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

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

Combining C-H functionalisation and flow photochemical heterocyclic metamorphosis (FP-HM) for the synthesis of benzo[1,3]oxazepines

C-H Activation/functionalisation and Flow Photochemical Heterocyclic Metamorphosis (FP-HM) have been combined to synthesize a library of benzo[1,3]oxazepines, a rarely described heterocyclic family. This combined protocol allows a range of arylated products to be made from simple starting materials, and the cheap flow photochemical system has proven effective for rapid synthesis of gram-quantities of benzo[1,3]oxazepines

Alcune osservazioni sull’iscrizione delle terme di Gebamondo (CIL VIII, 25362)

I due frammenti di architrave rivenuti a Tunisi nel 1894 e nel 1907 riportanti un’iscrizione in versi che loda le Terme di Gebamondo sono stati da sempre considerati parte di un singolo monumento. Tuttavia, il confronto paleografico tra i due mostra che sono stati realizzati da mani diverse. Sostengo che i frammenti appartengano a due copie della stessa iscrizione. Poi, ipotizzando che le due copie avessero una impaginazione simile, ho verificato se le varie integrazioni proposte per la parte centrale mancante siano compatibili con lo spazio della lacuna

Guido Canella ristrutturazione del Palazzo di Giustizia ad Ancona 1975-1989. Dentro un fragile "resto"

Appointed for carrying out the restoration of the Palace of Justice in Ancona, Guido Canella inserts, inside a fragile yet tenaciously preserved “ruin”, another architecture, a new measure set within the restored walls of the 19th century palace. On the inside a covered square with urban features overlooked from above by the courtrooms and other judiciary offices