Beyond lectures and practical courses: Teaching pharmacology using imaginative pedagogical tools.

Pharmacology has broadened its scope considerably in recent decades. Initially, it was of interest to chemists, doctors and pharmacists. In recent years, however, it has been incorporated into the teaching of biologists, molecular biologists, biotechnologists, chemical engineers and many health professionals, among others. Traditional teaching methods, such as lectures or laboratory work, have been superseded by the use of new pedagogical approaches to enable a better conceptualization and understanding of the discipline. In this article, we present several new methods that have been used in Spanish universities. Firstly, we describe a teaching network that has allowed the sharing of pedagogical innovations in Spanish universities. A European experience to improve prescribing safety is described in detail. The use of popular films and medical TV series in biomedical students shows how these audiovisual resources can be helpful in teaching pharmacology. The use of virtual worlds is detailed to introduce this new approach to teaching. The increasingly important area of the social aspects of pharmacology is also considered in two sections, one devoted to social pharmacology and the other to the use of learning based on social services to improve understanding of this important area. Finally, the use of Objective Structured Clinical Evaluation in pharmacology allows to know how this approach can help to better evaluate clinical pharmacology students. In conclusion, this article allows to know new pedagogical methods resources used in some Spanish universities that may help to improve the teaching of pharmacology.

Effect of substrate mismatch, orientation, and flexibility on heterogeneous ice nucleation.

Heterogeneous nucleation is the main path to ice formation on Earth. The ice nucleating ability of a certain substrate is mainly determined by both molecular interactions and the structural mismatch between the ice and the substrate lattices. We focus on the latter factor using molecular simulations of the mW model. Quantifying the effect of structural mismatch alone is challenging due to its coupling with molecular interactions. To disentangle both the factors, we use a substrate composed of water molecules in such a way that any variation on the nucleation temperature can be exclusively ascribed to the structural mismatch. We find that a 1% increase in structural mismatch leads to a decrease of ∼4 K in the nucleation temperature. We also analyze the effect of orientation of the substrate with respect to the liquid. The three main ice orientations (basal, primary prism, and secondary prism) have a similar ice nucleating ability. We finally assess the effect of lattice flexibility by comparing substrates where molecules are immobile to others where a certain freedom to fluctuate around the lattice positions is allowed. Interestingly, we find that the latter type of substrate is more efficient in nucleating ice because it can adapt its structure to that of ice.

Obtaining and evaluating of enzymatic extracts from hairless canary seed (CDC Maria) as gluten-free bread-improving agents.

In this study, enzyme extracts were obtained from hairless canary seeds (CDC Maria) and used as gluten-free bread improvers. The enzyme extraction was done with a specific protein buffer solution and subsequent centrifugation. The supernatant was called crude enzyme extract, a fraction of this extract was refrigerated (CE) and another fraction was lyophilized (CEL). The lyoprotective effect of corn fiber (CEL + CF), maltodextrin (CEL + M), and inulin (CEL + I) was evaluated. Each enzyme extract was added to a gluten-free bread at 0.25%, 0.5%, 0.75% and 1% (w/w). The quality of the gluten-free bread was determined by external and internal characteristically, physical and sensory analysis: analysis of the lamella thickness, the shape factor of pores, the final volume, the aeration percentage, the texture profile analysis, the pore size distribution and shelf-life time. The results showed that the formulation with CEL at 0.5% (w/w) significantly improved the gluten-free bread quality properties, generating an increase of the final volume and aeration percentage, a reduction of the firmness, chewiness and sample aging, and a finer and more uniform crumb structure when compared to a control sample (P < 0.001). This study revealed the potential of a food-improving additive obtained from a natural origin with a high-level production in Argentina.

Growth rate of CO2 and CH4 hydrates by means of molecular dynamics simulations.

CO2 and CH4 hydrates are of great importance both from an energetic and from an environmental point of view. It is therefore highly relevant to quantify and understand the rate with which they grow. We use molecular dynamics simulations to shed light on the growth rate of these hydrates. We put the solid hydrate phase in contact with a guest aqueous solution in equilibrium with the pure guest phase and study the growth of both hydrates at 400 bars with temperature. We compare our results with previous calculations of the ice growth rate. We find a growth rate maximum as a function of the supercooling in all cases. The incorporation of guest molecules into the solid structure strongly decelerates hydrate growth. Consistently, ice grows faster than either hydrate and the CO2 hydrate grows faster than the CH4 one because of the higher solubility of CO2. We also quantify the molecular motion required to build the solids under study and find that the distance traveled by liquid molecules exceeds by orders of magnitude that advanced by any solid. Less molecular motion is needed in order for ice to grow as compared to the hydrates. Moreover, when temperature increases, more motion is needed for solid growth. Finally, we find a good agreement between our growth rate calculations and experiments of hydrate growth along the guest-solution interface. However, more work is needed to reconcile experiments of hydrate growth toward the solution among each other and with simulations.

Homogeneous nucleation rate of methane hydrate formation under experimental conditions from seeding simulations.

In this work, we shall estimate via computer simulations the homogeneous nucleation rate for the methane hydrate at 400 bars for a supercooling of about 35 K. The TIP4P/ICE model and a Lennard-Jones center were used for water and methane, respectively. To estimate the nucleation rate, the seeding technique was employed. Clusters of the methane hydrate of different sizes were inserted into the aqueous phase of a two-phase gas-liquid equilibrium system at 260 K and 400 bars. Using these systems, we determined the size at which the cluster of the hydrate is critical (i.e., it has 50% probability of either growing or melting). Since nucleation rates estimated from the seeding technique are sensitive to the choice of the order parameter used to determine the size of the cluster of the solid, we considered several possibilities. We performed brute force simulations of an aqueous solution of methane in water in which the concentration of methane was several times higher than the equilibrium concentration (i.e., the solution was supersaturated). From brute force runs, we infer the value of the nucleation rate for this system rigorously. Subsequently, seeding runs were carried out for this system, and it was found that only two of the considered order parameters were able to reproduce the value of the nucleation rate obtained from brute force simulations. By using these two order parameters, we estimated the nucleation rate under experimental conditions (400 bars and 260 K) to be of the order of log10 (J/(m3 s)) = -7(5).

Trend changes in the incidence of benign parotid tumours in the last 30 years in a Spanish population.

PURPOSE: To study the demographics and clinical characteristics of benign parotid tumours, focusing on the evolution of the incidence of Warthin tumour (WT) in recent years. METHODS: A retrospective observational study is designed of patients diagnosed with a benign parotid tumour in a single tertiary hospital centre, from 1994 to 2021. The evaluation of the relationship between the different variables, and the changes in tumour incidence, is carried out using an analysis of standardized residuals. RESULTS: The study evaluated 279 patients, and the total of benign parotid tumours was 291. The most frequent type of tumour was pleomorphic adenoma (PA) (52.7%), followed by WT (37.6%). WT was more frequent in men (79%), and PA in women (55.8%). Smoking history is significantly high in patients with WT (83%), as well as mid-age, compared to PA. CONCLUSIONS: It seems to be an increase in the proportion of WT compared to PA in recent years. These changes can be concerning tobacco use and older patients at diagnosis in our series.

Phase diagram of the NaCl-water system from computer simulations.

NaCl aqueous solutions are ubiquitous. They can crystallize into ice, NaCl, or NaCl · 2H2O depending on the temperature-concentration conditions. These crystallization transitions have important implications in geology, cryopreservation, or atmospheric science. Computer simulations can help understand the crystallization of these solids, which requires a detailed knowledge of the equilibrium phase diagram. We use molecular simulations in which we put at contact the solution with the solid of interest to determine points of the solid-solution coexistence lines. We follow two different approaches, one in which we narrow down the melting temperature for a given concentration and the other in which we equilibrate the concentration for a given temperature, obtaining consistent results. The phase diagram thus calculated for the selected model (TIP4P/2005 for water molecules and Joung-Cheatham for the ions) correctly predicts coexistence between the solution and ice. We were only able to determine NaCl · 2H2O-solution coexistence points at higher temperatures and concentrations than in the experiment, so we could not establish a direct comparison in this case. On the other hand, the model underestimates the concentration of the solution in equilibrium with the NaCl solid. Our results, alongside other literature evidence, seem to indicate that ion-ion interactions are too strong in the model. Our work is a good starting point for the improvement of the potential model and for the study of the nucleation kinetics of the solid phases involved in the phase diagram.

Homogeneous nucleation of NaCl in supersaturated solutions.

The seeding method is an approximate approach to investigate nucleation that combines molecular dynamics simulations with classical nucleation theory. Recently, this technique has been successfully implemented in a broad range of nucleation studies. However, its accuracy is subject to the arbitrary choice of the order parameter threshold used to distinguish liquid-like from solid-like molecules. We revisit here the crystallization of NaCl from a supersaturated brine solution and show that consistency between seeding and rigorous methods, like Forward Flux Sampling (from previous work) or spontaneous crystallization (from this work), is achieved by following a mislabelling criterion to select such threshold (i.e. equaling the fraction of the mislabelled particles in the bulk parent and nucleating phases). This work supports the use of seeding to obtain fast and reasonably accurate nucleation rate estimates and the mislabelling criterion as one giving the relevant cluster size for classical nucleation theory in crystallization studies.

The Young-Laplace equation for a solid-liquid interface.

The application of the Young-Laplace equation to a solid-liquid interface is considered. Computer simulations show that the pressure inside a solid cluster of hard spheres is smaller than the external pressure of the liquid (both for small and large clusters). This would suggest a negative value for the interfacial free energy. We show that in a Gibbsian description of the thermodynamics of a curved solid-liquid interface in equilibrium, the choice of the thermodynamic (rather than mechanical) pressure is required, as suggested by Tolman for the liquid-gas scenario. With this definition, the interfacial free energy is positive, and the values obtained are in excellent agreement with previous results from nucleation studies. Although, for a curved fluid-fluid interface, there is no distinction between mechanical and thermal pressures (for a sufficiently large inner phase), in the solid-liquid interface, they do not coincide, as hypothesized by Gibbs.

Ice growth rate: Temperature dependence and effect of heat dissipation.

The transformation of liquid water into solid ice is arguably the most important phase transition on Earth. A key aspect of such transformation is the speed with which ice grows once it is nucleated. There are contradictory experimental results as to whether the ice growth rate shows a maximum on cooling. Previous simulation results point to the existence of such a maximum. However, simulations were performed at constant temperature with the aid of a thermostat that dissipates the heat released at the ice-water interface unrealistically fast. Here, we perform simulations of ice growth without any thermostat. Large systems are required to perform these simulations at constant overall thermodynamic conditions (pressure and temperature). We obtain the same growth rate as in previous thermostatted simulations. This implies that the dynamics of ice growth is not affected by heat dissipation. Our results strongly support the experiments predicting the existence of a maximum in the ice growth rate. By using the Wilson-Frenkel kinetic theory, we argue that such maximum is due to a competition between an increasing crystallization thermodynamic driving force and a decreasing molecular mobility on cooling.

MicroRNA (miR)-203 and miR-205 expression patterns identify subgroups of prognosis in cutaneous squamous cell carcinoma.

BACKGROUND: Cutaneous squamous cell carcinoma (CSCC) is the second most widespread cancer in humans and its incidence is rising. These tumours can evolve as diseases of poor prognosis, and therefore it is important to identify new markers to better predict its clinical evolution. OBJECTIVES: We aimed to identify the expression pattern of microRNAs (miRNAs or miRs) at different stages of skin cancer progression in a panel of murine skin cancer cell lines. Owing to the increasing importance of miRNAs in the pathogenesis of cancer, we considered the possibility that miRNAs could help to define the prognosis of CSCC and aimed to evaluate the potential use of miR-203 and miR-205 as biomarkers of prognosis in human tumours. METHODS: Seventy-nine human primary CSCCs were collected at the University Hospital of Salamanca in Spain. We identified differential miRNA expression patterns at different stages of CSCC progression in a well-established panel of murine skin cancer cell lines, and then selected miR-205 and miR-203 to evaluate their association with the clinical prognosis and evolution of human CSCC. RESULTS: miR-205 was expressed in tumours with pathological features recognized as indicators of poor prognosis such as desmoplasia, perineural invasion and infiltrative growth pattern. miR-205 was mainly expressed in undifferentiated areas and in the invasion front, and was associated with both local recurrence and the development of general clinical events of poor evolution. miR-205 expression was an independent variable selected to predict events of poor clinical evolution using the multinomial logistic regression model described in this study. In contrast, miR-203 was mainly expressed in tumours exhibiting the characteristics associated with a good prognosis, was mainly present in well-differentiated zones, and rarely expressed in the invasion front. Therefore, the expression and associations of miR-205 and miR-203 were mostly mutually exclusive. Finally, using a logistic biplot we identified three clusters of patients with differential prognosis based on miR-203 and miR-205 expression, and pathological tumour features. CONCLUSIONS: miR-205 and miR-203 tended to exhibit mutually exclusive expression patterns in human CSCC. This work highlights the utility of miR-205 and miR-203 as prognostic markers in CSCC.

Extended phenotype and clinical subgroups in unilateral Meniere disease: A cross-sectional study with cluster analysis.

OBJECTIVES: To define clinical subgroups by cluster analysis in patients with unilateral Meniere disease (MD) and to compare them with the clinical subgroups found in bilateral MD. DESIGN: A cross-sectional study with a two-step cluster analysis. SETTINGS: A tertiary referral multicenter study. PARTICIPANTS: Nine hundred and eighty-eight adult patients with unilateral MD. MAIN OUTCOME MEASURES: best predictors to define clinical subgroups with potential different aetiologies. RESULTS: We established five clusters in unilateral MD. Group 1 is the most frequently found, includes 53% of patients, and it is defined as the sporadic, classic MD without migraine and without autoimmune disorder (AD). Group 2 is found in 8% of patients, and it is defined by hearing loss, which antedates the vertigo episodes by months or years (delayed MD), without migraine or AD in most of cases. Group 3 involves 13% of patients, and it is considered familial MD, while group 4, which includes 15% of patients, is linked to the presence of migraine in all cases. Group 5 is found in 11% of patients and is defined by a comorbid AD. We found significant differences in the distribution of AD in clusters 3, 4 and 5 between patients with uni- and bilateral MD. CONCLUSIONS: Cluster analysis defines clinical subgroups in MD, and it extends the phenotype beyond audiovestibular symptoms. This classification will help to improve the phenotyping in MD and facilitate the selection of patients for randomised clinical trials.

On the time required to freeze water.

By using the seeding technique the nucleation rate for the formation of ice at room pressure will be estimated for the TIP4P/ICE model using longer runs and a smaller grid of temperatures than in the previous work. The growth rate of ice will be determined for TIP4P/ICE and for the mW model of water. Although TIP4P/ICE and mW have a similar melting point and melting enthalpy, they differ significantly in the dynamics of freezing. The nucleation rate of mW is lower than that of TIP4P/ICE due to its higher interfacial free energy. Experimental results for the nucleation rate of ice are between the predictions of these two models when obtained from the seeding technique, although closer to the predictions of TIP4P/ICE. The growth rate of ice for the mW model is four orders of magnitude larger than for TIP4P/ICE. Avrami's expression is used to estimate the crystallization time from the values of the nucleation and growth rates. For mW the minimum in the crystallization time is found at approximately 85 K below the melting point and its value is of about a few ns, in agreement with the results obtained from brute force simulations by Moore and Molinero. For the TIP4P/ICE the minimum is found at about 55 K below the melting point, but its value is about ten microseconds. This value is compatible with the minimum cooling rate required to avoid the formation of ice and obtaining a glass phase. The crossover from the nucleation controlled crystallization to the growth controlled crystallization will be discussed for systems of finite size. This crossover could explain the apparent discrepancy between the values of J obtained by different experimental groups for temperatures below 230 K and should be considered as an alternative hypothesis to the two previously suggested: internal pressure and/or surface freezing effects. A maximum in the compressibility was found for the TIP4P/ICE model in supercooled water. The relaxation time is much smaller than the crystallization time at the temperature at which this maximum occurs, so this maximum is a real thermodynamic feature of the model. At the temperature of minimum crystallization time, the crystallization time is larger than the relaxation time by just two orders of magnitude.

On the calculation of solubilities via direct coexistence simulations: Investigation of NaCl aqueous solutions and Lennard-Jones binary mixtures.

Direct coexistence molecular dynamics simulations of NaCl solutions and Lennard-Jones binary mixtures were performed to explore the origin of reported discrepancies between solubilities obtained by direct interfacial simulations and values obtained from the chemical potentials of the crystal and solution phases. We find that the key cause of these discrepancies is the use of crystal slabs of insufficient width to eliminate finite-size effects. We observe that for NaCl crystal slabs thicker than 4 nm (in the direction perpendicular to the interface), the same solubility values are obtained from the direct coexistence and chemical potential routes, namely, 3.7 ± 0.2 molal at T = 298.15 K and p = 1 bar for the JC-SPC/E model. Such finite-size effects are absent in the Lennard-Jones system and are likely caused by surface dipoles present in the salt crystals. We confirmed that µs-long molecular dynamics runs are required to obtain reliable solubility values from direct coexistence calculations, provided that the initial solution conditions are near the equilibrium solubility values; even longer runs are needed for equilibration of significantly different concentrations. We do not observe any effects of the exposed crystal face on the solubility values or equilibration times. For both the NaCl and Lennard-Jones systems, the use of a spherical crystallite embedded in the solution leads to significantly higher apparent solubility values relative to the flat-interface direct coexistence calculations and the chemical potential values. Our results have broad implications for the determination of solubilities of molecular models of ionic systems.

Computerized pharmaceutical intervention to reduce reconciliation errors at hospital discharge in Spain: an interrupted time-series study.

WHAT IS KNOWN AND OBJECTIVE: It is well known that medication reconciliation at discharge is a key strategy to ensure proper drug prescription and the effectiveness and safety of any treatment. Different types of interventions to reduce reconciliation errors at discharge have been tested, many of which are based on the use of electronic tools as they are useful to optimize the medication reconciliation process. However, not all countries are progressing at the same speed in this task and not all tools are equally effective. So it is important to collate updated country-specific data in order to identify possible strategies for improvement in each particular region. Our aim therefore was to analyse the effectiveness of a computerized pharmaceutical intervention to reduce reconciliation errors at discharge in Spain. METHODS: A quasi-experimental interrupted time-series study was carried out in the cardio-pneumology unit of a general hospital from February to April 2013. The study consisted of three phases: pre-intervention, intervention and post-intervention, each involving 23 days of observations. At the intervention period, a pharmacist was included in the medical team and entered the patient's pre-admission medication in a computerized tool integrated into the electronic clinical history of the patient. The effectiveness was evaluated by the differences between the mean percentages of reconciliation errors in each period using a Mann-Whitney U test accompanied by Bonferroni correction, eliminating autocorrelation of the data by first using an ARIMA analysis. In addition, the types of error identified and their potential seriousness were analysed. RESULTS AND DISCUSSION: A total of 321 patients (119, 105 and 97 in each phase, respectively) were included in the study. For the 3966 medicaments recorded, 1087 reconciliation errors were identified in 77·9% of the patients. The mean percentage of reconciliation errors per patient in the first period of the study was 42·18%, falling to 19·82% during the intervention period (P = 0·000). When the intervention was withdrawn, the mean percentage of reconciliation errors increased again to 27·72% (P = 0·008). The difference between the percentages of pre- and post-intervention periods was statistically significant (P = 0·000). Most reconciliation errors were due to omission (46·7%) or incomplete prescription (43·8%), and 35·3% of which could have caused harm to the patient. WHAT IS NEW AND CONCLUSION: A computerized pharmaceutical intervention is shown to reduce reconciliation errors in the context of a high incidence of such errors.

Staphylococcus pseudintermedius can be misdiagnosed as Staphylococcus aureus in humans with dog bite wounds.

The purpose of this study was to investigate whether S. pseudintermedius is misdiagnosed as S. aureus by clinical laboratories when isolated from humans with dog bite wounds. In addition, we attempted to determine whether S. pseudintermedius isolates related to dog bite wounds share phenotypic and genotypic traits. S. pseudintermedius was identified by PCR targeting the nuc gene. Isolates were tested for antibiotic susceptibility using VetMIC GP-mo microdilution panels. The occurrence of genes encoding leukocidins, exfoliatins, pyrogenic toxin superantigens and enterotoxins was determined by PCR. The relatedness of S. pseudintermedius isolates was investigated using Multi Locus Sequence Typing (MLST). Out of 101 isolates defined as S. aureus by human clinical microbiology laboratories, 13 isolates were re-identified as S. pseudintermedius and one isolate was confirmed to carry the mecA gene, i.e. methicillin-resistant (MRSP). The MRSP isolate was also defined as multi-resistant. Two methicillin-susceptible S. pseudintermedius isolates were also multi-resistant and five were susceptible to all antibiotics tested. With the exception of three S. pseudintermedius isolates belonging to multi locus sequence type (MLST) 158, all the isolates belonged to unique STs. All isolates contained lukS/F-I, siet and se-int, and expA were identified in two isolates and expB and sec canine-sel in one isolate respectively. S. pseudintermedius is frequently misdiagnosed as S. aureus from humans with dog bite wounds showing that it can act as an opportunistic pathogen in humans. No common phenotypic and genotypic traits shared by the S. pseudintermedius isolates could be identified.

Nucleation free-energy barriers with Hybrid Monte-Carlo/Umbrella Sampling.

The aim of this work is to evaluate nucleation free-energy barriers using molecular dynamics (MD). More specifically, we use a combination of Hybrid Monte Carlo (HMC) and an Umbrella Sampling scheme, and compute the crystallisation barrier of NaCl from its melt. Firstly the convergence and performance of HMC for different time-steps and the number of MD steps within a HMC cycle are assessed. The calculated potential energies and densities converge regardless of the chosen time-step. However the acceptance ratio of the Metropolis step within the HMC scheme strongly depends on the time-step and affects the performance. It is shown that the acceptance ratio is close to 100% for time-steps of the order of those commonly used in molecular dynamics runs. We then explore the results obtained with a "non-Metropolised" version of HMC where the MD trajectories are always accepted (omitting the Metropolis criteria) and conclude that they are satisfactory for time-steps below 5 fs. Next, HMC is combined with Umbrella Sampling (HMC/US) to compute the nucleation free-energy for both the standard and the "non-Metropolised" HMC (using a small time-step) and in both cases find excellent agreement with the reported values. To conclude, we explore approximations to the HMC/US technique implementing HMC with isothermal-isobaric MD trajectories. The computed nucleation free-energy curve is coincident, within the statistical error, with previous calculations.

The mold integration method for the calculation of the crystal-fluid interfacial free energy from simulations.

The interfacial free energy between a crystal and a fluid, γcf, is a highly relevant parameter in phenomena such as wetting or crystal nucleation and growth. Due to the difficulty of measuring γcf experimentally, computer simulations are often used to study the crystal-fluid interface. Here, we present a novel simulation methodology for the calculation of γcf. The methodology consists in using a mold composed of potential energy wells to induce the formation of a crystal slab in the fluid at coexistence conditions. This induction is done along a reversible pathway along which the free energy difference between the initial and the final states is obtained by means of thermodynamic integration. The structure of the mold is given by that of the crystal lattice planes, which allows to easily obtain the free energy for different crystal orientations. The method is validated by calculating γcf for previously studied systems, namely, the hard spheres and the Lennard-Jones systems. Our results for the latter show that the method is accurate enough to deal with the anisotropy of γcf with respect to the crystal orientation. We also calculate γcf for a recently proposed continuous version of the hard sphere potential and obtain the same γcf as for the pure hard sphere system. The method can be implemented both in Monte Carlo and Molecular Dynamics. In fact, we show that it can be easily used in combination with the popular Molecular Dynamics package GROMACS.

Homogeneous ice nucleation evaluated for several water models.

In this work, we evaluate by means of computer simulations the rate for ice homogeneous nucleation for several water models such as TIP4P, TIP4P/2005,TIP4P/ICE, and mW (following the same procedure as in Sanz et al. [J. Am. Chem. Soc. 135, 15008 (2013)]) in a broad temperature range. We estimate the ice-liquid interfacial free-energy, and conclude that for all water models γ decreases as the temperature decreases. Extrapolating our results to the melting temperature, we obtain a value of the interfacial free-energy between 25 and 32 mN/m in reasonable agreement with the reported experimental values. Moreover, we observe that the values of γ depend on the chosen water model and this is a key factor when numerically evaluating nucleation rates, given that the kinetic prefactor is quite similar for all water models with the exception of the mW (due to the absence of hydrogens). Somewhat surprisingly the estimates of the nucleation rates found in this work for TIP4P/2005 are slightly higher than those of the mW model, even though the former has explicit hydrogens. Our results suggest that it may be possible to observe in computer simulations spontaneous crystallization of TIP4P/2005 at about 60 K below the melting point.

Methicillin-resistant Staphylococcus aureus of lineage ST398 as cause of mastitis in cows.

UNLABELLED: The objective of this study was to analyse the prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in milk of cows with mastitis. The California mastitis test (CMT) was used to detect the presence of mastitis in all 100 cows of a farm in Brazil. The CMT was positive in milk of 115 mammary quarters from 36 cows (36%). MRSA isolates were recovered from 4 of these 36 cows with mastitis (11%), and they were further characterized (one MRSA/sample). The four MRSA isolates were typed as t011-ST398-agr1-SCCmecV and presented two different pulsed-field-gel-electrophoresis-ApaI patterns. These four MRSA isolates showed resistance to tetracycline, streptomycin and ciprofloxacin, carried the mecA, blaZ, tet(K), and tet(M) resistance genes, and presented the S84L and S80F amino acid substitutions in GyrA and GrlA proteins, respectively. Two ST398 isolates exhibited resistance to gentamicin and tobramycin [with aac(6)-aph(2") and ant(4)-Ia genes] and one isolate resistance to clindamycin [with lnu(B) and lsa(E) genes]; this latter isolate also carried the spectinomycin/streptomycin resistance genes spw and aadE. MRSA of lineage ST398 is worldwide spread, normally multidrug resistant and may be responsible for bovine mastitis. To our knowledge, this is the first detection of MRSA-ST398 in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies on the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) from bovine isolates have been performed in Brazil. MRSA of lineage ST398 is worldwide spread and associated with farm animals. Multidrug-resistant MRSA-ST398 isolates were recovered in 11% of mastitic cows from a single farm, with one isolate carrying the unusual lsa(E), spw and aadE genes. To our knowledge, this is the first detection of MRSA-ST398 isolates in milk samples of cows with mastitis in Brazil.