Assessment of the potential of phage-antibiotic synergy to induce collateral sensitivity in Salmonella Typhimurium.

This study was designed to evaluate the synergistic effect of phage and antibiotic on the induction of collateral sensitivity in Salmonella Typhimurium. The synergistic effects of Salmonella phage PBST32 combined with ciprofloxacin (CIP) against S. Typhimurium KCCM 40253 (STKCCM) were evaluated using a fractional inhibitory concentration (FIC) assay. The CIP susceptibility of STKCCM was increased when combined with PBST32, showing 16-fold decrease at 7 log PFU/mL. The combination of 1/2 × MIC of CIP and PBST32 (CIP[1/2]+PBST32) effectively inhibited the growth of STKCCM up to below the detection limit (1.3 log CFU/mL) after 12 h of incubation at 37 °C. The significant reduction in bacterial swimming motility was observed for PBST32 and CIP[1/4]+PBST32. The CIP[1/4]+PBST32 increased the fitness cost (relative fitness = 0.57) and decreased the cross-resistance to different classes of antibiotics. STKCCM treated with PBST32 alone treatment exhibited the highest coefficient of variation (90%), followed by CIP[1/4]+PBST32 (75%). These results suggest that the combination of PBST32 and CIP can be used to control bacterial pathogens.

Bacteriophages and antibiotic interactions in clinical practice: what we have learned so far.

Bacteriophages (phages) may be used as an alternative to antibiotic therapy for combating infections caused by multidrug-resistant bacteria. In the last decades, there have been studies concerning the use of phages and antibiotics separately or in combination both in animal models as well as in humans. The phenomenon of phage-antibiotic synergy, in which antibiotics may induce the production of phages by bacterial hosts has been observed. The potential mechanisms of phage and antibiotic synergy was presented in this paper. Studies of a biofilm model showed that a combination of phages with antibiotics may increase removal of bacteria and sequential treatment, consisting of phage administration followed by an antibiotic, was most effective in eliminating biofilms. In vivo studies predominantly show the phenomenon of phage and antibiotic synergy. A few studies also describe antagonism or indifference between phages and antibiotics. Recent papers regarding the application of phages and antibiotics in patients with severe bacterial infections show the effectiveness of simultaneous treatment with both antimicrobials on the clinical outcome.

PhageScore-based analysis of Acinetobacter baumannii infecting phages antibiotic interaction in liquid medium.

The growing interest in bacteriophages and antibiotics' combined use poses new challenges regarding this phenomenon's accurate description. This study aimed to apply the PhageScore methodology to assess the phage-antibiotic combination activity in liquid bacterial culture. For this purpose, previously described Acinetobacter infecting phages vB_AbaP_AGC01, Aba-1, and Aba-4 and antibiotics (gentamicin, ciprofloxacin, meropenem, norfloxacin, and fosfomycin) were used to obtain a lysis curve of bacteriophages under antibiotic pressure. The experimental data were analyzed using the Fractional Inhibitory Concentration Index (FICI) and PhageScore methodology. The results obtained by this method clearly show differences between phage lytic activity after antibiotic addition. Thus, we present the potential use of the PhageScore method as a tool for characterizing the phage antibiotic synergy in liquid culture. Further, the optimization of the PhageScore for this purpose can help compare antibiotics and their outcome on bacteriophage lytic activity.

Phage-Antibiotic Synergy via Delayed Lysis.

When phages infect bacteria cultured in the presence of sublethal doses of antibiotics, the sizes of the phage plaques are significantly increased. This phenomenon is known as phage-antibiotic synergy (PAS). In this study, the observation of PAS was extended to a wide variety of bacterium-phage pairs using different classes of antibiotics. PAS was shown in both Gram-positive and Gram-negative bacteria. Cells stressed with ß-lactam antibiotics filamented or swelled extensively, resulting in an increase in phage production. PAS was also sometimes observed in the presence of other classes of antibiotics with or without bacterial filamentation. The addition of antibiotics induced recA expression in various bacteria, but a recA deletion mutant strain of Escherichia coli also showed filamentation and PAS in the presence of quinolone antibiotics. The phage adsorption efficiency did not change in the presence of the antibiotics when the cell surfaces were enlarged as they filamented. Increases in the production of phage DNA and mRNAs encoding phage proteins were observed in these cells, with only a limited increase in protein production. The data suggest that PAS is the product of a prolonged period of particle assembly due to delayed lysis. The increase in the cell surface area far exceeded the increase in phage holin production in the filamented host cells, leading to a relatively limited availability of intracellular holins for aggregating and forming holes in the host membrane. Reactive oxygen species (ROS) stress also led to an increased production of phages, while heat stress resulted in only a limited increase in phage production.IMPORTANCE Phage-antibiotic synergy (PAS) has been reported for a decade, but the underlying mechanism has never been vigorously investigated. This study shows the presence of PAS from a variety of phage-bacterium-antibiotic pairings. We show that increased phage production resulted directly from a lysis delay caused by the relative shortage of holin in filamented bacterial hosts in the presence of sublethal concentrations of stress-inducing substances, such as antibiotics and reactive oxygen species (ROS).

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny.

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some ß-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.

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.

Isolation and characterization of a multidrug-resistant Staphylococcus aureus infecting phage and its therapeutic use in mice.

In recent years, the emergence of multidrug-resistant bacteria has limited the selection of drugs for treating bacterial infections, reduced clinical efficacy, and increased treatment costs and mortality. It is urgent to find alternative antibiotics. In order to explore a new method for controlling methicillin resistant Staphylococcus aureus (S. aureus) , this study isolated and purified a multi drug resistant S. aureus broad-spectrum phage JPL-50 from wastewater. JPL-50 belongs to the Siphoviridae family after morphological observation, biological characterization, and transmission electron microscopy (TEM) fragmentation spectrum analysis. It can cleave 84% of tested S. aureus (168/200) , in which 100% of tested mastitis-associated strains (48/48) and 72.04% of MRSA strains (67/93) were lysed. In addition, it has an optimal growth temperature of about 30°C, a high activity within a wide pH range (pH 3-10) , and an optimal multiplicity of infection of 0.01. The one-step growth curve shows a latent time of 20 minutes, an explosive time of 80 minutes. JPL-50 was 16, 927 bp in length and was encoded by double-stranded DNA, with no genes associated with bacterial resistance or virulence factors detected. In a therapeutic study, injection of the phage JPL-50 once and for 7 times in 7 days protected 40% and 60% of the mice from fatal S.aureus infection, respectively. More importantly, JPL-50-doxycycline combination could effectively inhibit host S.aureus in vitro and reduce the use of doxycycline within 8 hours. In conclusion, the bacteriophage JPL-50 has a wide lysis spectrum, high lysis rate, high tolerance to extreme environments, and moderate in vivo activity, providing ideas for developing multidrug-resistant S. aureus infections.

Phage-antibiotic synergy suppresses resistance emergence of Klebsiella pneumoniae by altering the evolutionary fitness.

Phage-antibiotic synergy (PAS) represents a superior treatment strategy for pathogen infections with less probability of resistance development. Here, we aim to understand the molecular mechanism by which PAS suppresses resistance in terms of population evolution. A novel hypervirulent Klebsiella pneumoniae (KP) phage H5 was genetically and structurally characterized. The combination of H5 and ceftazidime (CAZ) showed a robust synergistic effect in suppressing resistance emergence. Single-cell Raman analysis showed that the phage-CAZ combination suppressed bacterial metabolic activities, contrasting with the upregulation observed with phage alone. The altered population evolutionary trajectory was found to be responsible for the contrasting metabolic activities under different selective pressures, resulting in pleiotropic effects. A pre-existing wcaJ point mutation (wcaJG949A) was exclusively selected by H5, conferring a fitness advantage and up-regulated activity of carbohydrate metabolism, but also causing a trade-off between phage resistance and collateral sensitivity to CAZ. The wcaJ point mutation was counter-selected by H5-CAZ, inducing various mutations in galU that imposed evolutionary disadvantages with higher fitness costs, and suppressed carbohydrate metabolic activity. H5 and H5-CAZ treatments resulted in opposite effects on the transcriptional activity of the phosphotransferase system and the ascorbate and aldarate metabolism pathway, suggesting potential targets for phage resistance suppression. Our study reveals a novel mechanism of resistance suppression by PAS, highlighting how the complexity of bacterial adaptation to selective pressures drives treatment outcomes. IMPORTANCE: Phage-antibiotic synergy (PAS) has been recently proposed as a superior strategy for the treatment of multidrug-resistant pathogens to effectively reduce bacterial load and slow down both phage and antibiotic resistance. However, the underlying mechanisms of resistance suppression by PAS have been poorly and rarely been studied. In this study, we tried to understand how PAS suppresses the emergence of resistance using a hypervirulent Klebsiella pneumoniae (KP) strain and a novel phage H5 in combination with ceftazidime (CAZ) as a model. Our study reveals a novel mechanism by which PAS drives altered evolutionary trajectory of bacterial populations, leading to suppressed emergence of resistance. The findings advance our understanding of how PAS suppresses the emergence of resistance, and are imperative for optimizing the efficacy of phage-antibiotic therapy to further improve clinical outcomes.

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

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

Phage-antibiotic combinations in various treatment modalities to manage MRSA infections.

Introduction: The emergence of antibiotic resistance is a significant challenge in the treatment of bacterial infections, particularly in patients in the intensive care unit (ICU). Phage-antibiotic combination therapy is now being utilized as a preferred therapeutic option for infections that are multi-drug resistant in nature. Methods: In this study, we examined the combined impact of the staph phage vB_Sau_S90 and four antibiotics on methicillin-resistant Staphylococcus aureus (MRSA). We conducted experiments on three different treatment sequences: a) administering phages before antibiotics, b) administering phages and antibiotics simultaneously, and c) administering antibiotics before phages. Results: When the media was supplemented with sub-inhibitory concentrations of 0.25 µg/mL and 1 µg/mL, the size of the plaque increased from 0.5 ± 0.1 mm (in the control group with only the phage) to 4 ± 0.2 mm, 1.6 ± 0.1 mm, and 1.6 ± 0.4 mm when fosfomycin, ciprofloxacin, and oxacillin were added, respectively. The checkerboard analysis revealed a synergistic effect between the phages and antibiotics investigated, as indicated by a FIC value of less than 0.5. The combination treatment of phages and antibiotics demonstrated universal efficacy across all treatments. Nevertheless, the optimal effectiveness was demonstrated when the antibiotics were delivered subsequent to the phages. Utilizing the Galleria mellonella model, in vivo experiments showed that the combination of phage-oxacillin effectively eliminated biofilm-infected larvae, resulting in a survival rate of up to 80% in the treated groups. Discussion: Our findings highlight the advantages of using a combination of phage and antibiotic over using phages alone in the treatment of MRSA infections.