coli and effects of substances interacting with primosomal protein A on the resistant bacteria.

AIMS: The study was to identify the genes involved in phage resistance and to develop an effective biocontrol method to improve the lytic activity of phages against foodborne pathogens. METHODS AND RESULTS: A total of 3,909 single gene-deletion mutants of Escherichia coli BW25113 from the Keio collection were individually screened for genes involved in phage resistance. Phage S127BCL3 isolated from chicken liver, infecting both E. coli BW25113 and O157: H7, was characterized and used for screening. The 10 gene-deletion mutants showed increased susceptibility to phage S127BCL3. Among them, priA gene-deletion mutant strain showed significant susceptibility to the phages S127BCL3 and T7. Furthermore, we investigated the substances that have been reported to inhibit the function of primosomal protein A (PriA) and were used to confirm increased phage susceptibility in E. coli BW25113 (Parent strain) and O157: H7. CONCLUSION: PriA inhibitors at a low concentration showed combined effects with phage against E. coli O157: H7 and delayed the regrowth rate of phage-resistant cells.

K1 capsule-dependent phage-driven evolution in Escherichia coli leading to phage resistance and biofilm production.

AIMS: Understanding bacterial phage resistance mechanisms has implications for developing phage-based therapies. This study aimed to explore the development of phage resistance in Escherichia coli K1 isolates' to K1-ULINTec4, a K1-dependent bacteriophage. METHODS AND RESULTS: Resistant colonies were isolated from two different strains (APEC 45 and C5), both previously exposed to K1-ULINTec4. Genome analysis and several parameters were assessed, including growth capacity, phage adsorption, phenotypic impact at capsular level, biofilm production, and virulence in the in vivo Galleria mellonella larvae model. One out of the six resistant isolates exhibited a significantly slower growth rate, suggesting the presence of a resistance mechanism altering its fitness. Comparative genomic analysis revealed insertion sequences in the region 2 of the kps gene cluster involved in the capsule biosynthesis. In addition, an immunoassay targeting the K1 capsule showed a very low positive reaction compared to the control. Nevertheless, microscopic images of resistant strains revealed the presence of capsules with a clustered organization of bacterial cells and biofilm assessment showed an increased biofilm production compared to the sensitive strains. In the G. mellonella model, larvae infected with phage-resistant isolates showed better survival rates than larvae infected with phage-sensitive strains. CONCLUSIONS: A phage resistance mechanism was identified at the genomic level and had a negative impact on the K1 capsule production. The resistant isolates showed an increased biofilm production and a decreased virulence in vivo.

Correlation between the development of phage resistance and the original antibiotic resistance of host bacteria under the co-exposure of antibiotic and bacteriophage.

Bacteriophages (phages) are viruses capable of regulating the proliferation of antibiotic resistant bacteria (ARB). However, phages that directly cause host lethality may quickly select for phage resistant bacteria, and the co-evolutionary trade-offs under varying environmental conditions, including the presence of antibiotics, remains unclear as to their impact on phage and antibiotic resistance. Here, we report the emergence of phage resistance in three distinct E. coli strains with varying resistance to ß-lactam antibiotics, treated with different ampicillin (AMP) concentrations. Hosts exhibiting stronger antibiotic resistance demonstrated a higher propensity to develop and maintain stable phage resistance. When exposed to polyvalent phage KNT-1, the growth of AMP-sensitive E. coli K12 was nearly suppressed within 18 h, while the exponential growth of AMP-resistant E. coli TEM and super-resistant E. coli NDM-1 was delayed by 12 h and 8 h, respectively. The mutation frequency and mutated colony count of E. coli NDM-1 were almost unaffected by co-existing AMP, whereas for E. coli TEM and K12, these metrics significantly decreased with increasing AMP concentration from 8 to 50 µg/mL, becoming unquantifiable at 100 µg/mL. Furthermore, the fitness costs of phage resistance mutation and its impact on initial antibiotic resistance in bacteria were further examined, through analyzing AMP susceptibility, biofilm formation and EPS secretion of the isolated phage resistant mutants. The results indicated that acquiring phage resistance could decrease antibiotic resistance, particularly for hosts lacking strong antibiotic resistance. The ability of mutants to form biofilm contributes to antibiotic resistance, but the correlation is not entirely positive, while the secretion of extracellular polymeric substance (EPS), especially the protein content, plays a crucial role in protecting the bacteria from both antibiotic and phage exposure. This study explores phage resistance development in hosts with different antibiotic resistance and helps to understand the limitations and possible solutions of phage-based technologies.

Correlation of Pseudomonas aeruginosa Phage Resistance with the Numbers and Types of Antiphage Systems.

Phage therapeutics offer a potentially powerful approach for combating multidrug-resistant bacterial infections. However, to be effective, phage therapy must overcome existing and developing phage resistance. While phage cocktails can reduce this risk by targeting multiple receptors in a single therapeutic, bacteria have mechanisms of resistance beyond receptor modification. A rapidly growing body of knowledge describes a broad and varied arsenal of antiphage systems encoded by bacteria to counter phage infection. We sought to understand the types and frequencies of antiphage systems present in a highly diverse panel of Pseudomonas aeruginosa clinical isolates utilized to characterize novel antibacterials. Using the web-server tool PADLOC (prokaryotic antiviral defense locator), putative antiphage systems were identified in these P. aeruginosa clinical isolates based on sequence homology to a validated and curated catalog of known defense systems. Coupling this host bacterium sequence analysis with host range data for 70 phages, we observed a correlation between existing phage resistance and the presence of higher numbers of antiphage systems in bacterial genomes. We were also able to identify antiphage systems that were more prevalent in highly phage-resistant P. aeruginosa strains, suggesting their importance in conferring resistance.

Phage Mediated Biocontrol: A Promising Green Solution for Sustainable Agriculture.

In the current scenario of growing world population, limited cultivable land resources, plant diseases, and pandemics are some of the major factors responsible for declining global food security. Along with meeting the food demand, the maintenance of food quality is also required to ensure healthy consumption and marketing. In agricultural fields, pest infestations and bacterial diseases are common causes of crop damage, leading to massive yield losses. Conventionally, antibiotics and several pesticides have been used to manage and control these plant pathogens. However, the overuse of antibiotics and pesticides has led to the emergence of resistant strains of pathogenic bacteria. The bacteriophages are the natural predators of bacteria and are host-specific in their action. Therefore, the use of bacteriophages for the biocontrol of pathogenic bacteria is serving as a sustainable and green solution in crop protection and production. In this review, we have discussed the important plant pathogens and their impact on plant health and yield loss. Further, we have abridged the role of bacteriophages in the protection of crops from bacterial disease by discussing various greenhouse and field trials. Finally, we have discussed the impact of bacteriophages on the plant microbiome, phage resistance, and legal challenges in the registration and commercial production of bacteriophage-based biopesticides. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01204-x.

Defense and anti-defense mechanisms of bacteria and bacteriophages. / 细菌和噬菌体间的防御与反防御机制.

In the post-antibiotic era, the overuse of antimicrobials has led to a massive increase in antimicrobial resistance, leaving medical doctors few or no treatment options to fight infections caused by superbugs. The use of bacteriophages is a promising alternative to treat infections, supplementing or possibly even replacing antibiotics. Using phages for therapy is possible, since these bacterial viruses can kill bacteria specifically, causing no harm to the normal flora. However, bacteria have developed a multitude of sophisticated and complex ways to resist infection by phages, including abortive infection and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. Phages also can evolve and acquire new anti-defense strategies to continue predation. An in-depth exploration of both defense and anti-defense mechanisms would contribute to optimizing phage therapy, while we would also gain novel insights into the microbial world. In this paper, we summarize recent research on bacterial phage resistance and phage anti-defense mechanisms, as well as collaborative win-win systems involving both virus and host.

Erratum: Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa.

[This corrects the article DOI: 10.3389/fcimb.2023.1280265.].

Resistance to bacteriophage incurs a cost to virulence in drug-resistant Acinetobacter baumannii.

Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii.Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (107 p.f.u. ml-1), the effect was weak and not sufficient to save larvae from morbidity and mortality.Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.

Tradeoffs Between Evolved Phage Resistance and Antibiotic Susceptibility in a Highly Drug-Resistant Cystic Fibrosis-Derived Pseudomonas aeruginosa Strain.

Background: Multi-drug resistant pathogens pose significant challenges towards the effective resolution of bacterial infections. A promising alternative strategy is phage therapy in which limited applications has afforded lifesaving resolution from drug resistant pathogens. However, adoption of this strategy is hampered by narrow bacteriophage host ranges, and as with antibiotics, bacteria can acquire resistance to phage. Methods: To address these issues, we isolated 25 broad-host range phages against multiple cystic fibrosis (CF)-derived P. aeruginosa clinical strains thus promoting their application against conspecific pathogens. To investigate evolved resistance to phage in relation to antibiotic resistance, one CF-derived P. aeruginosa strain was exposed to a lytic phage over a short time scale. Results: Trade-offs were observed in which evolved phage resistant P. aeruginosa strains showed decreased resistance to antibiotics. These traits that likely reflect single nucleotide polymorphisms. Conclusion: Results suggest phage and antibiotics may be a combined approach to treat bacterial infections.

Phage-driven coevolution reveals trade-off between antibiotic and phage resistance in Salmonella anatum.

Phage therapy faces challenges against multidrug-resistant (MDR) Salmonella due to rapid phage-resistant mutant emergence. Understanding the intricate interplay between antibiotics and phages is essential for shaping Salmonella evolution and advancing phage therapy. In this study, MDR Salmonella anatum (S. anatum) 2089b coevolved with phage JNwz02 for 30 passages (60 days), then the effect of coevolution on the trade-off between phage and antibiotic resistance in bacteria was investigated. Our results demonstrated antagonistic coevolution between bacteria and phages, transitioning from arms race dynamics (ARD) to fluctuating selection dynamics (FSD). The fitness cost of phage resistance, manifested as reduced competitiveness, was observed. Bacteria evolved phage resistance while simultaneously regaining sensitivity to amoxicillin, ampicillin, and gentamicin, influenced by phage selection pressure and bacterial competitiveness. Moreover, the impact of phage selection pressure on the trade-off between antibiotic and phage resistance was more pronounced in the ARD stage than in the FSD stage. Whole genome analysis revealed mutations in the btuB gene in evolved S. anatum strains, with a notably higher mutation frequency in the ARD stage compared to the FSD stage. Subsequent knockout experiments confirmed BtuB as a receptor for phage JNwz02, and the deletion of btuB resulted in reduced bacterial competitiveness. Additionally, the mutations identified in the phage-resistant strains were linked to multiple single nucleotide polymorphisms (SNPs) associated with membrane components. This correlation implies a potential role of these SNPs in reinstating antibiotic susceptibility. These findings significantly advance our understanding of phage-host interactions and the impact of bacterial adaptations on antibiotic resistance.

Regulation of anti-phage defense mechanisms by using cinnamaldehyde as a quorum sensing inhibitor.

Background: Multidrug-resistant bacteria and the shortage of new antibiotics constitute a serious health problem. This problem has led to increased interest in the use of bacteriophages, which have great potential as antimicrobial agents but also carry the risk of inducing resistance. The objective of the present study was to minimize the development of phage resistance in Klebsiella pneumoniae strains by inhibiting quorum sensing (QS) and thus demonstrate the role of QS in regulating defense mechanisms. Results: Cinnamaldehyde (CAD) was added to K. pneumoniae cultures to inhibit QS and thus demonstrate the role of the signaling system in regulating the anti-phage defense mechanism. The QS inhibitory activity of CAD in K. pneumoniae was confirmed by a reduction in the quantitative expression of the lsrB gene (AI-2 pathway) and by proteomic analysis. The infection assays showed that the phage was able to infect a previously resistant K. pneumoniae strain in the cultures to which CAD was added. The results were confirmed using proteomic analysis. Thus, anti-phage defense-related proteins from different systems, such as cyclic oligonucleotide-based bacterial anti-phage signaling systems (CBASS), restriction-modification (R-M) systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, and bacteriophage control infection (BCI), were present in the cultures with phage but not in the cultures with phage and CAD. When the QS and anti-phage defense systems were inhibited by the combined treatment, proteins related to phage infection and proliferation, such as the tail fiber protein, the cell division protein DamX, and the outer membrane channel protein TolC, were detected. Conclusion: Inhibition of QS reduces phage resistance in K. pneumoniae, resulting in the infection of a previously resistant strain by phage, with a significant increase in phage proliferation and a significant reduction in bacterial growth. QS inhibitors could be considered for therapeutic application by including them in phage cocktails or in phage-antibiotic combinations to enhance synergistic effects and reduce the emergence of antimicrobial resistance.

Phage therapy: an alternative treatment modality for MDR bacterial infections.

The increasing global incidence of multidrug-resistant (MDR) bacterial infections threatens public health and compromises various aspects of modern medicine. Recognising the urgency of this issue, the World Health Organisation has prioritised the development of novel antimicrobials to combat ESKAPEE pathogens. Comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli, such pathogens represent a spectrum of high to critical drug resistance, accounting for a significant proportion of hospital-acquired infections worldwide. In response to the waning efficacy of antibiotics against these resilient pathogens, phage therapy (PT) has emerged as a promising therapeutic strategy. This review provides a comprehensive summary of clinical research on PT and explores the translational journey of phages from laboratory settings to clinical applications. It examines recent advancements in pre-clinical and clinical developments, highlighting the potential of phages and their proteins, alone or in combination with antibiotics. Furthermore, this review underlines the importance of establishing safe and approved routes of phage administration to patients. In conclusion, the evolving landscape of phage therapy offers a beacon of hope in the fight against MDR bacterial infections, emphasising the imperative for continued research, innovation and regulatory diligence to realise its full potential in clinical practice.

Escherichia coli CRISPR arrays from early life fecal samples preferentially target prophages.

CRISPR-Cas systems are defense mechanisms against phages and other nucleic acids that invade bacteria and archaea. In Escherichia coli, it is generally accepted that CRISPR-Cas systems are inactive in laboratory conditions due to a transcriptional repressor. In natural isolates, it has been shown that CRISPR arrays remain stable over the years and that most spacer targets (protospacers) remain unknown. Here, we re-examine CRISPR arrays in natural E. coli isolates and investigate viral and bacterial genomes for spacer targets using a bioinformatics approach coupled to a unique biological dataset. We first sequenced the CRISPR1 array of 1769 E. coli isolates from the fecal samples of 639 children obtained during their first year of life. We built a network with edges between isolates that reflect the number of shared spacers. The isolates grouped into 34 modules. A search for matching spacers in bacterial genomes showed that E. coli spacers almost exclusively target prophages. While we found instances of self-targeting spacers, those involving a prophage and a spacer within the same bacterial genome were rare. The extensive search for matching spacers also expanded the library of known E. coli protospacers to 60%. Altogether, these results favor the concept that E. coli's CRISPR-Cas is an antiprophage system and highlight the importance of reconsidering the criteria use to deem CRISPR-Cas systems active.

Phage resistance in Klebsiella pneumoniae and bidirectional effects impacting antibiotic susceptibility.

OBJECTIVES: Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilization of phage therapy has been severely compromised by the potential emergence of phage resistance. Although certain phage resistance mechanisms can restore bacterial susceptibility to certain antibiotics, a lack of knowledge of phage resistance mechanisms hinders optimal use of phages and their combination with antibiotics. METHODS: Genome-wide transposon screening was performed with a mutant library of Klebsiella pneumoniae MKP103 to identify phage pKMKP103_1-resistant mutants. Phage-resistant phenotypes were evaluated by time-kill kinetics and efficiency of plating assays. Phage resistance mechanisms were investigated with adsorption, one-step growth, and mutation frequency assays. Antibiotic susceptibility was determined with broth microdilution and population analysis profiles. RESULTS: We observed a repertoire of phage resistance mechanisms in K pneumoniae, such as disruption of phage binding (fhuA::Tn and tonB::Tn), extension of the phage latent period (mnmE::Tn and rpoN::Tn), and increased mutation frequency (mutS::Tn and mutL::Tn). Notably, in contrast to the prevailing view that phage resistance re-sensitizes antibiotic-resistant bacteria, we observed a bidirectional steering effect on bacterial antibiotic susceptibility. Specifically, rpoN::Tn increased susceptibility to colistin while mutS::Tn and mutL::Tn increased resistance to rifampicin and colistin. DISCUSSION: Our findings demonstrate that K pneumoniae employs multiple strategies to overcome phage infection, which may result in enhanced or reduced antibiotic susceptibility. Mechanism-guided phage steering should be incorporated into phage therapy to better inform clinical decisions on phage-antibiotic combinations.

Using phage to drive selections toward restoring antibiotic sensitivity in Pseudomonas aeruginosa via chromosomal deletions.

Phage therapy has re-emerged in modern medicine as a robust antimicrobial strategy in response to the increasing prevalence of antimicrobial-resistant bacteria. However, bacterial resistance to phages can also arise via a variety of molecular mechanisms. In fact, several clinical studies on phage therapy have reported the occurrence of phage-resistant variants, representing a significant concern for the successful development of phage-based therapies. In this context, the fitness trade-offs between phage and antibiotic resistance have revealed new avenues in the field of phage therapy as a countermeasure against phage resistance. This strategy forces to restore the antibiotic susceptibility of antimicrobial-resistant bacteria as compensation for the development of phage resistance. Here, we present the key achievements of these fitness trade-offs, notably focusing on the enhancement of antibiotic sensitivity through the induction of large chromosomal deletions by bacteriophage infection. We also describe the challenges of this strategy that need to be overcome to promote favorable therapeutic outcomes and discuss future directions. The insights gained from the trade-offs between phage and antibiotic sensitivity will help maximize the potential of phage therapy for the treatment of infectious diseases.

Isolation and Phenotypic Characterization of Virulent Bacteriophages Against Multidrug-Resistant Escherichia coli and Its Phage-Resistant Variant from Sewage Sources.

Purpose: The use of lytic bacteriophages for the control or elimination of pathogenic multidrug-resistant (MDR) bacteria is the promising alternative. However, the emergence of resistant bacterial variants after phage application may challenge its therapeutic benefit. In this study, we aimed to isolate candidate phages from sewage samples against two MDR Escherichia coli as well as their phage-resistant variant. Methods: MDR E. coli isolates (n = 10) obtained from Jimma Medical Center that had been properly identified and stored were used to isolate bacteriophages. Two lytic coliphages were isolated from hospital sewage samples following standard protocols. Upon single phage infection, phage-resistant variant quickly evolved serving as a new host for the isolation of a third lytic phage. This virulent phage's lytic activity against both its host and the wild host was investigated. The host infectivity of the various cocktails was assessed, and each phage's biological properties were studied. Results: Out of the first round of phage isolation process, two lytic phages were identified as VBO-E. coli 4307 and VBW-E. coli 4194. When exposed to VBO-E. coli 4307, the wild-type E. coli 4307 developed resistant variants. A third phage (VBA-E. coli 4307R) was isolated specific to this resistant variant (E. coli 4307R) under optimum condition. For VBO-E. coli 4307, VBW-E. coli 4194, and VBA-E. coli 4307R, the plaque assays generated under comparable conditions were 2.13 × 1010 PFU mL-1, 9.17 × 1012 PFU mL-1, and 3.3 × 1010 PFU mL-1, respectively. These phages have nearly identical stability and lytic ability but differ greatly in their host ranges for VBA-E. coli 4307R. Conclusion: While the wild-type MDR pathogen could easily evolve resistance when exposed to a single phage infection by VBO-E. coli 4307, it is still possible to isolate a novel bacteriophage from environmental samples that is effective against the phage-resistant variants. This indicates that it is possible to manage the effects of phage resistance pathogens even if they are MDR.