Prophylactic phage biocontrol prevents Burkholderia gladioli infection in a quantitative ex planta model of bacterial virulence.

Agricultural crop yield losses and food destruction due to infections by phytopathogenic bacteria such as Burkholderia gladioli, which causes devastating diseases in onion, mushroom, corn, and rice crops, pose major threats to worldwide food security and cause enormous damage to the global economy. Biocontrol using bacteriophages has emerged as a promising strategy against a number of phytopathogenic species but has never been attempted against B. gladioli due to a lack of quantitative infection models and a scarcity of phages targeting this specific pathogen. In this study, we present a novel, procedurally straightforward, and highly generalizable fully quantitative ex planta maceration model and an accompanying quantitative metric, the ex planta maceration index (xPMI). In utilizing this model to test the ex planta virulence of a panel of 12 strains of B. gladioli in Allium cepa and Agaricus bisporus, we uncover substantial temperature-, host-, and strain-dependent diversity in the virulence of this fascinating pathogenic species. Crucially, we demonstrate that Burkholderia phages KS12 and AH2, respectively, prevent and reduce infection-associated onion tissue destruction, measured through significant (P < 0.0001) reductions in xPMI, by phytopathogenic strains of B. gladioli, thereby demonstrating the potential of agricultural phage biocontrol targeting this problematic microorganism.IMPORTANCEAgricultural crop destruction is increasing due to infections caused by bacteria such as Burkholderia gladioli, which causes plant tissue diseases in onion, mushroom, corn, and rice crops. These bacteria pose a major threat to worldwide food production, which, in turn, damages the global economy. One potential solution being investigated to prevent bacterial infections of plants is "biocontrol" using bacteriophages (or phages), which are bacterial viruses that readily infect and destroy bacterial cells. In this article, we demonstrate that Burkholderia phages KS12 and AH2 prevent or reduce infection-associated plant tissue destruction caused by strains of B. gladioli, thereby demonstrating the inherent potential of agricultural phage biocontrol.

Bacteriophage steering of Burkholderia cenocepacia toward reduced virulence and increased antibiotic sensitivity.

Antibiotic resistance in bacteria is a growing global concern and has spurred increasing efforts to find alternative therapeutics, such as the use of bacterial viruses, or bacteriophages. One promising approach is to use phages that not only kill pathogenic bacteria but also select phage-resistant survivors that are newly sensitized to traditional antibiotics, in a process called "phage steering." Members of the bacterial genus Burkholderia, which includes various human pathogens, are highly resistant to most antimicrobial agents, including serum immune components, antimicrobial peptides, and polymixin-class antibiotics. However, the application of phages in combination with certain antibiotics can produce synergistic effects that more effectively kill pathogenic bacteria. Herein, we demonstrate that Burkholderia cenocepacia serum resistance is due to intact lipopolysaccharide (LPS) and membranes, and phage-induced resistance altering LPS structure can enhance bacterial sensitivity not only to immune components in serum but also to membrane-associated antibiotics such as colistin. IMPORTANCE Bacteria frequently encounter selection pressure from both antibiotics and lytic phages, but little is known about the interactions between antibiotics and phages. This study provides new insights into the evolutionary trade-offs between phage resistance and antibiotic sensitivity. The creation of phage resistance through changes in membrane structure or lipopolysaccharide composition can simultaneously be a major cause of antibiotic sensitivity. Our results provide evidence of synergistic therapeutic efficacy in phage-antibiotic interactions and have implications for the future clinical use of phage steering in phage therapy applications.

Genetic and Phenotypic Analysis of Phage-Resistant Mutant Fitness Triggered by Phage-Host Interactions.

The emergence of phage-resistant bacterial strains is one of the biggest challenges for phage therapy. However, the emerging phage-resistant bacteria are often accompanied by adaptive trade-offs, which supports a therapeutic strategy called "phage steering". The key to phage steering is to guide the bacterial population toward an evolutionary direction that is favorable for treatment. Thus, it is important to systematically investigate the impacts of phages targeting different bacterial receptors on the fitness of the bacterial population. Herein, we employed 20 different phages to impose strong evolutionary pressure on the host Pseudomonas aeruginosa PAO1 and examined the genetic and phenotypic responses of their phage-resistant mutants. Among these strains with impaired adsorptions, four types of mutations associated with bacterial receptors were identified, namely, lipopolysaccharides (LPSs), type IV pili (T4Ps), outer membrane proteins (OMPs), and exopolysaccharides (EPSs). PAO1, responding to LPS- and EPS-dependent phage infections, mostly showed significant growth impairment and virulence attenuation. Most mutants with T4P-related mutations exhibited a significant decrease in motility and biofilm formation ability, while the mutants with OMP-related mutations required the lowest fitness cost out of the bacterial populations. Apart from fitness costs, PAO1 strains might lose their resistance to antibiotics when counteracting with phages, such as the presence of large-fragment mutants in this study, which may inspire the usage of phage-antibiotic combination strategies. This work provides methods that leverage the merits of phage resistance relative to obtaining therapeutically beneficial outcomes with respect to phage-steering strategies.

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.

Isolation, characterization and application of noble bacteriophages targeting potato common scab pathogen Streptomyces stelliscabiei.

Bacteriophages have emerged as promising alternatives to pesticides for controlling bacterial pathogens in crops. Among these pathogens, Streptomyces stelliscabiei (syn. S. stelliscabiei) is a primary causative agent of potato common scab (PCS), resulting in substantial global economic losses. The traditional management methods for PCS face numerous challenges, highlighting the need for effective and environmentally friendly control strategies. In this study, we successfully isolated three novel bacteriophages, namely Psst1, Psst2, and Psst4, which exhibited a broad host range encompassing seven S. stelliscabiei strains. Morphological analysis revealed their distinct features, including an icosahedral head and a non-contractile tail. These phages demonstrated stability across a broad range of temperatures (20-50°C), pH (pH 3-11), and UV exposure time (80 min). Genome sequencing revealed double-stranded DNA phage with open reading frames encoding genes for phage structure, DNA packaging and replication, host lysis and other essential functions. These phages lacked genes for antibiotic resistance, virulence, and toxicity. Average nucleotide identity, phylogenetic, and comparative genomic analyses classified the three phages as members of the Rimavirus genus, with Psst1 and Psst2 representing novel species. All three phages efficiently lysed S. stelliscabiei in the liquid medium and alleviated scab symptom development and reduced pathogen abundance on potato slices. Furthermore, phage treatments of radish seedlings alleviated the growth inhibition caused by S. stelliscabiei with no disease symptoms. In soil potted experiments, phages significantly reduced disease incidence by 40%. This decrease is attributed to a reduction in pathogen density and the selection of S. stelliscabiei strains with reduced virulence and slower growth rates in natural environments. Our study is the first to report the isolation of three novel phages that infect S. stelliscabiei as a host bacterium. These phages exhibit a broad host range, and demonstrate stability under a variety of environmental conditions. Additionally, they demonstrate biocontrol efficacy against bacterial infections in potato slices, radish seedlings, and potted experiments, underscoring their significant potential as biocontrol agents for the effective management of PCS.

Phage steering of antibiotic-resistance evolution in the bacterial pathogen, Pseudomonas aeruginosa.

BACKGROUND AND OBJECTIVES: Antimicrobial resistance is a growing global concern and has spurred increasing efforts to find alternative therapeutics. Bacteriophage therapy has seen near constant use in Eastern Europe since its discovery over a century ago. One promising approach is to use phages that not only reduce bacterial pathogen loads but also select for phage resistance mechanisms that trade-off with antibiotic resistance-so called 'phage steering'. METHODOLOGY: Recent work has shown that the phage OMKO1 can interact with efflux pumps and in so doing select for both phage resistance and antibiotic sensitivity of the pathogenic bacterium Pseudomonas aeruginosa. We tested the robustness of this approach to three different antibiotics in vitro (tetracycline, erythromycin and ciprofloxacin) and one in vivo (erythromycin). RESULTS: We show that in vitro OMKO1 can reduce antibiotic resistance of P. aeruginosa (Washington PAO1) even in the presence of antibiotics, an effect still detectable after ca.70 bacterial generations in continuous culture with phage. Our in vivo experiment showed that phage both increased the survival times of wax moth larvae (Galleria mellonella) and increased bacterial sensitivity to erythromycin. This increased antibiotic sensitivity occurred both in lines with and without the antibiotic. CONCLUSIONS AND IMPLICATIONS: Our study supports a trade-off between antibiotic resistance and phage sensitivity. This trade-off was maintained over co-evolutionary time scales even under combined phage and antibiotic pressure. Similarly, OMKO1 maintained this trade-off in vivo, again under dual phage/antibiotic pressure. Our findings have implications for the future clinical use of steering in phage therapies. Lay Summary: Given the rise of antibiotic-resistant bacterial infection, new approaches to treatment are urgently needed. Bacteriophages (phages) are bacterial viruses. The use of such viruses to treat infections has been in near-continuous use in several countries since the early 1900s. Recent developments have shown that these viruses are not only effective against routine infections but can also target antibiotic resistant bacteria in a novel, unexpected way. Similar to other lytic phages, these so-called 'steering phages' kill the majority of bacteria directly. However, steering phages also leave behind bacterial variants that resist the phages, but are now sensitive to antibiotics. Treatment combinations of these phages and antibiotics can now be used to greater effect than either one independently. We evaluated the impact of steering using phage OMKO1 and a panel of three antibiotics on Pseudomonas aeruginosa, an important pathogen in hospital settings and in people with cystic fibrosis. Our findings indicate that OMKO1, either alone or in combination with antibiotics, maintains antibiotic sensitivity both in vitro and in vivo, giving hope that phage steering will be an effective treatment option against antibiotic-resistant bacteria.