Osteomyelitis is an inflammatory bone disorder caused by infection, leading to necrosis and destruction of bone. It can affect all ages, involve any bone, become a chronic disease and cause persistent morbidity. Treatment of osteomyelitis is challenging particularly when complex multiresistant bacterial biofilm has already been established. Bacteria in biofilm persist in a low metabolic phase, causing persistent infection due to increased resistance to antibiotics. Staphylococcus aureus and Staphylococcus epidermidis are the most common causative organism responsible for more than 50% of osteomyelitis cases. Osteomyelitis treatment implies the administration of high doses of antibiotics (AB) by means of endovenous and oral routes and should take a period of at least 6 weeks. Local drug delivery systems, using non-biodegradable (polymethylmethacrylate) or biodegradable and osteoactive materials such as calcium orthophosphates bone cements, have been shown to be promising alternatives for the treatment of osteomyelitis. These systems allow the local delivery of AB in situ with bactericidal concentrations for long periods of time and without the toxicity associated with other means of administration. This review examines the most recent literature evidence on the causes, pathogeneses and pharmacological treatment of osteomyelitis. A osteomielite é um processo inflamatório do tecido ósseo, de origem infecciosa, que resulta em destruição inflamatória, necrose e formação de novo osso. Pode aparecer em qualquer idade, afetar qualquer osso e tornar-se uma doença crônica com morbidade persistente. Apesar dos progressos na quimioterapia infecciosa, o tratamento da osteomielite é caro e difícil, em particular quando associada à presença de biofilmes bacterianos, especialmente de Staphylococcus aureus e Staphylococcus epidermidis. O tratamento da osteomielite inclui a administração de doses elevadas de antibióticos (AB) por via endovenosa e oral, durante um período de pelo menos 6 semanas. Os sistemas de veiculação localizada de fármacos, utilizando materiais não biodegradáveis (polimetilmetacrilato) ou biodegradáveis e osteoativos como os cimentos ósseos de ortofosfatos de cálcio e vidro bioativo, surgiram como uma alternativa promissora para o tratamento da osteomielite. Estes sistemas permitem a veiculação de AB in situ com concentrações bactericidas por longos períodos de tempo e sem a toxicidade associada às outras vias de administração. O presente trabalho propõe uma revisão da literatura relativa às causas, à patogenia e ao tratamento farmacológico da osteomielite. A metodologia do estudo da revisão consistiu numa pesquisa bibliográfica, nas bases de dados Google Scholar, Science Direct, Pubmed, Springer link, B-on. Foram revistos e analisados diversos artigos publicados desde o ano de 1979.Unitermos: Osteomielite/tratamento farmacológico. Osteomielite/terapia antimicrobial. Staphylococcus aureus/presença/osteomielite. Antibióticos/uso/tratamento da osteomielite.

Diabetic patients frequently develop diabetic foot ulcers (DFUs), particularly those patients vulnerable to Staphylococcus aureus opportunistic infections. It is urgent to find new treatments for bacterial infections. The antimicrobial peptide (AMP) nisin is a potential candidate, mainly due to its broad spectrum of action against pathogens. Considering that AMP can be degraded or inactivated before reaching its target at therapeutic concentrations, it is mandatory to establish effective AMP delivery systems, with the natural polysaccharide guar gum being one of the most promising. We analysed the antimicrobial potential of nisin against 23 S. aureus DFU biofilm-producing isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were determined for nisin diluted in HCl and incorporated in guar gum gel. Statistical analysis was performed using the Wilcoxon matched-pair test. Nisin was effective against all isolates, including some multidrug-resistant clinical isolates, independent of whether it is incorporated in guar gum. While differences among MIC, MBC and MBIC values were observed for HCl- and guar gum- nisin, no significant differences were found between MBEC values. Inhibitory activity of both systems seems to differ only twofold, which does not compromise guar gum gel efficiency as a delivery system. Our results highlight the potential of nisin as a substitute for or complementary therapy to current antibiotics used for treating DFU infections, which is extremely relevant considering the increase in multidrug-resistant bacteria dissemination. The guar gum gel represents an alternative, practical and safe delivery system for AMPs, allowing the development of novel topical therapies as treatments for bacterial skin infections.

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Diabetic foot ulcers (DFUs) are major complications of Diabetes mellitus being responsible for significant morbidity and mortality. DFUs frequently become chronically infected by a complex community of bacteria, including multidrug-resistant and biofilm-producing strains of Staphylococcus aureus and Pseudomonas aeruginosa. Diabetic foot infections (DFI) are often recalcitrant to conventional antibiotics and alternative treatment strategies are urgently needed. Antimicrobial Peptides (AMPs), such as pexiganan and nisin, have been increasingly investigated and reported as effective antimicrobial agents. Here, we evaluated the antibacterial potential of pexiganan and nisin used in combination (dual-AMP) to control the growth of planktonic and biofilm co-cultures of S. aureus and P. aeruginosa clinical strains, co-isolated from a DFU. A DFU collagen three-dimensional (3D) model was used to evaluate the distribution and efficacy of AMPs locally delivered into the model. The concentration of pexiganan required to inhibit and eradicate both planktonic and biofilm-based bacterial cells was substantially reduced when used in combination with nisin. Moreover, incorporation of both AMPs in a guar gum delivery system (dual-AMP biogel) did not affect the dual-AMP antimicrobial activity. Importantly, the application of the dual-AMP biogel resulted in the eradication of the S. aureus strain from the model. In conclusion, data suggest that the local application of the dual-AMPs biogel constitutes a potential complementary therapy for the treatment of infected DFU.

Freezing is an important operation in biotherapeutics industry. However, water crystallization in solution, containing electrolytes, sugars and proteins, is difficult to control and usually leads to substantial spatial solute heterogeneity. Herein, we address the influence of the geometry of freezing direction (axial or radial) on the heterogeneity of the frozen matrix, in terms of local concentration of solutes and thermal history. Solutions of hemoglobin were frozen radially and axially using small-scale and pilot-scale freezing systems. Concentration of hemoglobin, sucrose and pH values were measured by ice-core sampling and temperature profiles were measured at several locations. The results showed that natural convection is the major source for the cryoconcentration heterogeneity of solutes over the geometry of the container. A significant improvement in this spatial heterogeneity was observed when the freezing geometry was nonconvective, i.e., the freezing front progression was unidirectional from bottom to top. Using this geometry, less than 10% variation in solutes concentration was obtained throughout the frozen solutions. This result was reproducible, even when the volume was increased by two orders of magnitude (from 30 mL to 3 L). The temperature profiles obtained for the nonconvective freezing geometry were predicted using a relatively simple computational fluid dynamics model. The reproducible solutes distribution, predictable temperature profiles, and scalability demonstrate that the bottom to top freezing geometry enables an extended control over the freezing process. This geometry has therefore shown the potential to contribute to a better understanding and control of the risks inherent to frozen storage.

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Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice–liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as −15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near −10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (T CD ∼ −17 °C; calculated via isothermal chemical denaturation and Gibbs–Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near −5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.

Protein aggregation can follow different pathways, and these can result in different net aggregation rates and kinetic profiles. α-chymotypsinogen A (aCgn) was used as a model system to quantitatively and qualitatively assess an approach that combines ex situ size-exclusion chromatography (SEC) with in situ laser scattering (LS) to monitor aggregation vs. time. Aggregation was monitored for a series of temperatures and initial dimer (ID) levels for starting conditions that were primarily (> 97%) monomer, and under initial-rate conditions (limited to low monomer conversion-less than 20% monomer mass loss), as these conditions are of most to interest to many pharmaceutical and biotechnology applications. SEC results show that modest decreases of ID levels can greatly reduce monomer loss rates, but do not affect the effective activation energy for aggregation. The normalized aggregation rates determined from LS were typically ∼ 1 order of magnitude higher than the corresponding rates from SEC. Furthermore, LS signals vs. time became variable and highly nonlinear with decreasing ID level, temperature, and/or total protein concentration. Temperature-cycling LS experiments showed this corresponded to conditions where dimer/oligomer "seeding" was suppressed, and high levels of reversible oligomers ("prenuclei") were formed prior to "nucleation" and growth of stable aggregates. In those conditions, aggregation rates inferred from LS and SEC are greatly different, as the techniques monitor different stages of the aggregation process. Overall, the results illustrate an approach for interrogating non-native protein aggregation pathways, and potential pitfalls if one relies on a single method to monitor aggregation-this holds more generally than the particular methods here.