The tale of microencapsulated rifampicin: is it useful for the treatment of periprosthetic joint infection?

PURPOSE: Microencapsulation techniques have allowed the addition of rifampicin to bone cement, but its in vivo efficacy has not been proven. The aim of our study is to determine the superiority of cement containing gentamicin and rifampicin microcapsules in the treatment of PJI versus cement exclusively containing gentamicin. METHODS: An S. aureus PJI was induced in 15 NZW rabbits. A week after inoculation, the first stage of replacement was carried out, and the animals were divided into two groups: group R received a spacer containing gentamicin and rifampicin microcapsules, and group C received a spacer containing gentamicin. Intra-articular release curve of rifampicin and infection and toxicity markers were monitored for four weeks post-operatively, when microbiological analysis was performed. RESULTS: The microbiological cultures showed a significantly lower growth of S. aureus in soft tissue (2.3·104 vs 0; p = 0.01) and bone (5.7·102 vs 0; p = 0.03) in the group with rifampicin microcapsules. No differences were found in systemic toxicity markers. Rifampicin release from the cement spacer showed higher concentrations than the staphylococcal MIC throughout the analysis. CONCLUSION: The in vivo analyses demonstrated the superiority of cement containing gentamicin and rifampicin microcapsules versus the isolated use of gentamicin in the treatment of PJI in the rabbit model without serious side effects due to the systemic absorption of rifampicin. Given the increasing incidence of staphylococci-related PJI, the development of new strategies for intra-articular administration of rifampicin for its treatment has a high clinical impact.

3D printing in experimental orthopaedic surgery: do it yourself.

INTRODUCTION: Periprosthetic infection is considered an increasing incidence pathology whose therapeutic strategies can be defined as unsatisfactory. Currently, animal models are employed to study its physiopathology and strategic therapies, but non-species-specific materials are implanted as foreign bodies. The use of these implants implies intrinsic instability, which hinders the development of a biofilm on their surfaces and complicates the post-operative recovery of the animal. The objective of the present study is the design of a species-specific implant for the New Zealand white (NZW) rabbit by means of 3D printing. MATERIALS AND METHODS: A CT scan of the knee of a NZW rabbit was performed, and the tibial surface was reconstructed in order to fabricate a species-specific tibial plateau using Horos® and Autodesk® Meshmixer™ software. This implant was inserted in fifteen NZW rabbits, and the assessment of its stability was based on the position of the limb at rest and the animal weight-bearing capacity. Biofilm formation on the surface was demonstrated by crystal violet staining. RESULTS: A 1.81 cm × 1 cm × 1.24 cm stainless steel implant was designed. It consisted of a 4-mm-thick tibial plate with a rough surface and an eccentric metaphyseal anchoring. All of the animals exhibited hyperflexion of the operated limb immediately post-operative, and 100% could apply full weight bearing from day 5 after surgery. CONCLUSIONS: The species-specific design of implants in experimental surgery encourages rapid recovery of the animal and the development of a biofilm on their surfaces, making them ideal for the study of the physiopathology and for establishing possible therapeutic targets for prosthetic infection.

ß-Lactam Antibiotics Modify Root Architecture and Indole Glucosinolate Metabolism in Arabidopsis thaliana.

The presence of antibiotics in soils could be due to natural production by soil microorganisms or to the effect of anthropogenic activities. However, the impact of these compounds on plant physiology has not been thoroughly investigated. To evaluate the effect of ß-lactam antibiotics (carbenicillin and penicillin) on the growth and development of Arabidopsis thaliana roots, plants were grown in the presence of different amounts and we found a reduction in root size, an increase in the size of root hairs as well as an abnormal position closer to the tip of the roots. Those phenomena were dependent on the accumulation of both antibiotics inside root tissues and also correlated with a decrease in size of the root apical meristem not related to an alteration in cell division but to a decrease in cell expansion. Using an RNA sequencing analysis, we detected an increase in the expression of genes related to the response to oxidative stress, which would explain the increase in the levels of endogenous reactive oxygen species found in the presence of those antibiotics. Moreover, some auxin-responsive genes were misregulated, especially an induction of CYP79B3, possibly explaining the increase in auxin levels in the presence of carbenicillin and the decrease in the amount of indole glucosinolates, involved in the control of fungal infections. Accordingly, penicillin-treated plants were hypersensitive to the endophyte fungus Colletotrichum tofieldiae. These results underscore the risks for plant growth of ß-lactam antibiotics in agricultural soils, and suggest a possible function for these compounds as fungus-produced signaling molecules to modify plant behavior.

Limitations and Challenges in the Stability of Cysteamine Eye Drop Compounded Formulations.

Accumulation of cystine crystals in the cornea of patients suffering from cystinosis is considered pathognomonic and can lead to severe ocular complications. Cysteamine eye drop compounded formulations, commonly prepared by hospital pharmacy services, are meant to diminish the build-up of corneal cystine crystals. The objective of this work was to analyze whether the shelf life proposed for six formulations prepared following different protocols used in hospital pharmacies is adequate to guarantee the quality and efficacy of cysteamine eye drops. The long-term and in-use stabilities of these preparations were studied using different parameters: content of cysteamine and its main degradation product cystamine; appearance, color and odor; pH and viscosity; and microbiological analysis. The results obtained show that degradation of cysteamine was between 20% and 50% after one month of storage in the long-term stability study and between 35% and 60% in the in-use study. These data confirm that cysteamine is a very unstable molecule in aqueous solution, the presence of oxygen being the main degradation factor. Saturation with nitrogen gas of the solutions offers a means of reducing cysteamine degradation. Overall, all the formulae studied presented high instability at the end of their shelf life, suggesting that their clinical efficacy might be dramatically compromised.

Experimental reproduction of periprosthetic joint infection: Developing a representative animal model.

BACKGROUND: Infection after arthroplasty (prosthetic joint infection; PJI) is a devastating complication that can lead to functional loss of the affected limb. The purpose of the present study is to develop an animal model of PJI using a three-dimensional printed species-specific implant, which is a step forward for future research to develop new therapeutic strategies. METHODS: Fifteen New Zealand White rabbits were employed to reproduce PJI by intra-articular inoculation of 105 cfu/ml of Staphylococcus aureus ATCC® 29213. Three-dimensional printing technology was used to design a species-specific four-millimeter-thick implant maintaining the anatomical irregularities of the tibial-articular surface. Response to bacterial inoculation was monitored by clinical (weight and temperature), hematological (leukocyte, lymphocyte and platelet counts) and biochemical (erythrocyte sedimentation rate) analyses at the time of inoculation and seven days thereafter, when microbiological samples for culture were also taken. RESULTS: All animals recovered from surgery and all displayed full weight-bearing four days postoperatively. Fourteen of the 15 tested animals (93.3%) presented positive microbiological cultures. A statistically significant increase was found in the number of platelets and leukocytes, as well as a significant decrease in the percentage of lymphocytes, with P = 0.0001 in all cases. CONCLUSIONS: An experimental model faithfully reproducing the periprosthetic infection environment and achieving a high rate of infection has been designed. The use of three-dimensional printed species-specific implants allows rapid postoperative recovery of animals and the development of a stable biofilm. These characteristics make it an interesting model to study its pathogenesis and possible therapeutic strategies.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Educating in antimicrobial resistance awareness: adaptation of the Small World Initiative program to service-learning.

The Small World Initiative (SWI) and Tiny Earth are a consolidated and successful education programs rooted in the USA that tackle the antibiotic crisis by a crowdsourcing strategy. Based on active learning, it challenges young students to discover novel bioactive-producing microorganisms from environmental soil samples. Besides its pedagogical efficiency to impart microbiology content in academic curricula, SWI promotes vocations in research and development in Experimental Sciences and, at the same time, disseminates the antibiotic awareness guidelines of the World Health Organization. We have adapted the SWI program to the Spanish academic environment by a pioneering hierarchic strategy based on service-learning that involves two education levels (higher education and high school) with different degrees of responsibility. Throughout the academic year, 23 SWI teams, each consisting of 3-7 undergraduate students led by one faculty member, coordinated off-campus programs in 22 local high schools, involving 597 high school students as researchers. Post-survey-based evaluation of the program reveals a satisfactory achievement of goals: acquiring scientific abilities and general or personal competences by university students, as well as promoting academic decisions to inspire vocations for science- and technology-oriented degrees in younger students, and successfully communicating scientific culture in antimicrobial resistance to a young stratum of society.

Fate of classical faecal bacterial markers and ampicillin-resistant bacteria in agricultural soils under Mediterranean climate after urban sludge amendment.

The use of sewage sludge or biosolids as agricultural amendments may pose environmental and human health risks related to pathogen or antibiotic-resistant microorganism transmission from soils to vegetables or to water through runoff. Since the survival of those microorganisms in amended soils has been poorly studied under Mediterranean climatic conditions, we followed the variation of soil fecal bacterial markers and ampicillin-resistant bacteria for two years with samplings every four months in a split block design with three replica in a crop soil where two different types of biosolids (aerobically or anaerobically digested) at three doses (low, 40; intermediate, 80; and high, 160Mg·ha(-1)) were applied. Low amounts of biosolids produced similar decay rates of coliform populations than in control soil (-0.19 and -0.27log10CFUs·g(-1)drysoilmonth(-1) versus -0.22) while in the case of intermediate and high doses were close to zero and their populations remained 24months later in the range of 4-5log10CFUs·g(-1)ds. Enterococci populations decayed at different rates when using aerobic than anaerobic biosolids although high doses had higher rates than control (-0.09 and -0.13log10CFUs·g(-1)dsmonth(-1) for aerobic and anaerobic, respectively, vs -0.07). At the end of the experiment, counts in high aerobic and low and intermediate anaerobic plots were 1 log10 higher than in control (4.21, 4.03, 4.2 and 3.11log10CFUs·g(-1) ds, respectively). Biosolid application increased the number of Clostridium spores in all plots at least 1 log10 with respect to control with a different dynamic of decay for low and intermediate doses of aerobic and anaerobic sludge. Ampicillin-resistant bacteria increased in amended soils 4months after amendment and remained at least 1 log10 higher 24months later, especially in aerobic and low and intermediate anaerobic plots due to small rates of decay (in the range of -0.001 to -0.008log10CFUs·g(-1)dsmonth(-1) vs -0.016 for control). Aerobic plots had relative populations of ampicillin-resistant bacteria higher than anaerobic plots with different positive trends. Dose (22%) and time (13%) explained most of the variation of the bacterial populations. Dynamics of fecal markers did not correlate with ampicillin-resistant bacteria thus making necessary to evaluate specifically this trait to avoid possible risks for human and environmental health.

The MAP kinase Mkc1p is activated under different stress conditions in Candida albicans.

Candida albicans is an opportunistic pathogen that has adapted to live and grow in the human body as its natural environment. Under these conditions, this fungus faces numerous challenges, including oxidative, osmotic and enzymic processes that may damage external and internal structures. In view of the key role of MAP kinase signalling pathways in the physiology of C. albicans, the effect of agents mimicking in vivo environmental conditions on the activation of the p42-44 MAP kinases has been analysed. It has been found that Mkc1p is phosphorylated in the presence of oxidative stress, changes in osmotic pressure, cell wall damage and a decrease in the growth temperature. This phosphorylation is dependent on Pkc1p, indicating that both proteins operate in the same signalling pathway in C. albicans. Under some stimuli, the phosphorylation of Mkc1p required the presence of Hog1p, the MAP kinase of the high osmolarity glycerol (HOG) pathway. This suggests the existence of a new regulatory role, at least under some conditions, for these MAP kinase pathways in yeast.

A role for the MAP kinase gene MKC1 in cell wall construction and morphological transitions in Candida albicans.

The Candida albicans MKC1 gene encodes a mitogen-activated protein (MAP) kinase, which has been cloned by complementation of the lytic phenotype associated with Saccharomyces cerevisiae slt2 (mpk1) mutants. In this work, the physiological role of this MAP kinase in the pathogenic fungus C. albicans was characterized and a role for MKC1 in the biogenesis of the cell wall suggested based on the following criteria. First, C. albicans mkc1 delta/mkc1 delta strains displayed alterations in their cell surfaces under specific conditions as evidenced by scanning electron microscopy. Second, an increase in specific cell wall epitopes (O-glycosylated mannoprotein) was shown by confocal microscopy in mkc1 delta/mkc1 delta mutants. Third, the sensitivity to antifungals which inhibit (1,3)-beta-glucan and chitin synthesis was increased in these mutants. In addition, evidence for a role for the MKC1 gene in morphological transitions in C. albicans is presented based on the impairment of pseudohyphal formation of mkc1 delta/mkc1 delta strains on Spider medium and on the effect of its overexpression on Sacch. cerevisiae colony morphology on SLADH medium. Using the two-hybrid system, it was also demonstrated that MKC1 is able to interact specifically with Sacch. cerevisiae Mkk1p and Mkk2p, the MAP-kinase kinases of the PKC1-mediated route of Sacch. cerevisiae, and to activate transcription in Sacch. cerevisiae when bound to a DNA-binding element. These results suggest a role for this MAP kinase in the construction of the cell wall of C. albicans and indicate its potential relevance for the development of novel antifungals.

Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain.

Protein degradation by the ubiquitin (Ub) system controls the concentrations of many regulatory proteins. The degradation signals (degrons) of these proteins are recognized by the system's Ub ligases (complexes of E2 and E3 enzymes). Two substrate-binding sites of UBR1, the E3 of the N-end rule pathway in the yeast Saccharomyces cerevisiae, recognize basic (type 1) and bulky hydrophobic (type 2) N-terminal residues of proteins or short peptides. A third substrate-binding site of UBR1 targets CUP9, a transcriptional repressor of the peptide transporter PTR2, through an internal (non-N-terminal) degron of CUP9. Previous work demonstrated that dipeptides with destabilizing N-terminal residues allosterically activate UBR1, leading to accelerated in vivo degradation of CUP9 and the induction of PTR2 expression. Through this positive feedback, S. cerevisiae can sense the presence of extracellular peptides and react by accelerating their uptake. Here, we show that dipeptides with destabilizing N-terminal residues cause dissociation of the C-terminal autoinhibitory domain of UBR1 from its N-terminal region that contains all three substrate-binding sites. This dissociation, which allows the interaction between UBR1 and CUP9, is strongly increased only if both type 1- and type 2-binding sites of UBR1 are occupied by dipeptides. An aspect of autoinhibition characteristic of yeast UBR1 also was observed with mammalian (mouse) UBR1. The discovery of autoinhibition in Ub ligases of the UBR family indicates that this regulatory mechanism may also control the activity of other Ub ligases.

The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans.

Candida albicans mutants with mutations in mitogen-activated protein (MAP) kinase HOG1 displayed an increased sensitivity to agents producing reactive oxygen species, such as oxidants (menadione, hydrogen peroxide, or potassium superoxide), and UV light. Consistent with this finding, C. albicans Hog1 was activated not only in response to an increase in external osmolarity, as happens with its Saccharomyces cerevisiae homologue, but also in response to hydrogen peroxide. The Hog1-mediated response to oxidative stress was different from that of transcription factor Cap1, the homologue of S. cerevisiae Yap1, as shown by the different sensitivities to oxidants and the kinetics of cell death of cap1Delta, hog1, and hog1 cap1Delta mutants. Deletion of CAP1 did not influence the level of Hog1 phosphorylation, and deletion of HOG1 did not affect Cap1 nuclear localization. Moreover, we show that the HOG1 gene plays a role in chlamydospore formation, another oxygen-related morphogenetic event, as demonstrated by the fact that hog1 cells were unable to generate these thick-walled structures in several media through a mechanism different from that of the EFG1 regulator. This is the first demonstration of the role of the Hog1-mediated MAP kinase pathway in resistance to oxidative stress in pathogenic fungi, and it allows us to propose a molecular model for the oxidative stress response in C. albicans.