PRECLINICAL AND PROTEOMIC EVALUATION OF A STAPHYLOCOCCUS EPIDERMIDIS CLINICAL ISOLATE IN ORTHOPEDIC BIOFILM-RELATED INFECTIONS

Il termine pseudoartrosi \ue8 riferito a un tipo di frattura caratterizzata dall\u2019impossibilit\ue0 di una fisiologica guarigione. Le pseudoartrosi sono tra le complicazioni ortopediche pi\uf9 complesse non solo a livello clinico, ma anche a livello socio-economico incidendo drasticamente sia sulla qualit\ue0 di vita dei pazienti colpiti, sia sulle casse dei Sistema Sanitario Nazionale. Si stima che il 5-10% dei casi di frattura vada incontro allo sviluppo di pseudoartrosi e generalmente le ossa maggiormente interessate a questo fenomeno sono le ossa lunghe, tra le quali tibia, femore, omero, radio e ulna. Lo sviluppo di pseudoartrosi pu\uf2 conseguire la perdita di sostanza ossea, ad esempio a seguito di traumi o alla resezione di tumori, ma non solo, pu\uf2 anche derivare da una frattura che non riesce a consolidarsi. I fattori che possono maggiormente influenzare la corretta guarigione del tessuto osseo sono molteplici e spesso correlati allo stato di salute del paziente: la presenza di malattie metaboliche, l\u2019et\ue0, le abitudini alimentare e il fumo incidono drasticamente sul processo di guarigione della frattura. Un\u2019ulteriore causa dello sviluppo di pseudoartrosi \ue8 l\u2019insorgere di infezioni date da contaminazioni che possono susseguire fratture esposte o che possono avere luogo in sede operatoria. In questi casi si parla di pseudoartrosi settiche. \uc8 stato stimato che circa i due terzi dei casi clinici di pseudoartrosi settiche sono dovuti alla presenza di stafilococchi e pi\uf9 specificatamente alla presenza di Staphylococcus aureus and Staphylococcus epidermidis. In particolare, S. epidermidis \ue8 stato recentemente riconosciuto come un importante patogeno opportunista coinvolto nell\u2019aumento di infezioni nosocomiali, essendo comunemente presente sulla cute umana. S. epidermidis \ue8 un microrganismo commensale presenta fisiologicamente nella flora di pelle e mucose. A causa della sua presenza ubiquitaria \ue8 spesso difficile distinguere se un isolato clinico di S. epidermidis rappresenta l\u2019agente causale di un\u2019infezione o un contaminante non specifico della coltura. A differenza di S. aureus, S. epidermidis non esprime molteplici fattori di virulenza; \ue8 contraddistinto come patogeno dalla abilit\ue0 di formare biofilm sulla superficie di impianti o device introdotti all\u2019interno del nostro corpo (ad esempio protesi, pacemakers, cateteri ecc.). Il biofilm \ue8 una spessa matrice extracellulare secreta da batteri sessili irreversibilmente attaccati ad un substrato. All\u2019interno del biofilm i microrganismi creano comunit\ue0 organizzate in cui, a seconda delle influenze ambientali, la comunicazione tra cellula e cellula regola l\u2019espressione di geni coinvolti in meccanismi di sopravvivenza. Pertanto il biofilm costituisce una nicchia protettiva, un luogo in cui proliferare e sfuggire alle difese immunitarie dell\u2019ospite e ai trattamenti antibiotici, portando allo sviluppo di antibiotico resistenze. Un mezzo fondamentale e necessario per lo studio della patogenesi delle pseudoartrosi settiche sono i modelli preclinici, grazie ai quali \ue8 possibile indagare come i batteri interagiscano con l\u2019impianto e formino biofilm su di esso. In letteratura, ad oggi, non sono presenti modelli di pseudoartrosi settica causata da S. epidermidis e pertanto nella prima fase del progetto di dottorato \ue8 stato quindi generato un modello di pseudoartrosi settica in grado di mimare un\u2019infezione nosocomiale caratterizzata dalla presenza di un impianto metallico, al fine ultimo di indagare nuove strategie preventive e terapeutiche. Con l\u2019obiettivo di identificare la carica batterica minima in grado di portare allo sviluppo di pseudoartrosi settiche, i ratti sono stati sottoposti a una frattura femorale in cui, a seconda del gruppo sperimentale, sono state inoculate diverse concentrazioni di S. epidermidis meticillino-resistente (MRSE). Grazie a questo modello siamo stati in grado di definire tre differenti modelli preclinici. \uc8 stato infatti possibile dimostrare come un basso inoculo batterico (10^3 CFU/inoculo) \ue8 in grado di determinare un modello subclinico di pseudoartrosi settica, caratterizzata dalla sporadica presenza di pochi segni clinici di infezione. Abbiamo inoltre descritto come un inoculo intermedio (10^5 CFU/inoculo) sia in grado di determinare segni acuti di infezione e come un inoculo alto (10^8 CFU/inoculo) sia capace di portare ad una rapida formazione di biofilm sull\u2019impianto e quindi allo sviluppo di pseudoartrosi in tutti gli animali trattati. I successi ottenuti in questa prima fase del progetto hanno reso possibile la realizzazione della successiva fase sperimentale in cui, grazie al modello di infezione acuta, abbiamo potuto testare l\u2019efficacia di nuove strategie preventive. In letteratura sono descritti diversi approcci terapeutici per il trattamento di pseudoartrosi settiche e tante nuove ricerche stanno cercando di ottimizzare un trattamento che ancora ad oggi non \ue8 presente. L\u2019ingegneria tissutale, ad esempio, da anni studia l\u2019utilizzo di scaffold biologici o sintetici con propriet\ue0 osteoinduttive e antibatteriche per scagionare l\u2019instaurazione di infezioni favorendo allo stesso momento la rigenerazione del tessuto osseo. Tuttavia la maggior parte delle proposte sviluppate possiede delle limitazioni, legate, ad esempio, alla non biodegradabilit\ue0 del materiale che potrebbe in alcuni casi addirittura favorire l\u2019adesione batterica o legate al tipo sostanze antibiotiche caricabili nel materiale. In questo senso gli hydrogel rappresentano un giusto compromesso, poich\ue9 in grado di fornire localmente antibiotici, fattori di crescita o cellule, sfavorendo lo sviluppo di infezioni batteriche associate a impianti. \uc8 stato recentemente sviluppato un nuovo hydrogel a base di acido ialuronico e polilattico. Test in vitro e in vivo di questo rivestimento riassorbibile hanno dimostrato come esso sia in grado di prevenire la formazione di infezioni quando associato alla presenza di antibatterico. Tuttavia, non sono ancora note le propriet\ue0 osteoinduttive/osteoconduttive che si presume abbia questo innovativo hydrogel. Altri importanti traguardi dell\u2019ingegneria tissutale sono stati raggiunti grazie all\u2019utilizzo di terapie cellulari, dove il trapianto di cellule progenitrice ha portato a impressionanti risultati nella rigenerazione di organi e tessuti, nonch\ue9 nel trattamento di pseudoartrosi. Questo approccio mira a fornire cellule progenitrici sane in grado di produrre nuova matrice extracellulare all\u2019interno del tessuto danneggiato per ripristinare la perdita di funzione del tessuto danneggiato. Le cellule staminali mesenchimali (mesenchymal stem cells, MSCs) hanno attirato l\u2019attenzione dei ricercatori di questo settore come fonte facilmente accessibile di cellule autologhe in grado di differenziarsi in vitro in diversi tessuti di origine mesenchimale (ad esempio osseo, cartilagineo, adiposo). Inoltre, alcuni studi hanno descritto le propriet\ue0 immunomodulatorie e antimicrobiche di queste cellule, dimostrando come le MSCs possano limitare la crescita batterica in vivo e come possano attivamente modulare la risposta infiammatoria nel sito dell\u2019infezione. Tuttavia, il meccanismo d\u2019azione delle MSCs nella modulazione del processo infiammatorio non \ue8 ancora chiaro. Un\u2019altra importante caratteristica di queste cellule \ue8 la loro mancanza del complesso maggiore di istocompatibilit\ue0 II che le rende quindi candidate ideali per il trapianto allogenico. L\u2019obiettivo della seconda fase sperimentale di questo progetto \ue8 quello di valutare l\u2019efficacia di un innovativo hydrogel arricchito di vancomicina, nel prevenire lo sviluppo di una pseudoartrosi settica e allo stesso momento favorire la rigenerazione del tessuto osseo. L\u2019utilizzo locale dell\u2019hydrogel \ue8 stato comparato all\u2019uso di vancomicina per via sistemica, essendo un trattamento standard in clinica in caso di infezione da batteri meticillino-resistenti. Parallelamente all\u2019interno dello stesso disegno sperimentale, abbiamo valutato l\u2019efficacia dell\u2019utilizzo di BMSCs, cellule mesenchimale da midollo osseo, per il controllo della risposta infiammatoria e della diffusione della crescita batterica in vivo. In particolare, ratti immunocompetenti sono stati sottoposti a una frattura femorale infettata con MRSE e, successivamente, inoculati con BMSCs per via sistemica o locale a seconda del gruppo di appartenenza degli animali. Scopo dello studio non solo \ue8 stato quello di analizzare gli effetti immunomodulatori delle BMSCs attraverso la valutazione dei livelli di espressione di citochine infiammatorie, ma anche valutare le propriet\ue0 osteoinduttive delle BMSCs in grado di produrre matrice extracellulare nel tessuto danneggiato. Anche in questo caso l\u2019uso sistemico di cellule \ue8 stato paragonato a quello locale con il fine di stabilire una via di somministrazione sicura. Per tutta la prima fase del progetto di dottorato si \ue8 ricercata una strategia in grado di impedire la colonizzazione batterica dell\u2019impianto. Tuttavia, il nostro modello animale non \ue8 stato in grado di fornirci informazioni su come l\u2019infezione sia stata debellata dall\u2019organismo e quali sono stati i meccanismi attivati che hanno permesso l\u2019eradicazione dell\u2019infezione e della guarigione della frattura. Perch\ue9 i batteri non sono stati pi\uf9 in grado di formare biofilm in presenza dei trattamenti? Pertanto, per rispondere a questa domanda sperimentale, nell\u2019ultima fase del progetto di dottorato l\u2019attenzione si \ue8 spostata sull\u2019analisi del proteoma di S. epidermidis per capire i meccanismi molecolari che i batteri attivano quando formano e stabiliscono biofilm in vitro su inserti di titanio. Il percorso che regola la formazione del biofilm in vivo non \ue8 ancora completamente noto, ma \ue8 stato ampiamente studiato e descritto; diversamente troviamo in letteratura poche informazioni sul biofilm maturo. Pertanto, con lo scopo di individuare la chiave della stabilit\ue0 del biofilm maturo, le proteine espresse da due ceppi di S. epidermidis coltivati sia in forma planctonica sia in forma sessile sono state studiate. In particolare, abbiamo concentrato la nostra attenzione sul ceppo clinico GOI1153754-03-14 e comparato la sua espressione di proteine con quelle espresse da S. epidermidis ATCC 35984, considerato il ceppo standard e cui sequenza genomica \ue8 gi\ue0 stata studiata e depositata in banca dati. Infatti, punto cruciale dell\u2019analisi del proteoma dei batteri \ue8 la conoscenza del loro genoma. Perci\uf2, per prima cosa, l\u2019intera genoma di S. epidermidis GOI1153754-03-14 \ue8 stato sequenziato e successivamente depositato in banca dati. La presenza dell\u2019intera sequenza genomica di un isolato clinico \ue8 cruciale sia per le nostre successive analisi, ma permetter\ue0 anche di avere una maggiora conoscenza dei ceppi clinici coinvolti in infezioni ortopediche. A seguito di questa fase preliminare necessaria, S. epidermidis GOI1153754-03-14 e ATCC 35984 sono stati staticamente coltivati in forma planctonica e in biofilm formante per 72 ore. La coltura sessile \ue8 stata condotta su dischetti di titanio sabbiato per permettere l\u2019adesione dei batteri e la formazione di biofilm; al contrario la coltura planctonica \ue8 stata realizzata in agitazione per prevenire l\u2019aggregazione delle cellule. I batteri poi sono stati prelevati e dopo vari lavaggi, mirati a eliminare i residui del brodo di coltura, sono stati lisati per poter estrarre le proteine. Dopo la quantificazione, le proteine sono state prima divise secondo punto isoelettrico e poi secondo peso molecolare per ottenere mappe bidimensionali contenenti singoli spot associati a una singola proteina. Gli spot statisticamente differenti sono stati poi staccati e le proteine identificate per mezzo di analisi di spettrometria di massa. Le analisi hanno rivelato l\u2019incremento di espressione di geni legati allo stress cellulare in batteri coltivati in forma planctonica. La coltura a 72 ore ha reso possibile la formazione di biofilm sulla superficie del dischetto di titanio, ma allo stesso modo ha pregiudicato la crescita degli stessi in forma planctonica. La scelta del time point sperimentale in questo studio rappresenta la maggiore limitazione, ma allo stesso tempo un punto di partenza per valutare il proteoma di S. epidermidis a diversi tempi per lo studio dell\u2019attivazione/repressione di geni coinvolti nella formazione e maturazione della matrice. Lo scopo principale di quest\u2019ultima fase del progetto di dottorato e di futuri studi \ue8 quello di definire potenziali bersagli molecolari di innovative terapie o di definire nuovi biomarcatori diagnostici, tramite l\u2019analisi delle proteine espresse dai batteri in determinate condizioni di crescita.Non-union fractures, as a severe failure of bone healing, are among the most difficult and challenging orthopedic complications. Non-unions represent a clinical burden, as well as a socioeconomic encumbrance that decreases the quality of patients\u2019 lives and requires surgical treatment and long recovery times which increases the burden on the National Health Service. The percentage of fractures leading to non-union is between 5 and 10% and generally occurs in long bones like tibia, femur, humerus, radius, and ulna. Non-unions are strictly related to local bone loss caused, for example, by trauma or tumors; they may depend on patient health status (e.g. age, metabolic disease, comorbidities). In addition, they are frequently caused by bacterial infections established during surgical procedures (e.g. osteosynthesis, open fractures) and are referred to septic or infected non-unions. The most common bacteria involved in infected non-unions are Staphylococcus aureus and Staphylococcus epidermidis and bacterial contamination accounts for the two-thirds of the clinical cases. In particular, S. epidermidis is of utmost importance, being one of the most significant bacteria related to hospital-acquired infections. S. epidermidis is an inhabitant of healthy human skin and mucosal flora and it is a commensal bacterium characterized by a low pathogenic potential. However, this pathogen has emerged as a common cause of numerous nosocomial infections associated with medical devices (e.g. catheters, pacemaker, metal implants, etc.), because of its capability to create a protective niche on the surface of implanted orthopedic devices, called biofilm. Biofilm is a thick matrix of extracellular polymeric substances derived from sessile bacteria irreversibly attached to a substratum. Within the biofilm, microorganisms establish organized hierarchies similar to that of multicellular organisms; cell-to-cell signaling regulates the expression of genes involved in survival mechanisms, depending on environmental influences. Thus, biofilm confers a protective niche to pathogens in which they can grow and evade host immune defenses and antimicrobial treatments, leading to the development of antimicrobial-resistant strains, such as methicillin-resistant S. epidermidis (MRSE). Moreover, because of the ubiquitous prevalence of S. epidermidis as a commensal bacterium on human skin, it is often difficult to discern whether a clinical isolate represents the causative agent of an infection or an unspecific culture contaminant. In order to study S. epidermidis-associated infections in orthopedic implants, animal models are extremely useful to investigate the pathogenesis of biofilm-related non-union, with particular regard to subclinical infections. For the aforementioned reason and to fulfill the lack of knowledge in the literature, we established a preclinical model of methicillin-resistant S. epidermidis non-union in order to assess the role of subclinical infections in orthopedic and trauma surgery. Indeed, an animal model of infected nonunion able to resemble the features of a nosocomial implant-associated infection was generated, in order to investigate new preventive or therapeutic options. Thus, we evaluated the incidence of infected non-unions caused by dose-dependent concentrations of methicillin-resistant S. epidermidis (MRSE) in rats subjected to femoral fracture osteosynthesis with metal implants. At the end of the first experimental phase, we identified the lowest bacterial load of MRSE able to induce a clinical infected non-union. In particular, we determined that a low grade bacterial injection determines a subclinical infection, whereas the intermediate grade inoculum determines a clear acute clinical infection and the highest bacterial inoculum is able to form a visible biofilm upon the implant surface, impeding fracture healing in all the treated animals. The development of a valid animal model is crucial for the subsequent experimental phases, allowing us to study the complex physiopathology of non-unions caused by microorganisms in order to optimize therapeutic strategies. To treat non-unions, several therapeutic approaches are described in the literature, such as surgical debridement or local or systemic antibiotic therapies. Recently, also bone tissue engineers are developing biological or synthetic scaffolds with osteoinductive and antibacterial proprieties. However, most of these innovative materials used as drug delivery may have some drawbacks, including non-biodegradability, possible microbial adhesion and biofilm formation on their surfaces, restricted range of loadable antibiotics and long-lasting release with a potential increase of antibiotic resistance. Because of these limitations, hydrogels represent promising and potential alternative materials able to deliver antibiotics, growth factors or cells locally, while inducing osteogenesis and regulating bacterial bone infections associated with orthopedic devices. Recently, an innovative hydrogel composed of two biocompatible polymers (hyaluronic acid and poly-lactic acid) was tested in vitro and subsequently validated in vivo, as a bio-resorbable barrier against infections. However, it is not known yet if this hydrogel also possesses osteoinductive and/or osteoconductive properties. Moreover, in the last few years, bone tissue engineering and cell-based therapies reported some impressive results in the regeneration of organs or tissues, as well as in the treatment of non-septic non-unions. The cell therapy approach aims to deliver healthy cells able to produce new calcified matrix within the damaged tissue in order to fix the loss of function. Mesenchymal stromal cells (MSCs) have prompted significant interest in biomedical research and cell-based therapies due to their ability to self-renew, differentiate in vitro into mesenchymal tissues, such as bone, cartilage, or fat and as an easily accessible source of autologous cells. Furthermore, some recent studies demonstrated the immunomodulatory and antimicrobial features of MSCs. Finally, recent studies demonstrated that MSCs can limit bacterial growth in vivo thanks to the aforementioned proprieties able to modulate the inflammatory response and macrophage activation at the site of infection. Nevertheless, the mechanism of action of MSCs in the modulation of the inflammatory process is still unclear. Finally, the lack of the major histocompatibility complex II (MHC II) in MSCs could permit the allogeneic transplant of these cells. Aiming at testing innovative preventive and/or therapeutic strategies through our model of septic non-unions, the goal of the second experimental phase was to evaluate the efficacy of an innovative hydrogel, with presumed osteoinductive and antibacterial proprieties, to prevent and control the progression of bacterial biofilm formation in infected non-unions. Along with this goal, we investigated the feasibility and the efficacy of MSCs-based therapy to control both the inflammatory response and bacterial growth/spread in vivo. Specifically, immunocompetent rats, already exposed to a femoral fracture infected with MRSE, were inoculated with allogeneic mesenchymal stem cells isolated from the bone marrow (BMSCs) through two different administration routes \u2013 systemic (circulatory system) and local injection (fracture site). Through this study, we evaluated both the immunoregulatory effects of BMSCs, but also the osteoinductive proprieties of these cells able to produce calcified matrix in the damaged tissue. Similarly, we assessed the ability of an enriched hydrogel to prevent/treat infected non-unions by inhibiting biofilm formation and stimulating bone deposition due to its antibacterial and osteoinductive proprieties. The overall and anticipated goal of this first part of the project was to establish new therapeutic strategies for the prevention and the treatment of infected non-unions, which result in morbidity and, sometimes, limb loss in critical patients. These results may have an important impact on the treatment of the infected non-union in orthopedics. Moreover, these tested therapeutic strategies may also be used synergically to locally deliver cells due to the engineered hydrogel to optimize physiological pathways of fracture healing and to control the progress of local inflammations/infections. Another potential use of the therapies from this study would be the use of animal models affected with comorbidities (e.g. type I and II diabetes) instead of healthy animals, in order to translate the findings on complex infections that may occur in orthopedics. In the first experimental phase, all efforts were made to discourage bacterial attachment on the surface of metallic implants

Recent evidence on bioactive glass antimicrobial and antibiofilm activity : a mini-review

Bone defects caused by trauma or pathological events are major clinical and socioeconomic burdens. Thus, the efforts of regenerative medicine have been focused on the development of non-biodegradable materials resembling bone features. Consequently, the use of bioactive glass as a promising alternative to inert graft materials has been proposed. Bioactive glass is a synthetic silica-based material with excellent mechanical properties able to bond to the host bone tissue. Indeed, when immersed in physiological fluids, bioactive glass reacts, developing an apatite layer on the granule's surface, playing a key role in the osteogenesis process. Moreover, the contact of bioactive glass with biological fluids results in the increase of osmotic pressure and pH due to the leaching of ions from granules' surface, thus making the surrounding environment hostile to microbial growth. The bioactive glass antimicrobial activity is effective against a wide selection of aerobic and anaerobic bacteria, either in planktonic or sessile forms. Furthermore, bioglass is able to reduce pathogens' biofilm production. For the aforementioned reasons, the use of bioactive glass might be a promising solution for the reconstruction of bone defects, as well as for the treatment and eradication of bone infections, characterized by bone necrosis and destruction of the bone structure

Probiotics Streptococcus salivarius 24SMB and Streptococcus oralis 89a interfere with biofilm formation of pathogens of the upper respiratory tract

Background: Infections of the ears, paranasal sinuses, nose and throat are very common and represent a serious issue for the healthcare system. Bacterial biofilms have been linked to upper respiratory tract infections and antibiotic resistance, raising serious concerns regarding the therapeutic management of such infections. In this context, novel strategies able to fight biofilms may be therapeutically beneficial and offer a valid alternative to conventional antimicrobials. Biofilms consist of mixed microbial communities, which interact with other species in the surroundings and communicate through signaling molecules. These interactions may result in antagonistic effects, which can be exploited in the fight against infections in a sort of "bacteria therapy". Streptococcus salivarius and Streptococcus oralis are α-hemolytic streptococci isolated from the human pharynx of healthy individuals. Several studies on otitis-prone children demonstrated that their intranasal administration is safe and well tolerated and is able to reduce the risk of acute otitis media. The aim of this research is to assess S. salivarius 24SMB and S. oralis 89a for the ability to interfere with biofilm of typical upper respiratory tract pathogens. Methods: To investigate if soluble substances secreted by the two streptococci could inhibit biofilm development of the selected pathogenic strains, co-cultures were performed with the use of transwell inserts. Mixed-species biofilms were also produced, in order to evaluate if the inhibition of biofilm formation might require direct contact. Biofilm production was investigated by means of a spectrophotometric assay and by confocal laser scanning microscopy. Results: We observed that S. salivarius 24SMB and S. oralis 89a are able to inhibit the biofilm formation capacity of selected pathogens and even to disperse their pre-formed biofilms. Diffusible molecules secreted by the two streptococci and lowered pH of the medium revealed to be implied in the mechanisms of anti-biofilm activity. Conclusions: S. salivarius 24SMB and S. oralis 89a possess desirable characteristics as probiotic for the treatment and prevention of infections of the upper airways. However, the nature of the inhibition appear to be multifactorial and additional studies are required to get further insights

Putative Microbial Population Shifts Attributable to Nasal Administration of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a

Changes in bacterial composition of nasal microbiota may alter the host\u2019s susceptibility to several infectious and allergic diseases such as chronic rhinosinusitis and allergic rhinitis. The aim of this study was to evaluate the effects of 1-week administration of a probiotic product, composed by a combination of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a, on the nostril microbiota. Differences in the nasal microbiota composition were investigated by using a next-generation sequencing approach. A strong and significant decrease in Staphylococcus aureus abundance was detected immediately after the bacterial administration. Moreover, comparing the microbial networks of nostril microbiota before and 1 month after the end of treatment, we detected an increase in the total number of both bacterial nodes and microbial correlations, with particular regard to the beneficial ones. Furthermore, a less abundance of microbial genera commonly associated to potential harmful bacteria has been observed. These results suggest a potential ability of S. salivarius 24SMBc and S. oralis 89a to regulate and reorganize the nasal microbiota composition, possibly favoring those microorganisms that may be able to limit the overgrowth of potential pathogens

Probiotics Streptococcus salivarius 24SMB and Streptococcus oralis 89a interfere with biofilm formation of pathogens of the upper respiratory tract

Background: Infections of the ears, paranasal sinuses, nose and throat are very common and represent a serious issue for the healthcare system. Bacterial biofilms have been linked to upper respiratory tract infections and antibiotic resistance, raising serious concerns regarding the therapeutic management of such infections. In this context, novel strategies able to fight biofilms may be therapeutically beneficial and offer a valid alternative to conventional antimicrobials. Biofilms consist of mixed microbial communities, which interact with other species in the surroundings and communicate through signaling molecules. These interactions may result in antagonistic effects, which can be exploited in the fight against infections in a sort of "bacteria therapy". Streptococcus salivarius and Streptococcus oralis are \u3b1-hemolytic streptococci isolated from the human pharynx of healthy individuals. Several studies on otitis-prone children demonstrated that their intranasal administration is safe and well tolerated and is able to reduce the risk of acute otitis media. The aim of this research is to assess S. salivarius 24SMB and S. oralis 89a for the ability to interfere with biofilm of typical upper respiratory tract pathogens. Methods: To investigate if soluble substances secreted by the two streptococci could inhibit biofilm development of the selected pathogenic strains, co-cultures were performed with the use of transwell inserts. Mixed-species biofilms were also produced, in order to evaluate if the inhibition of biofilm formation might require direct contact. Biofilm production was investigated by means of a spectrophotometric assay and by confocal laser scanning microscopy. Results: We observed that S. salivarius 24SMB and S. oralis 89a are able to inhibit the biofilm formation capacity of selected pathogens and even to disperse their pre-formed biofilms. Diffusible molecules secreted by the two streptococci and lowered pH of the medium revealed to be implied in the mechanisms of anti-biofilm activity. Conclusions: S. salivarius 24SMB and S. oralis 89a possess desirable characteristics as probiotic for the treatment and prevention of infections of the upper airways. However, the nature of the inhibition appear to be multifactorial and additional studies are required to get further insights

Modulation of opportunistic species Corynebacterium diphtheriae, Haemophilus parainfluenzae, Moraxella catarrhalis, Prevotella denticola, Prevotella melaninogenica, Rothia dentocariosa, Staphylococcus aureus and Streptococcus pseudopneumoniae by intranasal administration of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a combination in healthy subjects

\u2013 OBJECTIVE: Probiotics S. salivarius 24SMBc and S. oralis 89a comprised in the nasal spray Rinogermina are known to exert inhibition of harmful pathogens and ameliorate the outcome of patients with chronic upper airways infections. In this study, for the first time, the effect of this formulation on the modulation of the microflora of healthy subjects was evaluated, with particular interest on pathobionts and pathogens present. PATIENTS AND METHODS: Metagenomic identification and quantification of bacterial abundances in healthy subjects were carried out by means of Ion Torrent Personal Machine. In particular, nasal swabs were sampled one, two and four weeks after seven days of treatment with Rinogermina. RESULTS: The modulation of the abundance of pathobionts and pathogenic species (i.e., Corynebacterium diphtheriae, Haemophilus parainfluenzae, Moraxella catarrhalis, Prevotella denticola, Prevotella melaninogenica, Rothia dentocariosa, Staphylococcus aureus and Streptococcus pseudopneumoniae) was characterized and a significant temporary decrease in their presence was identified. CONCLUSIONS: The beneficial effects of S. salivarius 24SMBc and S. oralis 89a nasal intake was assessed but seemed to be restricted in specific temporal windows. Thus it would be interesting to evaluate also this positive impact of longer administration of this probiotic formulation

Systemic and Local Administration of Antimicrobial and Cell Therapies to Prevent Methicillin-Resistant Staphylococcus epidermidis-Induced Femoral Nonunions in a Rat Model

S. epidermidis is responsible for biofilm-related nonunions. This study compares the response to S. epidermidis-infected fractures in rats systemically or locally injected with vancomycin or bone marrow mesenchymal stem cells (BMSCs) in preventing the nonunion establishment. The 50% of rats receiving BMSCs intravenously (s-rBMSCs) died after treatment. A higher cytokine trend was measured in BMSCs locally injected rats (l-rBMSCs) at day 3 and in vancomycin systemically injected rats (l-VANC) at day 7 compared to the other groups. At day 14, the highest cytokine values were measured in l-VANC and in l-rBMSCs for IL-10. \ub5CT showed a good bony bridging in s-VANC and excellent both in l-VANC and in l-rBMSCs. The bacterial growth was lower in s-VANC and l-VANC than in l-rBMSCs. Histology demonstrated the presence of new woven bone in s-VANC and a more mature bony bridging was found in l-VANC. The l-rBMSCs showed a poor bony bridging of fibrovascular tissue. Our results could suggest the synergic use of systemic and local injection of vancomycin as an effective treatment to prevent septic nonunions. This study cannot sustain the systemic injection of BMSCs due to high risks, while a deeper insight into local BMSCs immunomodulatory effects is mandatory before developing cell therapies in clinics

Modeling Staphylococcus epidermidis-Induced Non-Unions : Subclinical and Clinical Evidence in Rats

S. epidermidis is one of the leading causes of orthopaedic infections associated with biofilm formation on implant devices. Open fractures are at risk of S. epidermidis transcutaneous contamination leading to higher non-union development compared to closed fractures. Although the role of infection in delaying fracture healing is well recognized, no in vivo models investigated the impact of subclinical low-grade infections on bone repair and non-union. We hypothesized that the non-union rate is directly related to the load of this commonly retrieved pathogen and that a low-grade contamination delays the fracture healing without clinically detectable infection. Rat femurs were osteotomized and stabilized with plates. Fractures were infected with a characterized clinical-derived methicillin-resistant S. epidermidis (10(3), 10(5), 10(8) colony forming units) and compared to uninfected controls. After 56 days, bone healing and osteomyelitis were clinically assessed and further evaluated by micro-CT, microbiological and histological analyses. The biofilm formation was visualized by scanning electron microscopy. The control group showed no signs of infection and a complete bone healing. The 10(3) group displayed variable response to infection with a 67% of altered bone healing and positive bacterial cultures, despite no clinical signs of infection present. The 10(5) and 10(8) groups showed severe signs of osteomyelitis and a non-union rate of 83-100%, respectively. The cortical bone reaction related to the periosteal elevation in the control group and the metal scattering detected by micro-CT represented limitations of this study. Our model showed that an intra-operative low-grade S. epidermidis contamination might prevent the bone healing, even in the absence of infectious signs. Our findings also pointed out a dose-dependent effect between the S. epidermidis inoculum and non-union rate. This pilot study identifies a relevant preclinical model to assess the role of subclinical infections in orthopaedic and trauma surgery and to test specifically designed diagnostic, prevention and therapeutic strategies

Draft genome sequence of Staphylococcus epidermidis clinical strain GOI1153754-03-14 isolated from an infected knee prosthesis

We announce the draft genome sequence of Staphylococcus epidermidis clinical strain GOI1153754-03-14, isolated from an infected orthopedic prosthesis. The reported genomic sequence will provide valuable information concerning the mechanisms of the biofilm formation on metallic implants

Improving the bacterial recovery by using dithiothreitol with aerobic and anaerobic broth in biofilm-related prosthetic and joint infections

Biofilm-related infections are serious complications in the orthopaedic prosthetic field and an accurate, quick microbiological diagnosis is required to set up a specific antimicrobial therapy. It is well known that the diagnosis of these infections remains difficult due to the bacterial embedding within the biofilm matrix on the implant surfaces. Recently, the use of DL-dithiothreitol (DTT) has been proved effective in biofilm detachment from orthopaedic devices.The purpose of the study is to evaluate the efficacy of two DTT solutions enriched with specific broths for aerobic or anaerobic bacteria to dislodge pathogens from the biofilm, while supporting the bacterial recovery and viability. To do this, different experimental solutions were tested for efficacy and stability on strong biofilm producers: S. aureus and P. acnes. Mainly, we evaluate the capability of DTT dissolved in saline solution, brain heart infusion or thioglycollate broth to support the bacterial detachment from prosthetic materials and bacterial growth at different time points and storage conditions.We demonstrated that the use of DTT enriched with specific bacterial broths could be a suitable approach to optimize the bacterial detachment, recovery, growth and viability in the diagnosis of biofilm-related infections developed on orthopaedic prosthetic devices