Efficacy of different bioactive glass S53P4 formulations in biofilm eradication and the impact of pH and osmotic pressure.

AIM: The challenging properties of biofilm-associated infections and the rise of multidrug-resistant bacteria are prompting the exploration of alternative treatment options. This study investigates the efficacy of different bioactive glass (BAG) formulations - alone or combined with vancomycin - to eradicate biofilm. Further, we study the influence of BAG on pH and osmotic pressure as important factors limiting bacterial growth. METHOD: Different BAG S53P4 formulations were used for this study, including (a) powder (<45 µm), (b) granules (500-800 µm), (c) a cone-shaped scaffold and (d) two putty formulations containing granules with no powder (putty A) or with additional powder (putty B) bound together by a synthetic binder. Inert glass beads (1.0-1.3 mm) were included as control. All formulations were tested in a concentration of 1750 mg/ml in Müller-Hinton-Broth against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). Vancomycin was tested at the minimum-inhibitory concentration for each strain. Changes in pH and osmolality over time were assessed at 0 h, 24 h, 72 h and 168 h. RESULTS: All tested BAG formulations showed antibiofilm activity against MRSA and MRSE. Powder and putty B were the most effective formulations suppressing biofilm leading to its complete eradication after up to 168 h of co-incubation, followed by granules, scaffold and putty A. In general, MRSE appeared to be more susceptible to bioactive glass compared to MRSA. The addition of vancomycin had no substantial impact on biofilm eradication. We observed a positive correlation between a higher pH and higher antibiofilm activity. CONCLUSIONS: BAG S53P4 has demonstrated efficient biofilm antibiofilm activity against MRSA and MRSE, especially in powder-containing formulations, resulting in complete eradication of biofilm. Our data indicate neither remarkable increase nor decrease in antimicrobial efficacy with addition of vancomycin. Moreover, high pH appears to have a direct antimicrobial impact; the role of high osmolality needs further investigation.

Novel Stenotrophomonas maltophilia Bacteriophage as Potential Therapeutic Agent.

A novel bacteriophage CUB19 specific to the bacterial species Stenotrophomonas maltophilia was isolated from hospital sewage and characterized as a new species belonging to a proposed new phage genus 'Cubvirus' (Caudoviricetes). Its genome contains a total of 48,301 bp and 79 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified on the phage genome. CUB19 showed stability over a wide range of temperatures (-20 °C-60 °C) and pH values (pH 3-pH 13). Despite its narrow host range, this phage has potent observed antimicrobial and antibiofilm activity. A time-killing curve assay showed significant biofilm reduction after 24 h exposure to CUP19. Isothermal microcalorimetry assays investigating phage-antibiotic combinations revealed the effectiveness of CUB19 during co-administration with increasing antibiotic doses, regardless of the administration approach (simultaneous or staggered). These are encouraging indications for its application as a targeted therapeutic agent against resilient biofilm-associated Stenotrophomonas infections.

In Vitro Evaluation of Five Newly Isolated Bacteriophages against E. faecalis Biofilm for Their Potential Use against Post-Treatment Apical Periodontitis.

State-of-the-art treatment of root canal infection includes the use of mechanical debridement and chemical agents. This disinfection method is limited, and microorganisms can remain in the canal system. Enterococcus faecalis appears with a high prevalence in secondary and persistent root canal infections and can be linked to endodontic treatment failure due to its various resistance mechanisms. Here, we evaluated the activity of newly isolated bacteriophages against clinical isolates of E. faecalis (including one vancomycin- and gentamicin-resistant strain) as a single treatment or in combination with gentamicin and vancomycin. For the resistant strain, daptomycin and fosfomycin were tested. Sixteen E. faecalis strains were used to screen for the presence of bacteriophages in sewage. Five different bacteriophages were characterized in terms of virion morphology, host range and killing-kinetics against each E. faecalis host strain. To investigate the antibiofilm effect of antibiotic and phages, E. faecalis biofilm was grown on porous glass beads and treated with different antibiotic concentrations and with isolated bacteriophages alone or in staggered combinations. A strong biofilm reduction was observed when phages were combined with antibiotic, where combinations with gentamicin showed a better outcome compared to vancomycin. Regarding the resistant strain, daptomycin had a superior antibiofilm effect than fosfomycin.

Novel Bacteriophage Specific against Staphylococcus epidermidis and with Antibiofilm Activity.

Staphylococcus epidermidis has emerged as the most important pathogen in infections related to indwelling medical devices, and although these infections are not life-threatening, their frequency and the fact that they are extremely difficult to treat represent a serious burden on the public health system. Treatment is complicated by specific antibiotic resistance genes and the formation of biofilms. Hence, novel therapeutic strategies are needed to fight these infections. A novel bacteriophage CUB-EPI_14 specific to the bacterial species S. epidermidis was isolated from sewage and characterized genomically and phenotypically. Its genome contains a total of 46,098 bp and 63 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified in the phage genome. CUB-EPI_14 showed stability over a wide range of temperatures (from -20 °C to 50 °C) and pH values (pH 3-pH 12) and a narrow host range against S. epidermidis. Potent antimicrobial and antibiofilm activities were observed when the phage was tested against a highly susceptible bacterial isolate. These encouraging results open the door to new therapeutic opportunities in the fight against resilient biofilm-associated infections caused by S. epidermidis.

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.

On the function of biosynthesized cellulose as barrier against bacterial colonization of VAD drivelines.

Bacterial colonization of drivelines represents a major adverse event in the implantation of left ventricular assist devices (L-VADs) for the treatment of congestive heart failure. From the external driveline interface and through the skin breach, pathogens can ascend to the pump pocket, endangering the device function and the patient's life. Surface Micro-Engineered Biosynthesized cellulose (BC) is an implantable biomaterial, which minimizes fibrotic tissue deposition and promotes healthy tissue regeneration. The topographic arrangement of cellulose fibers and the typical material porosity support its potential protective function against bacterial permeation; however, this application has not been tested in clinically relevant animal models. Here, a goat model was adopted to evaluate the barrier function of BC membranes. The external silicone mantle of commercial L-VAD drivelines was implanted percutaneously with an intervening layer of BC to separate them from the surrounding soft tissue. End-point evaluation at 6 and 12 weeks of two separate animal groups revealed the local bacterial colonization at the different interfaces in comparison with unprotected driveline mantle controls. The results demonstrate that the BC membranes established an effective barrier against the bacterial colonization of the outer driveline interface. The containment of pathogen infiltration, in combination with the known anti-fibrotic effect of BC, may promote a more efficient immune clearance upon driveline implantation and support the efficacy of local antibiotic treatments, therefore mitigating the risk connected to their percutaneous deployment.

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.

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.

Antimicrobial activity of bioactive glass S53P4 against representative microorganisms causing osteomyelitis - Real-time assessment by isothermal microcalorimetry.

Bioactive glass (BAG) is a synthetic bone substitute with intrinsic antimicrobial properties, used for bone defect filling. We evaluated the antimicrobial activity of two formulations of BAG S53P4 against representative pathogens of osteomyelitis: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli and Candida albicans. Antimicrobial activity of BAG S53P4 was assessed by isothermal microcalorimetry, a highly sensitive assay measuring metabolic-related microbial heat production in real-time. Standard CFUs-counting was performed in parallel. BAG granules (diameter 500-800 µm) and powder (<45 µm) were evaluated in two concentrations (400 and 800 mg/ml). Isothermal microcalorimetry was performed in glass ampoules containing growth medium, BAG and test microorganism, heat production was measured for 24 h. BAG S53P4 inhibited heat production of most-tested microorganisms with heat reduction of 60%-98% compared to positive control after 24 h of exposure to the highest-tested concentration (800 mg/ml). BAG S53P4 in powder formulation (<45 µm) inhibited more microbial growth than in granule formulation (500-800 µm), with the exception of C. albicans for which both formulations presented similar inhibition rates ranging between 87 % and 97 %. The BAG inhibitory ratios estimated from the variation in the growth rate constants of each microorganism compared to the growth control ranged between 2.55 % and 100 %. Comparable results were obtained by CFUs-counting, with complete reduction in cell viability of most microorganisms after ≤ 24 h of microbial exposure to BAG S53P4 powder. In summary, BAG S53P4 demonstrated efficient inhibition of microbial growth, especially in powder formulation.

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.

In vitro antimicrobial activity against Abiotrophia defectiva and Granulicatella elegans biofilms.

OBJECTIVES: To determine the efficacy of different antibiotics (alone or in combination) against Abiotrophia defectiva and Granulicatella elegans biofilms and to investigate the anti-biofilm activity of gentamicin alone versus blood culture isolates from both species. METHODS: The activity of benzylpenicillin, clindamycin, daptomycin, fosfomycin, gentamicin, levofloxacin and rifampicin against 24-hour-old biofilms of A. defectiva and G. elegans was investigated in vitro by conventional microbiological methods and isothermal microcalorimetry. RESULTS: For planktonic bacteria, the MIC values of tested antibiotics ranged from 0.016 to 64 mg/L, as determined by microcalorimetry. Higher antibiotic concentrations, ranging from 1 to >1024 mg/L, were needed to produce an effect on biofilm bacteria. Gentamicin was an exception as it was active at 1 mg/L against both planktonic and biofilm G. elegans. A synergistic effect was observed when daptomycin was combined with benzylpenicillin, gentamicin or rifampicin against A. defectiva biofilms and when gentamicin was combined with rifampicin or levofloxacin against G. elegans biofilms. A. defectiva clinical isolates displayed greater variability in gentamicin susceptibility as compared with G. elegans strains. CONCLUSIONS: Antimicrobial susceptibility profiles vary widely between Abiotrophia and Granulicatella biofilms, and synergistic effects of the tested antibiotics were heterogeneous. The clinical relevance of these in vitro observations needs to be confirmed in experimental in vivo conditions and human trials, before guidelines for the treatment of A. defectiva and G. elegans infections are established. This study suggests the benefit of further clinical exploration of antibiotic combinations with anti-biofilm effect.

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.

Synergistic antibiotic activity against planktonic and biofilm-embedded Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis.

OBJECTIVES: To determine the antimicrobial activity against streptococcal biofilm in species mostly isolated from implant-associated infections and examine the effect of enzyme treatment of biofilm on the antimicrobial activity of different antibiotics. METHODS: The activities of fosfomycin, rifampicin, benzylpenicillin, daptomycin, gentamicin, levofloxacin, proteinase K and their combinations on planktonic and/or biofilm-embedded standard laboratory strains of Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis were investigated in vitro by standard methods and isothermal microcalorimetry. RESULTS: MIC values obtained for the tested antimicrobials against planktonic bacteria ranged from 0.016 to 128 mg/L for the three species tested. Higher antibiotic concentrations were usually required to reduce biofilm in comparison with planktonic bacteria, with the exception of gentamicin, for which similar concentrations (4-16 mg/L) exerted an effect on both planktonic and biofilm cells. A synergistic effect against the streptococcal biofilm of the three species was observed when gentamicin was combined with benzylpenicillin or with rifampicin. Moreover, antibiotic concentrations comparable to the MIC observed against planktonic cells induced a strong reduction of viable bacteria in proteinase K pre-treated biofilm. CONCLUSIONS: This study shows that the combination of gentamicin with either benzylpenicillin or rifampicin exerts a synergistic effect against biofilms produced by the tested streptococci strains in vitro. Our results also suggest that coupling a dispersal agent with conventional antibiotics may facilitate their access to the bacteria within the biofilm. In vivo and clinical studies are needed in order to confirm whether such a strategy may be effective in the treatment of implant-associated infections caused by streptococci.