Bacteriophages as an alternative strategy in the treatment and prevention of implant-associated infections

Bacterial biofilms growing on surfaces are highly refractory to the conventional antimicrobial therapy. Complete eradication of the biofilm consists of invasive surgical intervention combined with prolonged systemic antimicrobial administration. Hence, the development of new and alternative strategies to fight chronic implant-associated infections gained increasing interest. Due to their antimicrobial properties, bacteriophages have been revalued as a promising agents to treat bacterial implant-associated infections, especially related to multidrug resistant bacteria. The aim of this work was to investigate the antimicrobial activity of different bacteriophages, alone or in combination with conventional antibiotics as alternative approach, in order to improve the treatment of mono and dual-species implant-associated infections. Results revealed the wide potential of isothermal microcalorimetry (IMC) for screening and evaluating bacteriophage activity to predict a treatment success on demand for biofilm infections. Moreover, different methods such as sonication, confocal laser scanning and scanning electron microscopy were combined for the evaluation of anti-biofilm activities of bacteriophages. Higher phage titers and longer exposure of bacteriophages were required to kill biofilm bacteria in comparison with planktonic counterparts. However, in the experiments of biofilm prevention, our IMC results presented that the heat production was abolished in the presence of subinhibitory titers of phages within 24 hours. Additionally, tested phage formulations increased the survival of Galleria mellonella larvae Staphylococcus aureus infection compared to untreated control. We also determined that conventional antibiotics had high minimum biofilm eradicating concentration (MBEC) values (ranging from 128 to >4096 µg/mL) when investigated alone against biofilms. The co-administration of phages with antibiotics improved considerably the antibiotic efficacy against mono and dual-species biofilm, especially after staggered exposure, strongly reducing the MBEC values. Interestingly, staphylococcal bacteriophage Sb-1 demonstrated a dose-dependent reduction of the exopolysaccharide matrix, whereas PYO bacteriophage had no effect, suggesting that some phages could enhance the penetration of antibiotics to the deeper layers of biofilms. This work also showed that even lower Sb-1 phage titers can kill phage-treated Staphylococcus aureus-persistent cells, when metabolically inactive cells were inoculated in fresh medium and returned to a normally growing phenotype. Overall, this thesis generates new insights for preventing implant colonization and killing biofilm bacteria attached on a surface. Further pre-clinical and clinical trials are needed towards their clinical application.Bakterielle Biofilme, die auf Oberflächen wachsen, sind gegenüber der konventionellen antimikrobiellen Therapie sehr refraktär. Die vollständige Eradikation des Biofilms besteht aus einem invasiven chirurgischen Eingriff in Kombination mit einer längeren systemischen antimikrobiellen Verabreichung. Daher gewann die Entwicklung neuer und alternativer Strategien zur Bekämpfung chronischer implantatassoziierter Infektionen zunehmend an Interesse. Aufgrund ihrer antimikrobiellen Eigenschaften wurden Bakteriophagen als vielversprechende Agent zur Behandlung bakterieller implantat-assoziierter Infektionen, insbesondere im Zusammenhang mit multiresistenten Bakterien, neu bewertet. Ziel dieser Arbeit war es, die antimikrobielle Aktivität verschiedener Bakteriophagen, allein oder in Kombination mit konventionellen Antibiotika als alternativen Ansatz, zu untersuchen, um die Behandlung von Mono- und Dual-Spezies-Implantat-assoziierten Infektionen zu verbessern. Die Ergebnisse zeigten das breite Potenzial der isothermen Mikrokalorimetrie (IMK) für das Screening und die Bewertung der Aktivität von Bakteriophagen zur Vorhersage eines Behandlungserfolgs bei Biofilm-Infektionen auf Nachfrage. Darüber hinaus wurden verschiedene Methoden wie Sonikation, konfokales Laserscanning und Rasterelektronenmikroskopie kombiniert, um die Anti-Biofilm-Aktivitäten von Bakteriophagen zu bewerten. Höhere Phagentiter und eine längere Exposition der Bakteriophagen waren erforderlich, um Biofilm- Bakterien im Vergleich zu planktonischen Pendants abzutöten. In den Experimenten zur Biofilm-Prävention zeigten unsere IMK-Ergebnisse jedoch, dass die Wärmeproduktion in Gegenwart von subinhibitorischen Titern von Phagen innerhalb von 24 Stunden aufgehoben wurde. Darüber hinaus erhöhten getestete Phagenformulierungen die Überlebensrate einer Infektion mit Galleria mellonella Larven Staphylococcus aureus im Vergleich zur unbehandelten Kontrolle. Wir stellten auch fest, dass konventionelle Antibiotika hohe minimale Biofilm eradizierende Konzentration (MBEK) Werte (zwischen 128 und >4096 µg/mL) aufwiesen, wenn sie allein gegen Biofilme untersucht wurden. Die Verabreichung von Phagen mit Antibiotika verbesserte die Antibiotika-Wirksamkeit gegen Mono- und Doppelspezies-Biofilm erheblich, insbesondere nach gestaffelter Exposition, wodurch die MBEK-Werte stark reduziert wurden. Interessanterweise zeigte der Staphylokokken Bakteriophage Sb-1 eine dosisabhängige Reduktion der Exopolysaccharidmatrix, während PYO Bakteriophage keine Wirkung zeigte, was darauf hindeutet, dass einige Phagen das Eindringen von Antibiotika in die tieferen Schichten von Biofilmen verbessern könnten. Diese Arbeit zeigte auch, dass sogar noch niedrigere Sb-1- Phagen-Titer phagenbehandelte Staphylococcus aureus -persistente Zellen abtöten können, wenn metabolisch inaktive Zellen in frischem Medium beimpft wurden und zu einem normal wachsenden Phänotyp zurückkehren. Insgesamt generiert diese Arbeit neue Erkenntnisse zur Verhinderung der Besiedlung von Implantaten und zur Abtötung von auf einer Oberfläche anhaftenden Biofilmbakterien. Zu ihrer klinischen Anwendung sind weitere präklinische und klinische Studien erforderlich

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

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

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

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 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

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

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