Photocatalytic Quantum Dot-Armed Bacteriophage for Combating Drug-Resistant Bacterial Infection.

Multidrug-resistant (MDR) bacterial infection is one of the greatest challenges to public health, a crisis demanding the next generation of highly effective antibacterial agents to specifically target MDR bacteria. Herein, a novel photocatalytic quantum dot (QD)-armed bacteriophage (QD@Phage) is reported for combating green fluorescent protein-expressing Pseudomonas aeruginosa (GFP-P. aeruginosa) infection. The proposed QD@Phage nanosystem not only specifically binds to the host GFP-P. aeruginosa while preserving the infectivity of the phage itself, but also shows a superior capacity for synergistic bacterial killing by phage and by the photocatalytic localized reactive oxygen species (ROS) generated from anchored QD components. Notably, this highly targeted QD@Phage nanosystem achieves robust in vitro antibacterial elimination for both planktonic (over 99.9%) and biofilm (over 99%) modes of growth. In a mouse wound infection model, this system also shows remarkable activity in eliminating the wound infection and promoting its recovery. These results demonstrate that the novel QD@Phage nanosystem can diversify the existing pool of antibacterial agents and inspire the development of promising therapeutic strategies against MDR bacterial infection.

Bacteriophage therapy as a treatment option for complex cardiovascular implant infection: The German Heart Center Berlin experience.

Conventional antimicrobials have low or no activity against multidrug-resistant or chronic implant-associated infections. Lytic bacteriophages can rapidly and selectively kill bacteria, and can be combined with antibiotics. However, clinical experience of bacteriophage therapy in patients with cardiovascular infections is limited. We documented the outcome and safety of intravenous and local adjunctive bacteriophage therapy, to treat chronic relapsing cardiovascular implant infections at our institution.

Evaluation of Staphylococcal Bacteriophage Sb-1 as an Adjunctive Agent to Antibiotics Against Rifampin-Resistant Staphylococcus aureus Biofilms.

Rifampin plays a crucial role in the treatment of staphylococcal implant-associated infection, as it is the only antibiotic capable of eradicating Staphylococcus aureus biofilms. However, the emergence of rifampin resistance strongly limits its use. Combinatorial therapy of antibiotics and bacteriophages may represent a strategy to overcome the resistance. Here, we evaluated the activity of staphylococcal bacteriophage Sb-1 in combination with different antibiotics against the biofilms of 10 rifampin-resistant S. aureus clinical strains, including MRSA and MSSA. S. aureus biofilms formed on porous glass beads were exposed to antibiotics alone or combined with Sb-1 simultaneously or staggered (first Sb-1 for 24 h followed by antibiotic). Recovered bacteria were detected by measuring growth-related heat production at 37°C (isothermal microcalorimetry) and the biofilm eradication was assessed by sonication of beads and plating of the resulting sonication fluid. Minimum biofilm eradication concentration (MBEC) was defined as the lowest concentration of antibiotic required to kill all adherent bacteria, resulting in absence of growth after plating the sonication fluid. Tested antibiotics presented high MBEC values when administered alone (64 to > 1,024 µg/ml). The simultaneous or staggered combination of Sb-1 with daptomycin showed the highest activity against all MRSA biofilms, whereas the exposure to Sb-1 with vancomycin showed no improved anti-biofilm activity. Staggered administration of Sb-1 and flucloxacillin, cefazolin, or fosfomycin improved the antibiofilm activity in four out of six MSSA, whereas simultaneous exposure exhibited similar or lesser synergy. In conclusion, the combinatorial effect of Sb-1 and antibiotics enabled to eradicate rifampin-resistant S. aureus biofilms in vitro.

Adjunctive Use of Phage Sb-1 in Antibiotics Enhances Inhibitory Biofilm Growth Activity versus Rifampin-Resistant Staphylococcus aureus Strains.

Effective antimicrobials are crucial for managing Staphylococcus aureus implant-associated bone infections (IABIs), particularly for infections due to rifampin-resistant S. aureus (RRSA). Failure to remove the implant results in persistent infection; thus, prolonged suppressive antibiotic therapy may be a reasonable alternative. However, a high incidence of adverse events can necessitate the discontinuation of therapy. In this scenario, commercial Staphylococcal bacteriophage Sb-1 combined with antibiotics is an option, showing a promising synergistic activity to facilitate the treatment of biofilm infections. Therefore, we evaluated the efficacy of the inhibitory activity of five antibiotics (doxycycline, levofloxacin, clindamycin, linezolid, and rifampin) alone or combined with phage Sb-1 (106 PFU/mL) in a simultaneous and staggered manner, to combat five clinical RRSA strains and the laboratory strain MRSA ATCC 43300 in 72 h by isothermal microcalorimetry. The synergistic effects were observed when phage Sb-1 (106 PFU/mL) combined with antibiotics had at least 2 log-reduction lower concentrations, represented by a fractional biofilm inhibitory concentration (FBIC) of <0.25. Among the antibiotics that we tested, the synergistic effect of all six strains was achieved in phage/doxycycline and phage/linezolid combinations in a staggered manner, whereas a distinctly noticeable improvement in inhibitory activity was observed in the phage/doxycycline combination with a low concentration of doxycycline. Moreover, phage/levofloxacin and phage/clindamycin combinations also showed a synergistic inhibitory effect against five strains and four strains, respectively. Interestingly, the synergistic inhibitory activity was also observed in the doxycycline-resistant and levofloxacin-resistant profile strains. However, no inhibitory activity was observed for all of the combinations in a simultaneous manner, as well as for the phage/rifampin combination in a staggered manner. These results have implications for alternative, combined, and prolonged suppressive antimicrobial treatment approaches.

Bacteriophage-antibiotic combinations against ciprofloxacin/ceftriaxone-resistant Escherichia coli in vitro and in an experimental Galleria mellonella model.

Escherichia coli is the most common cause of Gram-negative prosthetic joint infections (PJIs) and ciprofloxacin is the first-line antibiofilm antibiotic. Due to the emergence of fluoroquinolone resistance, management of E. coli PJIs has become challenging and is associated with high treatment failure rates. We evaluated the efficacy of a newly isolated bacteriophage ɸWL-3 as a therapeutic agent in combination with ciprofloxacin, fosfomycin, gentamicin, meropenem or ceftriaxone against biofilm of a ciprofloxacin/ceftriaxone-resistant E. coli strain and the ATCC 25922 reference strain. ɸWL-3 was first characterised in terms of virion morphology, absorption rate, burst size and killing kinetics against both E. coli strains. The tested antibiotics presented high inhibitory concentrations (ranging from 16 to >1024 µg/mL) when tested alone against biofilms. Co-administration of ɸWL-3 with antibiotics improved the antibiotic efficacy against biofilm, especially after staggered exposure, reducing the minimum biofilm bactericidal concentration (MBBC) up to 512 times. The in vivo antimicrobial activity of ɸWL-3/fosfomycin combination against both E. coli strains was assessed in a Galleria mellonella invertebrate infection model. Treatment of infected larvae after lethal doses of E. coli resulted in enhanced survival rates when combinatorial therapy with ɸWL-3/fosfomycin was applied on E. coli ATCC 25922-infected larvae compared with monotherapy, but not for EC1-infected larvae, which we speculated could be due to higher release of endotoxins in a shorter period in EC1-infected larvae exposed to ɸWL-3. Our study provides new insights into the use of bacteriophages and antibiotics in the treatment of biofilm-associated infections caused by antibiotic-resistant bacteria.

Using Bacteriophages as a Trojan Horse to the Killing of Dual-Species Biofilm Formed by Pseudomonas aeruginosa and Methicillin Resistant Staphylococcus aureus.

Pseudomonas aeruginosa and Staphylococcus aureus are pathogens able to colonize surfaces and form together a mixed biofilm. Dual-species biofilms are significantly more resistant to antimicrobials than a monomicrobial community, leading to treatment failure. Due to their rapid bactericidal activity, the self-amplification ability and the biofilm degrading properties, bacteriophages represent a promising therapeutic option in fighting biofilm-related infections. In this study, we investigated the effect of either the simultaneous or staggered application of commercially available phages and ciprofloxacin versus S. aureus/P. aeruginosa dual-species biofilms in vitro. Biofilms were grown on porous glass beads and analyzed over time. Different techniques such as microcalorimetry, sonication and scanning electron microscopy were combined for the evaluation of anti-biofilm activities. Both bacterial species were susceptible to ciprofloxacin and to phages in their planktonic form of growth. Ciprofloxacin tested alone against biofilms required high concentration ranging from 256 to >512 mg/L to show an inhibitory effect, whereas phages alone showed good and moderate activity against MRSA biofilms and dual-species biofilms, respectively, but low activity against P. aeruginosa biofilms. The combination of ciprofloxacin with phages showed a remarkable improvement in the anti-biofilm activity of both antimicrobials with complete eradication of dual-species biofilms after staggered exposure to Pyophage or Pyophage + Staphylococcal phage for 12 h followed by 1 mg/L of ciprofloxacin, a dose achievable by intravenous or oral antibiotic administration. Our study provides also valuable data regarding not only dosage but also an optimal time of antimicrobial exposure, which is crucial in the implementation of combined therapies.

Antibacterial Efficacy of Two Commercially Available Bacteriophage Formulations, Staphylococcal Bacteriophage and PYO Bacteriophage, Against Methicillin-Resistant Staphylococcus aureus: Prevention and Eradication of Biofilm Formation and Control of a Systemic Infection of Galleria mellonella Larvae.

Sessile bacteria growing on surfaces are more resistant to standard antibiotics than their planktonic counterpart. Due to their antimicrobial properties, bacteriophages have re-emerged as a promising approach to treat bacterial biofilm-associated infections. Here, we evaluated the ability of two commercially available phage formulations, Staphylococcal bacteriophage (containing the monophage Sb-1) and PYO bacteriophage (a polyphage), in preventing and eradicating an in vitro biofilm of methicillin-resistant Staphylococcus aureus (MRSA) by isothermal microcalorimetry and high-resolution confocal laser scanning microscopy (CLSM). Moreover, to assess the potential in vivo efficacy of both phage preparations, a Galleria mellonella model of MRSA systemic infection was used. Microcalorimetry measurement showed that 107 PFU/ml (the highest tested titer) of both phage formulations were able to inhibit planktonic growth in a concentration-dependent manner. However, MRSA biofilm was eradicated only by co-incubation of 5-7 days with the highest phage titers, respectively. In the experiments of biofilm prevention, isothermal microcalorimetry revealed that the heat production was completely abolished in the presence of sub-inhibitory titers (104 PFU/ml) of phages. These data were also confirmed by confocal laser scanning microscopy. Both phage formulations increased the survival of G. mellonella larvae preventing or treating MRSA infection compared to untreated control. In conclusion, tested phage formulations are promising for preventing device colonization and killing biofilm bacteria attached on a surface. Novel strategies for direct coating and release of phages from material should be investigated.

Synergistic Activity of Fosfomycin, Ciprofloxacin, and Gentamicin Against Escherichia coli and Pseudomonas aeruginosa Biofilms.

Gram-negative (GN) rods cause about 10% periprosthetic joint infection (PJI) and represent an increasing challenge due to emergence of antimicrobial resistance. Escherichia coli and Pseudomonas aeruginosa are among the most common cause of GN-PJI and ciprofloxacin is the first-line antibiotic. Due to emergence of fluoroquinolone resistance, we evaluated in vitro the activity of fosfomycin, ciprofloxacin, and gentamicin, alone and in combinations, against E. coli and P. aeruginosa biofilms. Conventional microbiological tests and isothermal microcalorimetry were applied to investigate the anti-biofilm activity of the selected antibiotics against standard laboratory strains as well as clinical strains isolated from patients with prosthetic joint associated infections. The biofilm susceptibility to each antibiotic varied widely among strains, while fosfomycin presented a poor anti-biofilm activity against P. aeruginosa. Synergism of two-pair antibiotic combinations was observed against different clinical strains from both species. Highest synergism was found for the fosfomycin/gentamicin combination against the biofilm of E. coli strains (75%), including a gentamicin-resistant but fosfomycin-susceptible strain, whereas the gentamicin/ciprofloxacin combination presented synergism with higher frequency against the biofilm of P. aeruginosa strains (71.4%). A hypothetical bacteriolysis effect of gentamicin could explain why combinations with this antibiotic seem to be particularly effective. Still, the underlying mechanism of the synergistic effect on biofilms is unknown. In conclusion, combinatorial antibiotic application has shown to be more effective against biofilms compared to monotherapy. Further in vivo and clinical studies are essential to define the potential treatment regimen based on our results.

Bacteriophages as Adjuvant to Antibiotics for the Treatment of Periprosthetic Joint Infection Caused by Multidrug-Resistant Pseudomonas aeruginosa.

We documented the adjunctive bacteriophage therapy to treat a chronic relapsing periprosthetic joint infection of the knee and chronic osteomyelitis of the femur caused by multidrug-resistant Pseudomonas aeruginosa The combined antibiotic-phage treatment eradicated the infection, and no side effects to phages were observed.

Real-Time Antimicrobial Susceptibility Assay of Planktonic and Biofilm Bacteria by Isothermal Microcalorimetry.

Most antimicrobials currently used in the clinical practice are tested as growth inhibitors against free-floating microorganisms in a liquid suspension, rather than against sessile cells constituting biofilms. Hence, reliable, fast, and reproducible methods for assessing biofilm susceptibility to antimicrobials are strongly needed. Isothermal microcalorimetry (IMC) is a nondestructive sensitive technique that allows for the real-time monitoring of microbial viability in the presence or absence of antimicrobial compounds. Therefore, the efficacy of specific antimicrobials, alone or in combination, may be promptly validated supporting the development of new drugs and avoiding the administration of ineffective therapies. Furthermore, the susceptibility of both planktonic and biofilm cells to antimicrobials can be conveniently assessed without the need for elaborated staining procedures and under nontoxic working conditions. Quantitative data regarding the antimicrobial effect against different strains might be collected by monitoring the microbial cell replication, and, more importantly, a dose-dependent activity can be efficiently detected by measuring the delay and decrease in the heat flow peak of the treated samples. A limitation of IMC for anti-biofilm susceptibility test is the inability to directly quantify the non-replicating cells in the biofilm or the total biomass. However, as IMC is a nondestructive method, the samples can be also analyzed by using different techniques, acquiring more information complementary to calorimetric data. IMC finds application also for the investigation of antibiotic eluting kinetics from different biomaterials, as well as for studying bacteriophages activity against planktonic and biofilm bacteria. Thus, the wide applicability of this ultra-sensitive and automated technique provides a further advance in the field of clinical microbiology and biomedical sciences.

Bacteriophage Sb-1 enhances antibiotic activity against biofilm, degrades exopolysaccharide matrix and targets persisters of Staphylococcus aureus.

Most antibiotics have limited or no activity against bacterial biofilms, whereas bacteriophages can eradicate biofilms. We evaluated whether Staphylococcus aureus-specific bacteriophage Sb-1 could eradicate biofilm, both alone and in combination with different classes of antibiotics, degrade the extracellular matrix and target persister cells. Biofilm of methicillin-resistant S. aureus (MRSA) ATCC 43300 was treated with Sb-1 alone or in (simultaneous or staggered) combination with fosfomycin, rifampin, vancomycin, daptomycin or ciprofloxacin. The matrix was visualized by confocal fluorescent microscopy. Persister cells were treated with 104 and 107 plaque-forming units (PFU)/mL Sb-1 for 3 h in phosphate-buffered saline (PBS), followed by colony-forming units (CFU) counting. Alternatively, bacteria were washed and incubated in fresh brain heart infusion (BHI) medium and bacterial growth assessed after a further 24 h. Pretreatment with Sb-1 followed by the administration of subinhibitory concentrations of antibiotic caused a synergistic effect in eradicating MRSA biofilm. Sb-1 determined a dose-dependent reduction of matrix exopolysaccharide. Sb-1 at 107 PFU/mL showed direct killing activity on ≈ 5 × 105 CFU/mL persisters. However, even a lower titer had lytic activity when phage-treated persister cells were inoculated in fresh medium, reverting to a normal-growing phenotype. This study provides valuable data on the enhancing effect of Sb-1 on antibiotic efficacy, exhibiting specific antibiofilm features. Sb-1 can degrade the MRSA polysaccharide matrix and target persister cells and is therefore suitable for treatment of biofilm-associated infections.

Real-time assessment of bacteriophage T3-derived antimicrobial activity against planktonic and biofilm-embedded Escherichia coli by isothermal microcalorimetry.

Bacterial biofilms, highly resistant to the conventional antimicrobial therapy, remain an unresolved challenge pressing the medical community to investigate new and alternative strategies to fight chronic implant-associated infections. Recently, strictly lytic bacteriophages have been revalued as powerful agents to kill antibiotic-resistant bacteria even in biofilm. Here, the interaction of T3 bacteriophage and planktonic and biofilm Escherichia coli TG1, respectively, was evaluated using isothermal microcalorimetry. Microcalorimetry is a non-invasive and highly sensitive technique measuring growth-related heat production of microorganisms in real-time. Planktonic and biofilm E. coli TG1 were exposed to different titers of T3 bacteriophage, ranging from 102 to 107 PFU/ml. The incubation of T3 with E. coli TG1 showed a strong inhibition of heat production both in planktonic and biofilm already at lower bacteriophage titers (103 PFU/ml). This method could be used to screen and evaluate the antimicrobial potential of different bacteriophages, alone and in combination with antibiotics in order to improve the treatment success of biofilm-associated infections.