The controlled release and activity of antimicrobials from biomaterials

The increasing use of medical indwelling devices has triggered the development of difficult-to-treat infections due to biofilm formation on the implant surface. Nowadays, infection treatment consists of invasive surgical intervention combined with long-course systemic antimicrobial infusion. However, treatment failure due to ineffective surgical debridement and inappropriate antibiotic therapy is a troublesome reality. Hence, the development and formulation of novel smart biomaterials as scaffolds for the local release of drugs gained increasing interest. The aim of this work was to investigate the anti-biofilm and anti-persister activity of selected antimicrobials, to fine-tune the isothermal microcalorimetry (IMC) as antimicrobial susceptibility method and to study the formulation, release characteristics and antimicrobial activity of different biomaterials (including recently developed hydrogels) loaded with therapeutically relevant antimicrobials for the management of orthopedic implant-associated infections. Results showed that high concentrations of vancomycin enriched a Staphylococcus aureus biofilm in persister cells, which are mainly responsible for infection recalcitrance. It was reported that a deep analysis of IMC data enables the detection and identification in real-time of persister cells, proving the suitability of this method for the characterization of new anti-persister compounds and biomaterials. Interestingly, the combined use of the glycopeptide with daptomycin proved highly bactericidal against persisters. The lipopeptide was then successfully loaded into soft and fully degradable thermosensitive hydrogels, which released high concentrations of active drug (widely exceeding the minimum bactericidal concentration) in a controlled manner against S. aureus for at least 15 days. Similarly, high titers of bacteriophages were released from smart thermoresponsive hydrogels in a controlled manner for at least 7 days. As a comparison, also the gentamicin elution profile from bone graft substitutes was investigated, revealing a timely burst release of bactericidal concentrations. This work demonstrated that high doses of commonly used antibiotics may select for persister cells in biofilms. In fact, recalcitrance and extreme resistance of biofilm-associated infections affecting the musculoskeletal system are deeply influenced by metabolically inactive cells that, to the best of our knowledge, were here identified and characterized for the first time using IMC. Smart bioscaffolds may serve as drug reservoirs and offer optimal conditions for the release of high doses of anti-biofilm and anti-persister molecules in situ, providing relevant progress to the fast-growing field of biomaterials and advances towards their clinical application.Die zunehmende Verwendung von medizinischen Verweilvorrichtungen hat die Entwicklung von schwer zu behandelnden Infektionen durch Biofilmbildung auf den Oberflächen von Implantaten bestimmt. Heutzutage besteht die Behandlung von Infektionen in einer Kombination aus operativen Eingriffen und systemischen antimikrobiellen Infusionen. Behandlungsversagen aufgrund ineffizienten chirurgischen Debridements und unpasssende antibiotische Therapie sind jedoch eine problematische Realität. Daher hat die Entwicklung und Formulierung neuartiger intelligenter Biomaterialien als Gerüst für die lokale Freisetzung von Arzneimitteln zunehmendes Interesse gefunden. Das Ziel dieser Arbeit war die Untersuchung der Anti-Biofilm- und Anti-Persister-Aktivität ausgewählter antimikrobieller Mittel, die Feinabstimmung der isothermen Mikrokalorimetrie (IMC) als antimikrobielle Suszeptibilitätsmethode und die Untersuchung antimikrobieller Potenziale diverser Biomaterialien, die mit therapeutisch relevanten Mitteln zur Behandlung von orthopädischen implantatassoziierten Infektionen beladen sind. Die Ergebnisse zeigten, dass hohe Konzentrationen von Vancomycin einen Staphylococcus aureus-Biofilm in Persisterzellen angereichert haben, die hauptsächlich für die Problemhaftigkeit der Infektionen verantwortlich sind. Eine gründliche Analyse von Daten der isothermen Mikrokalorimetrie (IMC) ermöglicht den Nachweis und die Identifizierung von persistenten Zellen in Echtzeit, was die Eignung dieser Methode für die Charakterisierung neuer anti-persistenter Verbindungen und Biomaterialien belegt. Interessanterweise erwies sich die kombinierte Verwendung des Glycopeptids mit Daptomycin als stark bakterizid gegen Persister. Das Lipopeptid wurde erfolgreich in vollständig abbaubare wärmeempfindliche Hydrogele geladen, die nachweislich auf kontrollierte Weise hohe Konzentrationen an Wirkstoff freisetzen, die die minimale bakterizide Konzentration gegen S. aureus für mindestens 15 Tage weit überschreiten. In ähnlicher Weise wurden hohe Titer von Bakteriophagen mindestens 7 Tage lang kontrolliert aus intelligenten thermoresponsiven Hydrogelen freigesetzt. Zum Vergleich wurde auch das Gentamicin-Elutionsprofil von Knochentransplantatersatzmitteln untersucht, was eine zeitnahe Freisetzung bakterizider Konzentrationen aufzeigt. Diese Arbeit zeigte, dass häufig verwendete Antibiotika in hohen Dosen persistente Zellen in Biofilmen selektieren können. Tatsächlich werden Rekalzitranz und extreme Resistenz von Biofilm- assoziierten Infektionen im Bewegungsapparat stark durch metabolisch inaktive Zellen beeinflusst, die hier zum ersten Mal mit IMC identifiziert und charakterisiert wurden. Intelligente Bioscaffolds können als Reservoir für Arzneimittel dienen und bieten optimale Bedingungen für die Freisetzung hoher Dosen von Biofilm- und Anti-Persister-Molekülen in situ, was dem schnell wachsenden Biomaterialbereich und der klinischen Anwendung wichtige Fortschritte bringt

Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) 7> 256 mu g/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 mu g/ml] and rifampin (MBBC and MBEC = 128 mu g/ml). After the treatment of MRSA biofilm with 1024 mu g/ml vancomycin, similar to 5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells

Comparison of Sonication With Chemical Biofilm Dislodgement Methods Using Chelating and Reducing Agents: Implications for the Microbiological Diagnosis of Implant Associated Infection

The diagnosis of implant-associated infections is hampered due to microbial adherence and biofilm formation on the implant surface. Sonication of explanted devices was shown to improve the microbiological diagnosis by physical removal of biofilms. Recently, chemical agents have been investigated for biofilm dislodgement such as the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent dithiothreitol (DTT). We compared the activity of chemical methods for biofilm dislodgement to sonication in an established in vitro model of artificial biofilm. Biofilm-producing laboratory strains of Staphylococcus epidermidis (ATCC 35984), S. aureus (ATCC 43300), E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 53278) were used. After 3 days of biofilm formation, porous glass beads were exposed to control (0.9% NaCl), sonication or chemical agents. Quantitative and qualitative biofilm analyses were performed by colony counting, isothermal microcalorimetry and scanning electron microscopy. Recovered colony counts after treatment with EDTA and DTT were similar to those after exposure to 0.9% NaCl for biofilms of S. epidermidis (6.3 and 6.1 vs. 6.0 log10 CFU/mL, S. aureus (6.4 and 6.3 vs. 6.3 log10 CFU/mL), E. coli (5.2 and 5.1 vs. 5.1 log10 CFU/mL and P. aeruginosa (5.1 and 5.2 vs. 5.0 log10 CFU/mL, respectively). In contrast, with sonication higher CFU counts were detected with all tested microorganisms (7.5, 7.3, 6.2 and 6.5 log10 CFU/mL, respectively) (p <0.05). Concordant results were observed with isothermal microcalorimetry and scanning electron microscopy. In conclusion, sonication is superior to both tested chemical methods (EDTA and DTT) for dislodgement of S. epidermidis, S. aureus, E. coli and P. aeruginosa biofilms. Future studies may evaluate potential additive effect of chemical dislodgement to sonication

Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) &gt; 256 μg/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 μg/ml] and rifampin (MBBC and MBEC = 128 μg/ml). After the treatment of MRSA biofilm with 1024 μg/ml vancomycin, ∼5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells

In vitro anti-biofilm activity of a biphasic gentamicin-loaded calcium sulfate/hydroxyapatite bone graft substitute

Bone and implant-associated infections caused by microorganisms that grow in biofilms are difficult to treat because of persistence and recurrence of infection. Along with surgical debridement, the combination of systemic and local administration of antimicrobials represents the background for an efficient treatment strategy. Gentamicin is one of most used antibiotics for the local treatment of bone-related infections, alone or in combination, due to its bactericidal and broad-range activity. Gentamicin-loaded beads (GLBs), composed of calcium sulfate/hydroxyapatite, were assessed for their in vitro antimicrobial activity against planktonic and biofilm S. agalactiae, S. aureus, S. epidermidis, E. faecalis and E. coli, using standard methods and ultra-sensitive isothermal microcalorimetry. Gentamicin released from GLBs to clinically relevant concentrations (200–2500 μg/mL) within 1 h was able to kill planktonic S. agalactiae, S. epidermidis and E. coli at lower concentrations (MIC: ≤4 μg/mL). Moreover, 12 and 23 μg/mL of released gentamicin were able to prevent bacterial adhesion and suppress a 24 h-old biofilm of E. coli, respectively. Conversely, higher amounts of antibiotic, ranging from 171 to 1260 μg/mL, were needed to prevent and eradicate biofilms of gram-positive bacteria. Likewise, the emergence of resistance to GLBs in vitro and the bacterial attachment on the bone graft substitute, when the amount of gentamicin in the material is reduced, were also reported. This study provides further information regarding the in vitro anti-biofilm activity of the biphasic gentamicin-loaded bone graft substitute, suggesting the validity of this antibiotic-loaded material for the prophylaxis and treatment of bone and implant-associated infections

Injectable hyaluronic acid/PEG-p(HPMAm-lac)-based hydrogels dually cross-linked by thermal gelling and Michael addition

Fast in situ forming thermosensitive hydrogels consisted of vinyl sulfone bearing p(HPMAm-lac(1-2))-PEG-p(HPMAm-lac(1-2)) triblock copolymers and thiol modified hyaluronic acid were prepared via a dual cross-linking strategy based on thermal gelation at 37 degrees C and simultaneous Michael addition cross-linking between vinyl sulfone and thiol moieties. The formation of a chemical network was varied within a time period of 9-60 min by controlling the degree of vinyl sulfone derivatization, the triblock copolymer concentration and the degree of thiolation. The extent of thiol substitution on the polysaccharidic hyaluronan chain markedly affected the physical and mechanical properties, as well as the swelling and degradation behavior of the resulting networks, as confirmed by rheology, water uptake experiments And degradation tests. In addition, the developed hydrogels showed a good cytocompatibility in vitro during a timeframe of 21 days both for mouse bone marrow stromal cell and for NIH 3T3 mouse fibroblasts. The developed hydrogels showed potential as promising injectable biomaterials with tunable gelation kinetics, adjustable mechanical properties, swelling and degradation times. These biomaterials could find application both as a regenerative cell matrix and as controlled drug delivery system. (C) 2015 Elsevier Ltd. All rights reserved

Daptomycin-loaded biodegradable thermosensitive hydrogels enhance drug stability and foster bactericidal activity against Staphylococcus aureus

A drug delivery system based on fully biodegradable thermosensitive hydrogels enabling controlled antibiotic release may support the management of implant-associated infections. In this work, the lipopeptide antibiotic daptomycin was encapsulated in hydrogel networks consisting of vinyl sulfonated triblock copolymers of PEG-p(HPMAm-lac1,2) and thiolated hyaluronic acid. High concentrations of active daptomycin exceeding the minimum biofilm eradicating concentration were sustainably eluted from the biodegradable carrier

Detection of drug resistance mutations at low plasma HIV-1 RNA load in a European multicentre cohort study

Guidelines indicate a plasma HIV-1 RNA load of 500-1000 copies/mL as the minimal threshold for antiretroviral drug resistance testing. Resistance testing at lower viral load levels may be useful to guide timely treatment switches, although data on the clinical utility of this remain limited. We report here the influence of viral load levels on the probability of detecting drug resistance mutations (DRMs) and other mutations by routine genotypic testing in a large multicentre European cohort, with a focus on tests performed at a viral load <1000 copies/mL